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WO2026006809A1 - Multispecific molecules binding tnfr2 and cd25 and uses thereof - Google Patents

Multispecific molecules binding tnfr2 and cd25 and uses thereof

Info

Publication number
WO2026006809A1
WO2026006809A1 PCT/US2025/035805 US2025035805W WO2026006809A1 WO 2026006809 A1 WO2026006809 A1 WO 2026006809A1 US 2025035805 W US2025035805 W US 2025035805W WO 2026006809 A1 WO2026006809 A1 WO 2026006809A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
cdr3
cdr2
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/035805
Other languages
French (fr)
Inventor
Luigi Franchi
Anthony W. Opipari
Lee Kim SWEE
Paul-Albert KÖNIG
Laura PREISS
Ferdinand Huber
Annegrit SEIFRIED
Julia KNOPF
Tomasz PRÓCHNICKI
Tobias STUWE
Stephen Michael SOISSON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Odyssey Therapeutics Inc
Original Assignee
Odyssey Therapeutics Inc
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Filing date
Publication date
Application filed by Odyssey Therapeutics Inc filed Critical Odyssey Therapeutics Inc
Publication of WO2026006809A1 publication Critical patent/WO2026006809A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to multispecific antigen-binding proteins, and methods of use thereof.
  • the multispecific antigen-binding proteins including bispecific molecules, may comprise one or more antigen-binding domains that specifically bind tumor necrosis factor receptor 2 (TNFR2), and one or more antigen-binding domains that specifically bind cluster of differentiation 25 (CD25).
  • TNFR2 tumor necrosis factor receptor 2
  • CD25 cluster of differentiation 25
  • Tregs regulatory T cells
  • Tregs are a subset of T ceils that play a crucial role in peripheral self-tolerance and the prevention of autoimmunity. Due to their potent immunosuppressive function, Tregs can be targeted for the treatment of autoimmunity. Current strategies seeking to increase or modulate Tregs in autoimmune patients are based on the ex vivo expansion of Tregs prior to autologous transfer.
  • Tregs can support immune homeostasis under normal, healthy conditions, and their activation can be beneficial in the context of autoimmune disease, during proliferative diseases (e.g., cancer), Tregs can accumulate within the tumor microenvironment where they can hamper antitumor responses mounted by infiltrating immune cells, effectively protecting the cancer cells from immune attack.
  • Tregs are capable of suppressing most types of immune cells including CD4+ and CDS-i- T cells, B cells, and antigen-presenting cells (APCs) (e.g., dendritic cells macrophages and monocytes), natural killer (NK) cells, and NKT cells.
  • APCs antigen-presenting cells
  • Tregs are higher in tumors and peripheral blood mononuclear cells (PBMCs) of many cancer patients, and high Treg levels can be associated with poor prognosis, e.g., in solid tumors including breast, cervical, renal, melanomas, ovarian, hepatocellular, gastric, and pancreatic cancers.
  • PBMCs peripheral blood mononuclear cells
  • a multispecific antigen-binding protein comprising one or more binding domains that specifically bind tumor necrosis factor receptor 2 (TNFR2), and one or more binding domains that specifically bind cluster of differentiation 25 (CD25).
  • the multispecific antigen-binding protein further comprises one or more binding domains that binds to another antigen, e.g., human serum albumin.
  • the multispecific antigen-binding protein is multivalent, e.g., bivalent, trivalent, tetravalent, pentavalent, hexavalent, septivalent, or octavalent for both antigens.
  • the multispecific antigen-binding protein comprises at least one of the anti-TNFR2 antigen-binding domains described herein. In some embodiments, the multispecific antigenbinding protein comprises two of the anti-TNFR2 antigen-binding domains described herein. In some embodiments, the multispecific antigen-binding protein comprises three of the anti-TNFR2 antigenbinding domains described herein. In some embodiments, the multispecific antigen-binding protein comprises four of the anti-TNFR2 antigen-binding domains described herein. In some embodiments, the multispecific antigen-binding protein comprises five of the anti-TNFR2 antigen-binding domains described herein. In some embodiments, the multispecific antigen-binding protein comprises six of the anti-TNFR2 antigen-binding domains described herein.
  • the multispecific antigen-binding protein comprises at least one of the anti-CD25 antigen-binding domains described herein. In some embodiments, the multispecific antigenbinding protein comprises two of the anti-CD25 antigen-binding domains described herein, in some embodiments, the multispecific antigen-binding protein comprises three of the anti-CD25 antigenbinding domains described herein. In some embodiments, the multispecific antigen-binding protein comprises four of the anti-CD25 antigen-binding domains described herein.
  • the multispecific antigen-binding protein comprises five of the anti-CD25 antigen-binding domains described herein, in some embodiments, the multispecific antigen-binding protein comprises six of the anti-CD25 antigen-binding domains described herein.
  • the one or more anti-TNFR2 antigen-binding domains and/or the one or more anti-CD25 antigen-binding domains are linked by a flexible linker. In some embodiments of the multispecific antigen-binding protein described herein, the one or more anti-TNFR2 antigen-binding domains and/or the one or more anti- CD25 antigen-binding domains are linked by a rigid linker,
  • the one or more anti-TNFR2 antigen-binding domains may bind to the same epitope on TNFR2. In other embodiments, the one or more anti-TNFR2 antigen-binding domains may bind to different epitopes on TNFR2.
  • the one or more anti-CD25 antigen-binding domains may bind to the same epitope on CD25. In other embodiments, the one or more anti-CD25 antigen-binding domains may bind to different epitopes on CD25,
  • the present disclosure provides one or more antigen-binding domains that specifically bind TNFR2, comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from a) (Y/F) ⁇ Q(S/A)LS(T/S)(P/A)N(Y/F)GQ(V/T)F (SEQ ID NO: 60); b) AADSDL(S/R)TV(V/T)(V/T)GPHDY (SEQ ID NO: 61); c) AKDAG(S/G)WG(T/R)GPFG(Y/F)(E/D) ⁇ D ⁇ (SEQ ID NO: 62); d) A(A/G)(T/A/S)(P/L)(S/T)GKAY(T/S)Y (SEQ ID NO: 10387); e) ATPGPY(T/S/M)YCAPYGSSWSRG ⁇ DY (SEQ ID NO: 64);
  • the CDR3 of the one or more anti-TNFR2 antigen-binding domains comprises an amino acid sequence a). (T/A/V)(A/G)(S/A)(A/P)(A/T/S)(A/G)(A/R) A(A/F)(T/N/A)(A/Y); or b). (A/G)(A/G)(T/A/S)(A/P/L)(A/S/T)(A/G)(A/K)(A/G)(A/Y)(A/T/S)(A/Y).
  • the CDR3 of the one or more anti ⁇ TNFR2 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 3, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 63, 6635, 6639, 6643, 7093, 7099, 7289-7292, 7333, 10374, 10410-10433, 10435-10455, and 10811.
  • the one or more anti-TNFR2 antigen-binding domains further comprise a CDR1 comprising an amino acid sequence selected from a) GSI(V/F)(R/S)(T/A)(N/D)(S/G/A); b) GFT ⁇ F/L)DD(I/Y)A (SEQ ID NO: 69); c) GFTFS(S/R/G)YA (SEQ ID NO: 70); d) GRTFSDYG (SEQ ID NO: 16); e) G(L/F)TLDYYA (SEQ ID NO: 71); f) GF(T/N)FSM ⁇ S (SEQ ID NO: 72); g) GRTF(G/R/S)(N/S)(Y/L)(T/F) (SEQ ID NO: 73); h) GASLSRNA (SEQ ID NO: 40); i) GS(I/T)FRFPP (SEQ ID NO: 74);
  • the one or more anti-TNFR2 antigen-binding domains comprise a CORI comprising an amino acid sequence selected from SEQ ID NOs: 1, 5, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 6633, 6637, 6641, 7089, and 7281-7284.
  • the one or more anti-TNFR2 antigen-binding domains comprise a CDR2 comprising an amino add sequence selected from a) IRSDGF(T/I) (SEQ ID NO: 75); b) l(Y/F)SY(S/G)(S/P)NT (SEQ ID NO: 76); c) l(Y/S)(S/D)DGS(E/D)T (SEQ ID NO: 77); d) INWS(N/Q/E/S)(G/A)RT (SEQ ID NO: 10409); e) l(S/N)(V/T)(S/G)DGST (SEQ ID NO: 78); f) IDT(R/G)GST (SEQ ID NO: 79); g) IR(W/R/Y)(T/P)G(G/L)(S/I)T (SEQ ID NO: 80); h) IYDDGET (SEQ ID NO:
  • the one or more anti-TNFR2 antigen-binding domains comprise a CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 2, 9, 13, 17, 21, 25, 23, 33, 37, 41, 45, 6634, 6638, 6642, 7096, 7268, and 7285-7288, and 10388-10393.
  • the one or more anti-TNFR2 antigen-binding domains comprise i)a CDR1 comprising an amino acid sequence of GSI(V/F)(R/S)(T/A)(N/D)(S/G/A), a CDR2 comprising an amino acid sequence of SEQ iD NO: 75, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 60; ii)a CDR1 comprising an amino acid sequence of SEQ iD NO: 69, a CDR2 comprising an amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 61; iii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 70, a CDR2 comprising an amino acid sequence of SEQ ID NO: 77, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 62; iv)a CDR1 comprising an amino acid sequence of GSI(V/F
  • the one or more anti-TNFR2 antigen-binding domains comprise a)a CDR1 comprising an amino acid sequence of SEQ ID NO: 69, a CDR2 comprising an amino acid sequence of SEQ iD NO: 76, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 61; b)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10387; c)a CDR1 comprising an amino acid sequence of SEQ ID NO: 73, a CDR2 comprising an amino acid sequence of SEQ iD NO: 80, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 66; d)a CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 61; b
  • ATPGPYTYCAPYGSSWSRGYDY (SEQ ID NO: 22); vi)a CDR1 comprising an amino acid sequence of GF(T/N)FSMYS (SEQ ID NO: 72), a CDR2 comprising an amino acid sequence of IDT(R/G)GST (SEQ ID NO: 79), and a CDR3 comprising an amino acid sequence of ARV(G/R)G(T/A)PYEY(N/G)Y (SEQ ID NO: 7273); vii)a CDR1 comprising an amino acid sequence of GRTF(G/S)S(Y/L)(T/F) (SEQ ID NO: 7274), a CDR2 comprising an amino acid sequence of IR(W/R/Y)(T/P)G(G/L)(S/I)T (SEQ ID NO: 80), and a CDR3 comprising an amino acid sequence of (A/V)A(A/S)PTGRAF(T/N)Y (SEQ ID NO
  • the one or more anti-TNFR2 antigen-binding domains comprise a)a CDR1 comprising an amino acid sequence of GFT(F/L)DD(I/Y)A (SEQ ID NO: 69), a CDR2 comprising an amino acid sequence of l( ⁇ /F)S ⁇ (S/G)(S/P)NT (SEQ ID NO: 76), and a CDR3 comprising an amino acid sequence of AADSDLSTW(V/T)GPHDY (SEQ ID NO: 7271); b)a CDR1 comprising an amino acid sequence of GRTFSDYG (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of INWSN(G/A)RT (SEQ ID NO: 7268), and a CDR3 comprising an amino acid sequence of A(A/G)(T/A/S)(P/L)(S/T)GKAY(T/S)Y (SEQ ID NO: 10387); or
  • the one or more anti-TNFR2 antigen-binding domains comprise i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 3; ii) a CDR1 comprising an amino acid sequence of SEQ iD NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6; iii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10; iv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 12, a CDR2 comprising an amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising an amino acid sequence of SEQ ID NO:
  • Iviiija CDRi comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10429; lix)a CDRI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10430; lx)a CDRI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10431; lxi)a CDRI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NQ: 104
  • Ixvixja CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10440; lxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10441; lxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10442;
  • Ixxiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10443; lxxiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10444;
  • Ixxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10445; lxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10446;
  • CDR1 comprising an amino acid sequence of SEQ ID NO: 16
  • CDR2 comprising an amino acid sequence of SEQ ID NO: 10389
  • CDR3 comprising an amino acid sequence of SEQ ID NO: 10447
  • Ixxviija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino add sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10448; Ixxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10449; ixxvix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10450; lxxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ
  • Ixxxija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10452; lxxxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ !D NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10453;
  • Ixxxiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10454; or
  • Ixxxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 16
  • a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389
  • a CDR3 comprising an amino acid sequence of SEQ ID NO: 10455.
  • the one or more anti-TNFR2 antigen-binding domains comprise a)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; b)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; c)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7099; d)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ ID NO:
  • the one or more anti-TNFR2 antigen-binding domains comprise a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1128-1686, 6745-6806, and 7102-7125; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1687- 2245, 6807-6868, and 7126-7149; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 2246-2804, 6869-6930, and 7150-7173.
  • the one or more anti-TNFR2 antigen-binding domains is a singie-domain antibody.
  • the single-domain antibody is a VHH, a VNAR, or a VH domain.
  • the VHH of the one or more anti-TNFR2 antigen-binding domains is a cameiid VHH.
  • the VHH of the one or more anti-TNFR2 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 93-640, 2805-3363, 6636, 6640, 6644, 7090, 6651-6697, 6931-6992, 7174-7197, 10380, 10381, 10383, 10385, and 10386, or a sequence having at least 75% identity thereto, in some embodiments, the VHH of the one or more anti-TNFR2 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 10380, 10381, 10383
  • the VHH of the one or more anti-TNFR2 antigen-binding domains is a humanized VHH.
  • the VHH of the one or more anti-TNFR2 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 6648, 6649, 6650, 7092, 7095, 7098, 7101,7293-7296, 641-1127, 6698-6744, 10375, 10382, 10384, 10394-10408, 10457-10502, 10720-10727, and 10812, or a sequence having at least 75% identity thereto.
  • the VHH of the one or more anti-TNFR2 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 6648, 6649, 6650, 7092, 7095, 7098, 7101, 7293-7296, 10375, 10382, 10384, 10394-10408, 10457-10502, 10720-10727, and 10812 or a sequence having at least 75% identity thereto.
  • the one or more anti-TNFR2 antigen-binding domains of the present disclosure comprise TNF or a variant thereof.
  • the one or more anti-TNFR2 antigen-binding domains bind to human TNFR2. in some embodiments, the one or more anti-TNFR2 antigen-binding domains bind to human TNFR2 with a KD of less than about 3xl0’ 6 M. in some embodiments, the one or more anti-TNFR2 antigen-binding domains bind to human TNFR2 with a KD of about IxiO" 10 to 5xW s M.
  • the one or more anti-TNFR2 antigen-binding domains bind to cyno TNFR2. In some embodiments, the one or more anti-TNFR2 antigen-binding domains bind to cyno TNFR2 with a KD of less than about 3xl0" 6 M.
  • the one or more anti-TNFR2 antigen-binding domains bind to cyno TNFR2 with a K D of about IxlO’ 9 to 2xW 7 M, [0035)1 n some embodiments, the one or more anti-TNFR2 antigen-binding domains bind to the same epitope(s) on TNFR2 as antibody clone MR2-1. In some embodiments, the one or more anti-TNFR2 antigen-binding domains do not bind to the same epitope(s) on TNFR2 as antibody clone MR2-1.
  • the one or more anti-TNFR2 antigen-binding domains have an agonist effect upon binding to TNFR2. In some embodiments, the one or more anti-TNFR2 antigen-binding domains have an agonist effect upon binding to TNFR2 with an EC50 of about 1-100 nM (e.g., l-10nM, 10-100 nM).
  • the present disclosure provides one or more antigen-binding domains that specifically binds CD25, comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from a) NAL(G/L/P/Q/W) ⁇ (SEQ ID NO: 4131); b) NALR(D/H/N/F) (SEQ ID NO: 4134); c) (K/S/T)TLRY (SEQ ID NO: 4136); d).
  • CDR3 complementarity determining region 3
  • AA(S/T)(D/N/Y/K)(F/V)(L/P)(I/L)A(T/I/A)(T/S/A)IS(A/G)(Y/H)DY (SEQ ID NO: 10308); f) AAYVYPDYYCS(D/E)YVLL(K/R)YD ⁇ (SEQ ID NO: 7363); g) NIYR(P/S)QVP(P/S/T)TR ⁇ S (SEQ ID NO: 7365); and h) AAKRLGP(M/I/A/L)VH(Q/R)YSLEVLTPLFLDEYDY (SEQ ID NO: 9423), wherein one or more non-alanine residues in the CDR3 sequence are optionally replaced with an alanine, and/or one or more alanine residues in the CDR3 sequence are optionally replaced with a glycine.
  • the CDR3 comprises an amino acid sequence a).(A/V/S)(A/K/T)(A/G)(A/R/K)(A/G/H/N/R)(A/S)(A/G)(A/S/G)(A/Y)(A/P)(A/W/F/L)(A/D/E)(A/D/E)(A/ Y/V); or b).
  • the CDR3 comprises an amino acid sequence selected from a). NAL(G/L/P/Q/W)Y (SEQ ID NO: 4131); b). NALR(D/H/N/F) (SEQ ID NO: 4134); c). (K/S/T)TLRY (SEQ ID NO: 4136); d). AKGR(H/N)SGSYYPWD(D/E)Y (SEQ ID NO: 4139); e). (A/V)KGR(G/H/N)SGSYYP(W/F)D(D/E)Y (SEQ ID NO: 9440); f).
  • AA(S/T)(D/N/Y)FL(i/L)ATTIS(A/G)YDY (SEQ ID NO: 4141); g). AAYVYPDYYCS(D/E)YVLL(K/R)YDY (SEQ ID NO: 7363); h). NIYR(P/S)QVP(P/S/T)TRYS (SEQ ID NO: 7365); and i). AAKRLGPMVH(Q/R)YSLEVLTPLFLDEYDY (SEQ ID NO: 7367).
  • the CDR3 of the one or more anti ⁇ CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4103, 4107, 4111, 4115, 4119, 4139,
  • the CDR3 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4103, 4107, 4111, 4115, 4119, 5337, 5339, 5371, 5375, 5398, 5401, 5431, 5515, 5519, 5521, 5528, 5532, 5542, 5544, 5545, 5547, 5548, 7344, 7347, 7349, 7350, 9411-9416, 9436, 9887, 9966, 9975, 9978, 9979, 9980, and 10504-10544.
  • the CDR3 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4103, 4107, 4111, 4115, 4119, 7344, 7347, 7349, 7350, 9411-9416, 9436, and 10504-10544.
  • the one or more anti-CD25 antigen-binding domains described herein may further comprise a CDR1 comprising an amino acid sequence selected from a) GR(K/R/S)FSTLI (SEQ ID NG: 4137); b) GFTFS(N/S)YA (SEQ ID NO: 4140); c) GRTF(A/S)(S/W/D)(F/N/Y)G (SEQ ID NO: 10309); d) GFTLDYYA (SEQ ID NO: 7342); and e) G(I/M)P(F/-)(A/-)L(P/V/Y)A (SEQ ID NO: 7366).
  • a CDR1 comprising an amino acid sequence selected from a) GR(K/R/S)FSTLI (SEQ ID NG: 4137); b) GFTFS(N/S)YA (SEQ ID NO: 4140); c) GRTF(A/S)(S/W/D)(
  • the CDR1 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4101, 4105, 4109, 4113, 4117, 4132,
  • the CDR1 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4101, 4105, 4109, 4113, 4117, 4132, 7342, and 7345.
  • the one or more anti-CD25 antigen-binding domains described herein may further comprise a CDR2 comprising an amino acid sequence selected from a) (l/V)(D/E)R(D/G)(D/G)T(A/P/T); b) l ⁇ SD(G/S)SGT (SEQ ID NO: 9441); c) IS(Q/R/G)(S/G)GGRT (SEQ ID NO: 10310); d) IS(R/S)(D/S)G(D/G)ST (SEQ ID NO: 7364); e) ISSGGNT ⁇ SEQ ID NO: 7346); and f) ISSTDGRT (SEQ ID NO: 7348).
  • a CDR2 comprising an amino acid sequence selected from a) (l/V)(D/E)R(D/G)(D/G)T(A/P/T); b) l ⁇ SD(G/S)SGT (SEQ ID NO: 9441
  • the CDR2 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence seiected from a) (l/V)(D/E)R(D/G)GT(A/P/T); b) l(D/E)RDGT(T/P) (SEQ ID NO: 4135); c) l(D/E)R(D/G)(D/G)T(P/T); d) IYSDGSGT (SEQ ID NO: 4114); e) ISQSGGRT (SEQ ID NO: 4118) f). !S(R/S)(D/S)G(D/G)ST (SEQ ID NO: 7364); g). ISSGGNT (SEQ ID NO: 7346); and h). ISSTDGRT (SEQ ID NO: 7348).
  • the CDR2 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence seiected from SEQ ID NOs: 4102, 4106, 4110, 4114, 4118, (l/V)(D/E)R(D/G)GT(A/P/T), 4135, l(D/E)R(D/G)(D/G)T(P/T), 5042, 5046, 5059, 5067, 5092, 5214, 5215, 5216, 1117, 7343, 7346, 7348, and 9435.
  • the CDR2 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4102, 4106, 4110, 4114, 4118, 7343, 7346, 7348, and 9435.
  • the one or more anti-CD25 antigen-binding domains comprise i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G](D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; in) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V) ⁇ D/E)R(D/G)(D/G)T(A/
  • a CORI comprising an amino acid sequence of SEQ iD NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)( D/E) R(D/G)GT(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ iD NO: 4131; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4135, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 4134; vi) a CDR1 comprising an amino acid sequence of SEQ iD NO: 4137, a CDR2 comprising an amino acid sequence of l(D/E)R(D/G)(D/G)T(P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4136; vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a
  • a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9441, and a CDR3 comprising an amino acid sequence of ⁇ A/V/S)(A/K/T)(A/G)(A/R/K)(A/G/H/N/RHA/S) ⁇ A/G)(A/S/G)(A/Y)(A/Y)(A/P)(A/W/F/L) ⁇ A/D/E)(A/D/E)(A/Y/ V); or xxi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of (A/G)(A/G)(A/K)(A/R)(A/L)(A/G)(A/P)(M/I/
  • the one or more CD25 antigen-binding domains comprise i) a CDR1 comprising an amino add sequence of SEQ ID NO: 4101, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4102, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4103; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4106, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4107; ill) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4109, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4110, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4111; ivj a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ
  • a CDR1 comprising an amino acid sequence of SEQ iD NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino add sequence of SEQ ID NO: 9966;
  • a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9436;
  • a CDR1 comprising an amino acid sequence of SEQ ID NO: 4109, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4110, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4111;
  • a CDR1 comprising an amino acid sequence of SEQ, ID NO: 4918, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5215, and a CDR3 comprising an amino acid sequence of SEQ, ID NO: 5528; xli) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5532; xlii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5542; xliii) a CDR1 comprising an amino acid sequence of SEQ ID NG: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR1 comprising an amino
  • xlviija CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ ID NG: 10504; xlviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10505; xl ix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10506; l)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an
  • Iviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10515; lix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10516; lx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10517; lxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of
  • Ixivja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10521; lxv)a CDR1 comprising an amino add sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10522;
  • Ixvija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10523;
  • Ixxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10530;
  • Ixxvja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10531; lxxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10532;
  • CDR1 comprising an amino acid sequence of SEQ ID NO; 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10533;
  • Ixxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10534; lxxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ !D NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10535; ixxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10536; lxxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ iD NO:
  • Ixxxiija CDR1 comprising an amino add sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10538;
  • Ixxxiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10539;
  • Ixxxivja CDRI comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10540;
  • Ixxxvja CDRI comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10541;
  • Ixxxvija CDRI comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10542;
  • the one or more anti-CD25 antigen-binding domains comprise a CDRI comprising an amino acid sequence selected from any one of SEQ ID NOs: 4726-5030, 7931-8226, and 9660-9770; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5031- 5335, 8227-8522, and 9771-9880; and/or a CDR3 comprising
  • the one or more anti-CD25 antigen-binding domains include a singledomain antibody.
  • the single-domain antibody is a VHH, a VNAR, or a VH domain.
  • the VHH of the one or more anti-CD25 antigen-binding domains is a camelid VHH.
  • the VHH of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, 4143- 4442, 5641-5945, 7351-7354, 7368-7659, 8819-9114, 9437, 9442-9551, 9992-10102, and 10246-10276, or a sequence having at least 75% identity thereto.
  • the VHH of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, 7351- 7354, 9437, and 10246-10276, or a sequence having at least 75% identity thereto.
  • the humanized VHH of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 4126-4130, 4443- 4725, 7359-7362, 7660-7930, 9417-9422, 9439, 9552-9659, 10214-10245, and 10545-10585, or a sequence having at least 75% identity thereto.
  • the humanized VHH of the one or more anti ⁇ CD25 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ, ID NOs: 4126-4130, 7359- 7362, 9417-9422, 9439, 10214-10245, and 10545-10585, or a sequence having at least 75% identity thereto.
  • the one or more anti-CD25 antigen-binding domains of the present disclosure comprise IL-2 or a variant thereof.
  • the one or more anti-CD25 antigen-binding domains bind to human CD25.
  • the one or more anti-CD25 antigen-binding domains bind to human CD25 with a KD of less than about 3.5 x!0 ⁇ 7 M.
  • the one or more anti-CD25 antigen-binding domains bind to human CD25 with a KD of about lxl0“ M to about 5xl0 ⁇ 8 M.
  • the one or more antl-CD25 antigen-binding domains bind to cyno CD25. In some embodiments, the one or more anti-CD25 antigen-binding domains bind to cyno CD25 with a KD of less than about l*10 ⁇ 6 M, In some embodiments, the one or more anti-CD25 antigen-binding domains bind to cyno CD25 with a KD of about lxl0“ 9 to about 2xl0“ 7 M.
  • the one or more anti-CD25 antigen-binding domains bind to the same epitope(s) on CD25 as i L-2, In some embodiments, the one or more anti-CD25 antigen-binding domains compete for binding to CD25 with IL-2. In some embodiments, the one or more anti-CD25 antigenbinding domains have an antagonistic effect upon binding to CD25. In some embodiments, the one or more anti-CD25 antigen-binding domains do not bind to the same epitope(s) as IL-2. In some embodiments, the one or more anti-CD25 antigen-binding domains do not compete for binding to CD25 with IL-2.
  • the one or more anti-TNFR2 antigen-binding domains and/or the one or more CD25 antigen-binding domains comprise one or more modifications that reduce binding of said antigen-binding protein by pre-existing antibodies found in human blood or serum.
  • the anti-TNFR2 single-domain antibody and/or the anti-CD25 singledomain antibody comprises one or more modifications at the amino-terminus and/or the carboxyterminus.
  • the anti-TNFR2 single-domain antibody and/or the anti ⁇ CD25 singledomain antibody comprises the amino acid sequence VPAG (SEQ ID NO: 7267) or VAGG (SEQ ID NO: 7266) at the carboxy-terminus starting from position ill according to Chothia.
  • the anti-TNFR2 single-domain antibody and/or the anti-CD25 singledomain antibody comprises a substitution of amino acid residue Glu with Asp (EID) at the first position of the amino-terminus.
  • the one or more anti-TNFR2 antigen-binding domains and/or the one or more CD25 antigen-binding domains are one or more single-domain antibodies.
  • the one or more single-domain antibodies are one or more VHHs.
  • the multispecific antigen-binding protein further comprises an immunoglobulin Fc region, in some embodiments, the immunoglobulin Fc region is an Fc region of a human immunoglobulin. In some embodiments, the immunoglobulin Fc region is an Fc region of human IgGl, lgG2, lgG3 or lgG4, or a variant thereof.
  • the immunoglobulin Fc region is an Fc region of human IgGl, or a variant thereof.
  • the Fc region of human IgGl comprises one or more mutations selected from Leu234Ala (L234A), Leu234Gly (L234G), Leu234Ser (L234S), Leu234Thr (L234T), Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Leu235Ser (L235S), Leu235Thr (L235T), Leu235Val (L235V), Leu235Gln (L235Q), Gly236Arg (G236R), Met252Tyr (M252Y), Ser254Thr (S254T), Thr256Glu (T256E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser
  • the immunoglobulin Fc region is an Fc region of human IgGl comprising L234A, L235A, and P329A.
  • the immunoglobulin Fc region is an Fc region of human lgG4, or a variant thereof.
  • the Fc region of human lgG4 comprises one or more mutations selected from Ser228Pro (S228P), Leu235Glu (L235E), Leu235Ala (L235A), Phe234Ala (F234A), and/or Pro329Gly (P329G) according to EU numbering.
  • the Fc region of human lgG4 comprises a set of mutations selected from:
  • the immunoglobulin Fc region is an Fc region of human lgG4 comprising S228P and L235E.
  • the multispecific antigen-binding protein comprises at least one linker, optionally selected from the group consisting of SEQ ID Nos: 3969-4027, and 6261-6362.
  • the at least one linker is a rigid linker.
  • the at least one rigid linker is selected from the group consisting of PAPAPAPAPAPAPAPAPAP (SEQ ID NO: 4009), GGGGSPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 4012), GGGGSPAPAPAPAPGGGGS (SEQ ID NO: 6361), GGGGSPAPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 6362), and A(EAAAK)nA (SEQ ID NO: 4027), where n is any integer, e.g., 1, 2, 3, 4, 5, 6, or 7, 8, 9 or 10,
  • the at least one linker is a flexible linker.
  • the at least one flexible linker is selected from the group consisting of G n S (SEQ ID NO: 4013), SG n (SEQ ID NO: 4014), where n is any integer, e.g., 1, 2, 3, 4, 5, 6, or 7, 8, 9 or 10, and (GGGGS)n (SEQ ID NO: 4015), where n is any integer, e.g., 1, 2, 3, 4, 5, 6, or 7, 8, 9 or 10.
  • the multispecific antigen-binding protein comprises the amino acid sequence of any one of SEQ ID NOs: 6401-6520 and 10792-10810, or a sequence having at least 70% identity thereto.
  • a conjugate comprising the multispecific antigen-binding protein described herein, wherein the multispecific antigen-binding protein is conjugated to a second moiety.
  • the second moiety is selected from a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytokine, a cytotoxic agent, a chemotherapeutic agent, and a diagnostic agent, or a combination thereof,
  • the polynucleotide molecule comprises a nucleotide sequence encoding any of the amino acid sequences selected from SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 93-640, 2805-3363, 6636, 6640, 6644, 7090, 6651-6697, 6931-6992, 7174-7197, 10380, 10381, 10383, 10385, and 10386, encoding an anti-TNFR2 VHH, or a sequence having at least 70% identity thereto, in some embodiments, the polynucleotide molecule comprises encoding any of the amino acid sequences selected from SEQ ID NOs: 81-92, 6648, 6649, 6650, 7092, 7095, 7098, 7101,7293-7296, 641-1127, 6698-6744, 10375, 10382, 10384, 10394-10408, 10457-10502
  • the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 48-59, 3364-3922, 6645-6647, 7091, 7094, 7097, 7100, 6993-7054, 7198-7221, and 10611-10661, encoding an anti-TNFR2 VHH, or a sequence having at least 70% identity thereto.
  • the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 48-59, 6645-6647, 7091, 7094, 7097, and 7100, and 10611-10661, encoding an anti-TNFR2 VHH, or a sequence having at least 70% identity thereto.
  • the polynucleotide molecule comprises a nucleotide sequence encoding any one of the amino acid sequences selected from SEQ, ID NOs: 4104, 4108, 4112, 4116, 4120, 4143- 4442, 5641-5945, 7351-7354, 7368-7659, 8819-9114, 9437, 9442-9551, 9992-10102, and 10246-10276, encoding an anti-CD25 VHH, or a sequence having at least 70% identity thereto.
  • the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 4121-4125, 5946-6250, 7355-7358, 9115-9410, 9438, and 10103-10213, encoding an anti-CD25 VHH, or a sequence having at least 70% identity thereto.
  • the polynucleotide molecule comprises the nucleotide sequence encoding an amino acid sequence selected from SEQ ID NOs: 4126-4130, 4443-4725, 7359- 7362, 7660-7930, 9417-9422, 9439, 10214-10245, and 9652-9659, encoding a humanized anti-CD25 VHH, or a sequence having at least 70% identity thereto.
  • the polynucleotide molecule comprises the nucleotide sequence encoding an amino acid sequence selected from SEQ ID NOs: 9438, 10277-10307, and 10662- 10703 encoding a humanized anti-CD25 VHH, or a sequence having at least 70% identity thereto.
  • the polynucleotide molecule comprises a nucleotide sequence selected from SEQ ID NOs: 6521-6632, or a similar sequence thereof having at least 70% identity thereto.
  • the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs- 6521-6632 encoding a multispecific antigen-binding protein.
  • a recombinant vector comprising the polynucleotide molecule described herein.
  • a host cell comprising the polynucleotide molecule described herein, or the recombinant vector described herein.
  • kits comprising the multispecific antigen-binding protein, the conjugate, the polynucleotide molecule, the recombinant vector, or the host cell described herein, and optionally, instructions and/or packaging for the same.
  • composition comprising the multispecific antigen-binding protein, the conjugate, the polynucleotide molecule, or the recombinant vector described herein, and a pharmaceutically acceptable carrier and/or excipient.
  • a method for preparing a multispecific antigen-binding protein that specifically binds TNFR2 and CD25 comprising the steps of:
  • a method for promoting proliferation, activating and/or enhancing suppressive function, and/or stabilizing immunosuppressive phenotype of a population of regulatory T cells (Treg) comprising contacting the population of Treg with the multispecific antigenbinding protein or the conjugate described herein.
  • said contacting occurs in vitro, in some embodiments, said contacting occurs in vim.
  • the method further comprises administering the multispecific antigen-binding protein or the conjugate into a subject in need thereof.
  • a method of treating or preventing a disease or disorder in a subject in need thereof comprising administering to the subject an effective amount of the multispecific antigen-binding protein or the conjugate described herein.
  • the disease or disorder is an immunological disease, inflammatory disease, cancer, cardiovascular disease, or an infertility and pregnancy-associated disease.
  • the immunological disease is selected from an autoimmune disease, a neurological condition, an allergy, asthma, macular degeneration, muscular atrophy, a disease related to miscarriage, atherosclerosis, bone loss, a musculoskeletal disease, obesity, a graft-versus-host disease, and an allograft rejection.
  • the autoimmune disease is selected from lupus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpastures disease, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), I
  • the lupus is systemic lupus erythematosus (SLE), cutaneous lupus, lupus nephritis, neonatal lupus, or drug-induced lupus.
  • the cutaneous lupus is acute cutaneous lupus, chronic cutaneous lupus erythematosus, discoid lupus erythematosus (DLE), or subacute cutaneous lupus erythematosus.
  • the autoimmune disease is atopic dermatitis, psoriasis, systemic lupus erythematosus, or arthritis.
  • the neurological condition is selected from a brain tumor, a brain metastasis, a spinal cord injury, schizophrenia, epilepsy, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Huntington's disease, Parkinson's disease, and stroke.
  • the allergy is selected from food allergy, seasonal allergy, pet allergy, hives, hayfever, allergic conjunctivitis, poison ivy allergy oak allergy, mold allergy, drug allergy, dust allergy, cosmetic allergy, and chemical allergy.
  • the allograft rejection is selected from skin graft rejection, bone graft rejection, vascular tissue graft rejection, ligament graft rejection, and organ graft rejection, [0103]!
  • the ligament graft rejection is selected from cricothyroid ligament graft rejection, caudal cruciate ligament graft rejection, periodontal ligament graft rejection, suspensory ligament of the lens graft rejection, palmar radiocarpal ligament graft rejection, dorsal radiocarpal ligament graft rejection, ulnar collateral ligament graft rejection, radial collateral ligament graft rejection, suspensory ligament of the breast graft rejection, anterior sacroiliac ligament graft rejection, posterior sacroiliac ligament graft rejection, sacrotuberous ligament graft rejection, sacrospinous ligament graft rejection, inferior pubic ligament graft rejection, superior pubic ligament graft rejection, anterior cruciate ligament graft rejection,
  • the organ graft rejection is selected from heart graft rejection, lung graft rejection, kidney graft rejection, liver graft rejection, pancreas graft rejection, intestine graft rejection, and thymus graft rejection.
  • the graft-versus-host disease arises from a bone marrow transplant or one or more blood cells selected from B-cells, T-cells, basophils, common myeloid progenitor cells, common lymphoid progenitor cells, dendritic cells, eosinophils, hematopoietic stem cells, neutrophils, natural killer cells, megakaryocytes, monocytes, or macrophages.
  • blood cells selected from B-cells, T-cells, basophils, common myeloid progenitor cells, common lymphoid progenitor cells, dendritic cells, eosinophils, hematopoietic stem cells, neutrophils, natural killer cells, megakaryocytes, monocytes, or macrophages.
  • the Inflammatory disease is acute or chronic inflammation.
  • the inflammatory disease is selected from osteoarthritis, atopic dermatitis, endometriosis, polycystic ovarian syndrome, inflammatory bowel disease, fibrotic lung disease, and cardiac inflammation.
  • the cancer is selected from adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith- Wiedemann syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dube syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant mel
  • the infertility and pregnancy-associated diseases is selected from recurrent pregnancy loss, pre-eclampsia, preterm labor, fetal growth restriction, and intrauterine growth restriction.
  • a method of regenerating a tissue or organ comprising one or more TNFR2+ and/or CD25+ cells comprising contacting the tissue or organ with an effective amount of the multispecific antigen-binding protein or the conjugate described herein.
  • provided herein is a method of regenerating a tissue or organ comprising one or more TNFR24- cells, said method comprising contacting the tissue or organ with an effective amount of the multispecific antigen-binding protein or the conjugate described herein.
  • a method of regenerating a tissue or organ comprising one or more CD25+ cells said method comprising contacting the tissue or organ with an effective amount of the multispecific antigen-binding protein or the conjugate described herein.
  • the tissue or organ is selected from pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerve, eye, thymus, tongue, bone, liver, small intestine, large intestine, gastrointestinal, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structures, embryo, and testes tissue.
  • said contacting occurs m vitro.
  • said contacting occurs in vivo.
  • the method further comprises administering the multispecific antigen-binding protein or the conjugate into a subject in need thereof.
  • the subject is a mammal.
  • the mammal is human.
  • a method for inducing tolerance to a foreign agent and/or preventing or reducing immune response to a foreign agent in a subject in need thereof comprising an administering to the subject an effective amount of the multispecific antigen-binding protein, or the conjugate described herein.
  • the foreign agent is a therapeutic protein, a peptide, a vector, a biochemical vector, a lipid, a carbohydrate, a nucleic acid, a sperm, an oocyte, or an embryo.
  • the vector is a viral vector, a bacterial vector, or a fungal vector.
  • the viral vector is a DNA or RNA vector.
  • [OllSjFsgure 1 shows the therapeutic rational of the multispecific antigen-binding proteins of the present disclosure.
  • An exemplary bispecific format is depicted.
  • Fsgure 2A depicts an exemplary general panning strategy for isolation of tumor necrosis factor (INF) receptor type 2 (TNFR2)-specific variable domain of heavy chain (VHH) antibodies, also referred to herein as V-bodies (Vbs). Binders to human and rodent TNFR2 were enriched from VHH immune libraries by two rounds of phage display.
  • BM bone marrow.
  • Figure 2B shows VHH immune library selection for next-generation sequencing (NGS) across the phage display process.
  • NGS next-generation sequencing
  • F8gure 3 shows a schematic diagram of an exemplary NGS workflow. Following phage display, the VHH region of the phage eluate was amplified via polymerase chain reaction (PCR). Unique and samplespecific barcodes were then fused, and NGS was subsequently performed using the Illumina NovaSeq platform (Genewiz). The raw data were de-multiplexed, and then processed by the NGS analysis pipeline. Forward and reverse sequence pairs were merged via overlapping regions and the VHHs, including complementarity determining regions (CDRs) were annotated.
  • PCR polymerase chain reaction
  • CDRs complementarity determining regions
  • V-body sequences were clustered, thereby allowing for detailed analysis of, e.g,, V-body enrichment during phage display, sequence diversity, CDR3 length distribution, and cluster abundance. Based on such analyses, more than 600 candidates were selected for DNA synthesis (Twist) and further characterization.
  • FIG. 4A F8gure ⁇ 4A ⁇ 4B illustrate human TNFR2 (hTNFR2) V-body binding validation at a fixed concentration of 1 pM V-body.
  • the bar histogram ( Figure 4A) and table ( Figure 4B) show the percentage of Alexa488-positive cells for all VHHs tested.
  • the lower dotted line indicates background staining
  • the upper dotted line indicates two times the background level.
  • a V-body having a signal-to-noise ratio greater than 2 is considered as a "binder”. Shading within the table indicates binders to hTNFR2.
  • Figures 5A-5B illustrate human TNFR2 (hTNFR2) V-body binding validation at a fixed concentration of 100 nM V-body.
  • the bar histogram ( Figure 5A) and table ( Figure SB) show the percentage of Alexa488-positive cells for all VHHs tested.
  • the lower dotted line indicates background staining ( ⁇ 5%), and the upper dotted line indicates two times the background level.
  • a V-body having a signal-to-noise ratio greater than 2 is considered as a "binder".
  • Figures SA-6C depict cross-specificity of V-body binding to mouse TNFR2 (mTNFR2) ( Figure SA) and cynomolgus TNFR2 (cTNFR2) ( Figure SB) at a fixed concentration of WO nM.
  • the bar histograms ( Figure 6A-6B) and table ( Figure 6C) show the percentage of Aiexa488-positive cells for all VHHs tested. For the bar histogram, the lower dotted line indicates background staining, and the upper dotted line indicates two times the background level.
  • Figure 7 shows testing of human TNFR2 V-body binding across a range of concentrations for V- bodies T-002, T-014, T-001, T-006, T-007, T-003.
  • V-bodies were tested at molar concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.12 nM and 1.55 nM.
  • Figure 8 shows a schematic diagram of an exemplary experimental setup for determination of binding affinities of the V-bodies for their respective target via surface plasmon resonance (SPR) (left panel) and a corresponding table describing the V-body candidates analyzed (right panel).
  • SPR surface plasmon resonance
  • Figures 9A-9F depict surface plasmon resonance (SPR) sensorograms of VHH binding to human, cynomolgus, and mouse TNFR2, Fitted binding curves and calculated dissociation constants (KD) are included.
  • SPR surface plasmon resonance
  • Fsgure 10 shows a summary of binding affinities of 16 selected anti-TNFR2 V-bodies to human, cynomolgus and mouse TNFR2. Cyno, cynomolgus.
  • Fsgure 11 demonstrates that some humanized anti-TNFR2 V-bodies targeted the epitope recognized by a MR2-1 bivalent agonist.
  • T-013hul and T-018hul may recognize the same epitope as MR2-1.
  • MR2-1 binding enhanced binding of T-015hul, T-017hul and T-Ollhul to TNFR2.
  • [O13O]F8gures 14A-14C shows HEK293 TNFR2 NF-KB (LUC) reporter gene assay samples and assay controls.
  • a description of reporter gene assay samples and assay controls is shown in Figure 14A.
  • a bar graph showing protein concentration (mg/mL) for the V-body constructs and respective controls is depicted in Figure MB.
  • a bar graph showing RLUs for V-body constructs and respective controls tested on a PCL control is depicted in Figure 14C.
  • Flgures 1SA-1SC show exemplary dot plots of RLUs measured across increasing concentrations (mol/L) of control (control 12) and tetravalent V-body fusion constructs comprising four V-bodies mounted onto the fragment crysta lliza ble (Fc) region of a lgG4 variant comprising S228P, L235E and P329G mutations.
  • Figures 17A-17F show exemplary dot plots of RLUs measured across increasing concentrations (mol/L) of control (control 10) and an alternative design of tetravalent V-body fusion constructs comprising four V-bodies mounted onto the Fc region of a lgG4 variant comprising S228P, L235E and P329G mutations.
  • Figures 18A-18C show exemplary dot plots of RLUs measured across increasing concentrations (mol/L) of control (control 2) and bivalent V-body fusion constructs. Limit of detection, LOD.
  • Figures 29A-19C show exemplary dot plots of RLUs measured across increasing concentrations (mol/L) of control (control 13) and IL-2 N88D V-body fusion constructs. Limit of detection, LOD.
  • Figure 20 depicts a comparison of RLUs measured across increasing concentrations (mol/L) of monospecific construct 10 (tetravalent Fc) and construct 12 (Vb-Fc-Vb) tested on NF-xB reporter (Luc) HEK293 reporter cell-line stably expressing TNFR2 (clone 8).
  • F8gure 21 depicts an exemplary experimental timeline of TNFR2 stimulation by multivalent V-body fusion constructs (e.g., tetravalent Fc, Vb-Fc-Vb, rigid bivalent no Fc) on primary human peripheral blood mononuclear cells (PBMCs) and cluster of differentiation 4 positive (CD4 + ) CD25* CD127dim regulatory T cells (Tregs).
  • PBMCs peripheral blood mononuclear cells
  • CD4 + CD25* CD127dim regulatory T cells
  • Fsgure 22 shows a bar graph of an overview of in-assay concentrations (nM) of multivalent V-body fusion constructs first wave binders. Concentrations (nM) of the VHH constructs is also shown.
  • Fjgure 23 illustrates an exemplary gating strategy applied for Treg markers.
  • Treg Donor 1 is shown as an example and an identical strategy was used for Treg Donor 1 and Donor 3.
  • Live cell and CD4 gating were based on Fluorescence Minus One (FMO) FMO control determination of the cut-off point between background fluorescence and positive cell populations.
  • FMO Fluorescence Minus One
  • Forkhead box P3 (FoxP3), Human Leukocyte Antigen, DR isotype (HLA-DR), chemokine motif (C-C motif) receptor 8 (CCR8), and OX-40 gating was based on the CD4 subset of IgG control-stained sample from the same donor.
  • FoxP3 the gate was set at approximately 0.2%.
  • Figures 24A-24B demonstrate multivalent anti-TNFR2 V-body fusion constructs increased expression of the Treg suppression marker HLA-DR and CCR8. Histograms displaying expression of lreg suppression marker HLA-DR for specific T-003 binders compared to control formats ( Figure 24A). Density plots displaying expression of Treg suppression marker HLA-DR and CCR8 for specific T-003 binders compared to control formats ( Figure 24B). Fluorescein isothiocyanate, FITC; Phycoerythrin, PE.
  • Figure 25A-25B demonstrate tetravalent anti-TNFR2 V-body fusion constructs strongly increased expression of Treg suppression marker HLA-DR on FoxP3 + Tregs.
  • the bar graphs show HLA-DR mean fluorescent intensity (MFI) measured for each of the tetravalent Fc, Vb-Fc-Vb, and rigid bivalent no Fc V- body fusion formats relative to control formats.
  • MFI mean fluorescent intensity
  • Figure 26 shows dose-response curves based on HLA-DR MFI values of CD4 + FoxP3 + Tregs for construct T-003 and control 10 for Donor 2.
  • Fsgure 27 shows dose-dependent induction of Treg suppression marker HLA-DR expression across various concentrations of tetravalent anti-TNFR2 V-body Fc fusion construct 10 for Donor 1 (top panel) and Donor 2 (bottom panel).
  • Figure 30 shows exemplary design of multivalent anti-TNFR2 V-body fusion constructs
  • Anti- TNFR2 V-bodies are shown as ovals
  • linkers are shown with flexible (e.g., GS linkers) as curved lines
  • rigid linkers e.g., proline linker
  • Fc domains as dimeric bars.
  • Figure discloses SEQ ID NO: 7280.
  • F8gure 31 shows assessment of tetravalent-Fc VHH activity on naive CD4+CD25-rCD45RA+ human Treg. HLA-DR and CCR8 expression on CD4+FQXIP3+ and expansion after 5 day stimulation with anti- CD3/IL-2 plus VHH or MR2-1 are shown.
  • Proportion of Treg (CD4+ CD25+ F0XP3+) in spleen expressing activation markers (CCR8, ICAM-1, or ICOS) 5 days after a single injection of control VHH or TNFR2-specific VHH T-007__2xVHH-Fc is shown.
  • Figure 38 shows that TNFR2 agonist VHH selectively expands Treg in the spleen. Cell subsets as percentage of CD45+ cells in the spleen 5 days after single injection of TNFR2-specific VHH T- 007__2xVHH-Fc or control VHH are shown.
  • Figure 46 shows VHH immune library selection for next-generation sequencing (NGS) across the phage display process.
  • NGS next-generation sequencing
  • [OlSSjFsgure 47 shows a schematic diagram of an exemplary NGS workflow. Following phage display, the VHH region of the phage eluate was amplified via polymerase chain reaction (PCR). Unique and sample-specific barcodes were then fused, and NGS was subsequently performed using the Illumina NovaSeq platform (Genewiz). The raw data were de-multiplexed, and then processed by the MGS analysis pipeline. Forward and reverse sequence pairs were merged via overlapping regions and the VHHs, including complementarity determining regions (CDRs) were annotated.
  • PCR polymerase chain reaction
  • CDRs complementarity determining regions
  • a multispecific antigen-binding protein of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure from N- terminus to C-terminus: anti-TNFR2 VHH - first linker - Fc region- second linker - anti-CD25 VHH.
  • the multiple linkers used in the multispecific antigen -binding protein are not necessarily the same or different.
  • the one or more antl-TNFR2 antigen-binding domains e.g., VHHs
  • one or more anti CD25 antigen-binding domains e.g., VHHs
  • the adjacent antigen-binding domains may be optionally operably linked to one another via a linker.
  • the linkers may be a flexible Sinker or a rigid linker.
  • a multispecific antigen-binding protein of the present disclosure may comprise the following structure from N-terminus to C-terminus: (anti-TNFR2 VHH),,-- linker - (anti- CD25 VHH) m , wherein n and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc.).
  • each anti-TNFR2 VHH may be optionally operably linked to another anti-TNFR2 VHH via a linker.
  • each anti-CD25 VHH may be optionally operably linked to another anti-CD25 VHH via a linker.
  • the multiple linkers used in the multispecific antigen-binding protein are not necessarily the same or different.
  • a multispecific antigen-binding protein of the present disclosure may comprise the following structure from N-terminus to C-terminus: (anti-CD25 VHH)n- linker - (anti-TNFR2 VHH) m , wherein n and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc.).
  • n>2 each anti-CD25 VHH may be optionally operably linked to another anti-CD25 VHH via a linker.
  • each anti-TNFR2 VHH may be optionally operably linked to another anti-TNFR2 VHH via a linker.
  • the multiple linkers used in the multispecific antigen-binding protein are not necessarily the same or different.
  • an antigen-binding domain that binds to TNFR2 or CD25 is a single-domain antibody (also termed as "sdAb").
  • the single-domain antibodies of the present disclosure can be derived from numerous sources, including but not limited to VHHs, VNARs, or VH domains (naturally occurring or engineered VH domains), VHHs can be generated from camelid heavy chain only antibodies and libraries thereof. VNARs can be generated from cartilaginous fish heavy chain only antibodies and libraries thereof.
  • Various methods have been implemented to generate monomeric sdAbs from conventionally heterodimeric VH and VL domains, including interface engineering and selection of specific germline families.
  • sdAbs of the present invention are human or humanized.
  • Binding affinity of a molecular Interaction between two molecules can be measured via various techniques, such as surface plasmon resonance (SPR), bio-layer interferometry (BLI ), enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, fluorescent-activated cell sorting (FACS), or flow cytometry binding assays and the like.
  • SPR surface plasmon resonance
  • BLI bio-layer interferometry
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescent-activated cell sorting
  • flow cytometry binding assays and the like.
  • Surface plasmon resonance is a biosensor technique that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions (see e.g., Ober et al. 2001, Intern. Immunology 13: 1551- 1559). SPR can for example be performed using the BIACORE® system or Carterra ISA system.
  • Another biosensor technique that can be used to determine affinities of biomolecular interactions is bio-layer interferometry (Bid) (see e.g., Abdiche et al, 2008, Anal Biochem.
  • affinities can be measured in Kinetic Exclusion Assay (KinExA) (see e.g., Drake et al. 2004, Anal. Biochem., 328: 35-43), which is a solution-based method to measure true equilibrium binding affinity and kinetics of unmodified molecules.
  • KinExA Kinetic Exclusion Assay
  • Equilibrated solutions of an antibody/antigen complex are passed over a column with beads precoated with antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished with a fluorescently labeled protein binding the antibody (or antigen).
  • SEQ ID NO: 60 ii)a CDR1 comprising an amino acid sequence of SEQ iD NO: 69, a CDR2 comprising an amino acid sequence of SEQ iD NO: 76, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 61; iii)a CDR1 comprising an amino acid sequence of SEQ iD NO: 70, a CDR2 comprising an amino acid sequence of SEQ iD NO: 77, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 62; iv)a CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 63; v)a CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a)a CDR1 comprising an amino acid sequence of SEQ ID NO: 69, a CDR2 comprising an amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 61; b)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 63; c)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7333; - d)a CDR1 comprising an amino acid sequence of SEQ ID NO: 73, a CDR
  • anti-TNFR2 antigen-binding domains e.g., antibodies such as single-domain antibodies
  • CDR1-CDR2-CDR3 a set of three CDRs contained within any of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 21, or Table 13.
  • Ivija CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10428; lvii)a CDR1 comprising an amino add sequence of SEQ ID NO: 32, a CDR2 comprising an amino add sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino add sequence of SEQ ID NO: 34;
  • a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10429; lixja CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10430; lx)a CDR1 comprising an amino add sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10431; lxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ
  • IxliiJCDRl comprising an amino acid sequence of SEQ iD NO: 32, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10811; lxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10435; lxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10436; lxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising
  • Ixviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10439; lxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10440; lxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10441; lxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ
  • CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10448; lxxviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ !D NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10449;
  • Ixxxiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10454; or lxxxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10455.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 10414, 10421, 10422, 10423, 10429, and 10811.
  • anti-TNFR2 antigen-binding domainsje comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within a VHH amino acid sequence as defined by any of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13.
  • variable domains comprising the amino acid sequence of SEQ ID NO: 10405; ss) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10406; tt) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10407; uu) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10408; w) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10457; ww) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10458; xx) a variable domain
  • variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10473; mmm) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10474; nnn) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino add sequence of SEQ ID NO: 10475; ooo) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10476; ppp) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10477; qqq) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the
  • variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10499; mmmm) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10500; nnnn) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10501; oooo) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10502; pppp) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10720; qqq) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10
  • VHH comprising the amino acid sequence of SEQ ID NO: 10408; ggja variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10457; hh)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10458; ii)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10459; jj)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10460; kk)a variable domain that comprises a CDR1, CDR2 ?
  • VHH comprising the amino acid sequence of SEQ ID NO: 10461; ll)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10462; mm)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10463; nn)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10464; oo)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10465; pp)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10466; qq)a variable domain that comprises a CDR1, CDR2,
  • VHH comprising the amino acid sequence of SEQ ID NO: 10720; bbbb)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10721; cccc)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10722; dddd)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10723; eeee)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10724; ffff)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10725; gggg)a
  • an anti-TNFR2 antigen-binding domain e.g., antibody such as a single-domain antibody
  • an anti-TNFR2 antigen-binding domain of the present disclosure can include a VHH amino acid sequence selected from SEQ ID Nos: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 93-640, 2805-3363, 6651-6697, 6931-6992, 7174-7197, 7222-7254, 10380, 10381, 10383, 10385, and 10386, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain e.g., antibody such as a single-domain antibody
  • an anti-TNFR2 antigen-binding domain e.g., antibody such as a single-domain antibody
  • an anti-TNFR2 antigen-binding domain of the present disclosure can Include a VHH amino acid sequence selected from SEQ ID Nos: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 7222-7254, 10380, 10381, 10383, 10385, and 10386, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID Nos: 81-92, 6648, 6649, 6650, 7092, 7095, 7098, 7101, 7293-7296, 641-1127, 6698-6744, 10375, 10382, 10384, 10394-10408, 10457-10502, 10720-10727, 10812, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain e.g., antibody such as a single-domain antibody
  • an anti-TNFR2 antigen-binding domain of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID Nos: 10461, 10468, 10469, 10470, 10477, and 10812, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure can include a VHH amino acid sequence selected from SEQ ID NOs: 19, 6644, 6650, 7095, 7098, 7101, 7293-7296, 7222-7254, 10375, 10382, 10384, 10380, 10381, 10383, 10385, and 10386, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ, ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 6643.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 6650 or 10720.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino add sequence of SEQ ID NO: 6643.
  • an anti-TNFR2 antigenbinding domain comprises the amino add sequence of SEQ ID NO: 6650 or 10720, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289.
  • an anti ⁇ TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10394.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10394, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 7294.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 7294, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289.
  • an anti ⁇ TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10395.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10395, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10396.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10396, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289.
  • an anti ⁇ TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10397.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 7289.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10397, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10398.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10398, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10399.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10399, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10400.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10400, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10401.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10401, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10391.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10402.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10391, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10402, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10392.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10403.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10392, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10403, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10393.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10404.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10393, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10404, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 18.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10405.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10405, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 18.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10406.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10406, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 18.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10407.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10407, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10391.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10408.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10391, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10408, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10410.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10457.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10410.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10457, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10411.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10458.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10411.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10458, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10412.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10459.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10412.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10459, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10413.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10460.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10413.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10460, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10414.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10461.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10414.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10461, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10415.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10462.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10415.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10462, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain e.g., antibody such as single-domain antibody
  • an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10463.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10416.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10463, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10417.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10464.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10417.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10464, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10418.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10465.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10418.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10465, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10419.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10466.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10419.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10466, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10420.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10467 or 10721
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10420.
  • an anti-TNFR2 antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 10467 or 10721, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10421.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10468 or 10722.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10421.
  • an anti-TNFR2 antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 10468 or 10722, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10422.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10469 or 10723.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10422.
  • an anti-TNFR2 antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 10469 or 10723, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10423.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10470 or 10724.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10423.
  • an anti-TNFR2 antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 10470 or 10724, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10424.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10471 or 10725.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10424.
  • an anti-TNFR2 antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 10471 or 10725, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10425.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10472.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10425.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10472, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10426.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10473.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10426.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10473, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10427.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10474 or 10726.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10427.
  • an anti-TNFR2 antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 10474 or 10726, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10428.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10475.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10428.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10475, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 34.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10476.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10476, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10429.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10477 or 10727.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10429.
  • an anti-TNFR2 antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 10477 or 10727, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10430.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10478.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10430.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10478, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10431.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10479.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10431.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10479, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10432.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10480.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10432.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10480, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10433.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10481.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10433.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10481, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10811.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10812.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10811.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10812, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10435.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10482.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10435.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10482, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10436.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10483.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10436.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10483, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7099.
  • an anti ⁇ TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 7101.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 7099.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 7101, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10437.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10484.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10437.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10484, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10438.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10485.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10438.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10485, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10439.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10486.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10439.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10486, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10440.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10487.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10440.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10487, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10441.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10488.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10441.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10488, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10442.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10489.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10442.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10489, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10443.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10490.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10443.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10490, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10444.
  • an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10491.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10444.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10491, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10445.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10492.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10445.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10492, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen- domain protein (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10446.
  • an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10493.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10446.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10493, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10447.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10494.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10447.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10494, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10448.
  • an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10495.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10448.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10495, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10449.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10496.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10449.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10496, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10450.
  • an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10497.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10450.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10497, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10451.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10498.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10451.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10498, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10452.
  • an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10499.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10452.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10499, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10453.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10500.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10453.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10500, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10454.
  • an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10501.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10454.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10501, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10455.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10502.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10455.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ, ID NO: 10502, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16.
  • an anti-TNFR2 antigembinding domain (e,g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10375.
  • an anti- TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO 10374.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10375, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 8.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 9.
  • an anti- TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 7095 or 10380.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a CDRS comprising an amino acid sequence of SEQ ID NO: 7093.
  • an anti-TNFR2 antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 7095 or 10380, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: S634.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10381 or 10382
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10381 or 10382, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10383 or 10384.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10383 or 10384, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7281.
  • an antl-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289.
  • an anti ⁇ TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 7293 or 10385.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7281, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 7289.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 7293 or 10385, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7283.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7287.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7291.
  • an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (I.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 7295 or 10386.
  • an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 7283, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7287, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7291.
  • an anti-TNFR2 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 7295 or 10386, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the present disclosure also includes an anti ⁇ TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) that competes for binding to TNFR2 with any one of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13.
  • an anti ⁇ TNFR2 antigen-binding domain e.g., antibody such as a single-domain antibody
  • the present disclosure also includes an anti-TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) that binds to the same epitope on TNFR2 as any one of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13.
  • an anti-TNFR2 antigen-binding domain e.g., antibody such as a single-domain antibody
  • binds to the same epitope on TNFR2 as any one of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13.
  • the present disclosure also includes an anti-TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) that comprises a means for binding an epitope within human TNFR2 bound by any one of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13.
  • an anti-TNFR2 antigen-binding domain e.g., antibody such as a single-domain antibody
  • the present disclosure provides one or more anti-CD25 antigen-binding domains (e.g., antibodies, such as single-domain antibodies) that bind to CD25.
  • anti-CD25 antigen-binding domains e.g., antibodies, such as single-domain antibodies
  • CD25 also called interleukin-2 receptor subunit alpha
  • IL-2Ra or IL2RA interleukin-2 receptor subunit alpha
  • IL-2Ro is a single pass type-1 transmembrane protein with a total length of 251 amino acids.
  • the receptor subunit consists of two sushi or elbow domains that are connected via an unordered loop region (Wang et al., Science 310, 1159--1163. 2005).
  • the C-terminal domain of the protein is a long, disordered region that is needed to allow CD25 forming a cap like structure in the IL-2 receptor complex but still being anchored in the membrane.
  • the actual structure and positioning of the loop has not been resolved in any of the available crystal structures.
  • the sushi domains of CD25 form five stranded beta sheet sandwiches that are related to each other in a pseudo-2-fold symmetry.
  • Sushi domain 1 accounts for most of the interactions with IL-2 (82%) while Sushi domain 2 contributes significantly less (Stauber et al., Proc Natl Acad Sci U S A 103, 2788-2793. 2006).
  • the structure of CD25 is stabilized by several intradomain and two interdomain disulfide bonds, in addition, CD25 carries several glycans with one N-glycosylation located at the C-terminus of Sushi domain 2 and four O-glycans located in the C-terminal unordered region.
  • [0372JCD25 interacts with IL-2 in a tight manner. It is postulated that the IL-2 receptor complex forms in a stepwise manner starting with IL-2 binding to CD25/IL-2Ro, then engaging subunit p and finally interacting with the y receptor subunit (Stauber et al., Proc Natl Acad Sci U S A 103, 2788-2793. 2006). Interestingly, it has been reported that CD25 can present IL-2 in cis and in trans (Liao et aL, Immunity 38, 13-25. 2013; Wuest et aL, Nat Med 17, 604-609. 2011), both resulting in IL-2 receptor complex assembly.
  • the CD25/IL-2Ra has the largest interface with 11-2 within the complex, which is reflected in the very high affinity between IL-2 and CD25 (Liao et al., 2013).
  • CD25/IL- 2Ra makes no direct contact with the other two subunits P or y.
  • Deglycosylation experiments of the individual subunits were found to impact the complex formation with the y subunit aggregating, while the subunits a and P were still able to bind to IL-2 (Stauber et al., Proc Natl Acad Sci U S A 103, 2788- 2793. 2006).
  • the glycosylation on CD25/IL-2Ra is not essential for the interaction with IL-2.
  • IL-2- Ry and I L-2Rf are also part of other interleukin receptor complexes while CD25/IL-2Ra is exclusively found in the IL-2 receptor complex (Liao et aL, Immunity 38, 13-25. 2013).
  • CD25 can be found in the human serum (Pedersen and Lauritsen, Scand J Immunol 70, 40-43. 2009). This soluble form of CD25 can result from a shedding event of the membrane anchored protein, producing a truncated CD25 with a molecular weight (MW) of ⁇ 20kDa.
  • antigen-binding domains bind to human CD25.
  • the human CD25 protein is encoded by the human interleukin-2 receptor subunit alpha (IL2RA) gene (NCBI Gene ID: 3559) and has the amino acid sequence of MDSYLLMWGLLTFIMVPGCQAELCDDDPPEiPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWD NQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFWGQMVYYQC VQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAAT METSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI (UniProt
  • antigen-binding domains bind to cynomolgus monkey (“cyno") CD25.
  • the cyno CD25 protein is encoded by the cyno interleukin-2 receptor subunit alpha (IL2RA) gene (NCBI Gene ID: 102123605) and has the amino acid sequence of MDPYLLMWGLLTFITVPGCQAELCDDDPPKITHATFKAVAYKEGTMLNCECKRGFRRIKSGSPYMLCTGNSSHSSWDN QCQCTSSAARNTTKQVTPQPEEQKERKTTEMQSQMQLADQVSLPGHCREPPPWENEATERIYHFVVGQTVYYQCVQ GYRALHRGPAESICKMTHGKTRWTQPQLICTGETEPSQFPGEEEPQASPDGLPESETSRLVTTTDFRIQTEVAATMETFI FTTEYQVAVAGCVFL
  • antigen-binding domains bind to mouse CD25.
  • the mouse CD25 protein is encoded by the mouse interleukin 2 receptor subunit alpha (Il2ra) gene (NCBI Gene ID: 16184) and has the amino acid sequence of MEPRLLMLGFLSLTIVPSCRAELCLYDPPEVPNATFKALSYKNGTILNCECKRGFRRLKELVYMRCLGNSWSSNCQCTSN SHDKSRKQVTAQLEHQKEQQTTTDMQKPTQSMHQENLTGHCREPPPWKHEDSKRIYHFVEGQSVHYECIPGYKALQ RG PAISI CKM KCG KTG WTQPQLTCVDEREH H RFLASEESQGSRNSSPESETSCPITTTDFPQPTETTAMTETFVLTM EYK VAVASCLFLLISILLLSGLTWQHRWRKSRRTI (UniPro
  • the one or more anti-CD25 antigen-binding domains of the present disclosure comprise IL-2 or a variant thereof.
  • anti-CD25 antigen-binding domains of the present disclosure upon binding to CD25 do not impair the binding of its cognate ligand, interleukin-2 (IL-2), to CD25.
  • anti-CD25 antigen-binding domains of the present disclosure do not have overlapping epitopes with IL-2.
  • anti-CD25 antigen-binding domains of the present disclosure upon binding to CD25 may impair the binding of IL-2 to CD25.
  • anti-CD25 antigen-binding domains of the present disclosure may have overlapping epitopes with IL-2.
  • the anti-CD25 antigen-binding domains may impair IL-2 binding to CD25.
  • the anti-CD25 antigen-binding domains may compete for binding to CD25 with IL-2.
  • anti ⁇ CD25 antigen-binding domains of the present disclosure may have an antagonistic effect (e.g., a blocking effect) upon binding to CD25.
  • An antagonistic CD25 binder can block or decrease activation of CD25 and/or attenuate one or more signal transduction pathways mediated by CD25.
  • Antagonistic CD25 binders may block or decrease CD2S activation by binding CD25, e.g., to induce a conformational change that renders the receptor biologically inactive.
  • antagonistic CD25 binders may prevent the trimerization of an IL-2 receptor complex as can occur due to the interaction between CD25 and its cognate ligand, IL-2, thus impairing CD25-mediated signaling.
  • when the anti-CD25 antigen-binding domains of the present disclosure have overlapping epitopes with IL-2, such anti-CD25 antigen-binding domains may have an antagonistic effect upon binding to CD25.
  • anti-CD25 antigen-binding domains of the present disclosure may have an agonistic effect (e.g., a stimulatory effect) upon binding to CD25.
  • An agonistic CD25 binder can stimulate or enhance activation of CD25 and/or strengthen one or more signal transduction pathways mediated by CD25.
  • Agonistic CD25 binders may stimulate or enhance CD25 activation by binding CD25, e.g., to induce a conformational change that renders the receptor biologically active.
  • agonistic CD25 binders may promote the trimerization of an IL-2 receptor complex as can occur due to the interaction between CD25 and its cognate ligand, IL-2, thus promoting CD25-mediated signaling.
  • when the anti-CD25 antigen-binding domains of the present disclosure have overlapping epitopes with IL-2, such anti ⁇ CD25 antigen-binding domains may have an agonistic effect upon binding to CD25.
  • anti-CD25 antigen-binding domains of the present disclosure bind to human CD25.
  • anti-CD25 antigen-binding domains (e.g., antibodies such as singledomain antibodies) of the present disclosure may bind to human CD25 with a Ko of less than about IxlO" 6 M, for example, less than about 5xlO ⁇ 7 M, less than about 3xl0" 7 M, less than about IxlO" 7 M, less than about 3xl0" 8 M, less than about 5xl0" 8 M, less than about 3xl0" 8 M, less than about IxlO" 8 M, less than about 8xl0" 9 M, less than about 5xl0“ 9 M, less than about 3xl0 ⁇ 9 M, less than about IxlO" 9 M, about IxlO" 10 to IxlO" 9 M, about lxlQ ⁇ w to 5xl0" 9 M, about IxlO" 10 to IxlO" 9 M, about lxl
  • an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 1.6 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 7.6 nM. In one embodiment, an anti- CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KB of about 9.4 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 10 nM.
  • an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 11 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 12 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 13 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 14 nM. In one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KB of about 17 nM.
  • an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about IS nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 19 nM. In one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KB of about 20 nM. In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 21 nM. In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 22 nM.
  • an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KD of about 26 nM. in one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 31 nM. In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 35 nM. in one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KD of about 49 nM. In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 50 nM.
  • an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 58 nM. In one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a of about 61 nM. In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 62 nM, In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 66 nM. In one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KD of about 73 nM.
  • an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 76 nM, In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 97 nM. In one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KD of about 102 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 107 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 149 nM.
  • an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KD of about 241 nM. in one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 348 nM.
  • anti-CD25 antigen-binding domains of the present disclosure bind to cynomolgus monkey ("cyno") CD25.
  • anti-CD25 antigen-binding domains (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno CD25 with a KD of less than about IxlO -6 M, for example, less than about 5xlO -7 M, less than about 3xl£T 7 M, less than about IxlO” 7 M, less than about 8xl0” 8 M, less than about 5xl0” 8 M, less than about 3xl0’ 8 M, less than about IxlO" 8 M, less than about 8xl0” 9 M, less than about 5xl0 ⁇ 9 M, less than about 3xl0 ⁇ 9 M, less than about IxlO’ 9 M, about IxlO’ 10 to IxlO” 3 M, about IxlO” 10
  • an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 706 pM, in one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 793 pM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 1.5 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 73 nM.
  • an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 34 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 48 nM. In one embodiment, an anti- CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 49 nM.
  • an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 52 nM, In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 57 nM. in one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to cyno CD25 with a KD of about 70 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 79 nM.
  • an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 97 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ka of about 107 nM. In one embodiment, an anti- CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 112 nM. in one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KB of about 115 nM.
  • an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 117 nM. In one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to cyno CD25 with a KD of about 119 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 121 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ka of about 131 nM.
  • an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 136 nM. in one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KB of about 142 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 146 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 148 nM.
  • an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 149 nM, In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 162 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KB of about 163 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 186 nM.
  • an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 191 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 211 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 235 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KES of about 283 nM.
  • an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 339 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 380 nM, In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 411 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KES of about 956 nM. In one embodiment, an antl-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KB of about 2.1 pM.
  • anti-CD25 antigen-binding domains of the present disclosure bind to mouse CD25.
  • anti-CD25 antigen-binding domains of the present disclosure may bind to mouse CD25 with a KD of less than about IxlO” 6 M, for example, less than about 5xl0" 7 M, less than about 3xl0’ 7 M, less than about IxlO’ 7 M, less than about 8x10 8 M, less than about 5xlO’’ 8 M, less than about 3xl0’ 8 M, less than about IxlO’ 8 M, less than about 8xl0’ 3 M, less than about 5xl0’ s M, less than about 3xl0" 9 M, less than about IxlO" 9 M, about IxlO" 10 to IxlO’ 3 M, about IxlO" 10 to 5xl0’ 9 M, about IxlO’ 10 to IxlO" 8 M, about 1x10 ” 10
  • anti-CD25 antigen-binding domains of the present disclosure do not bind to mouse CD25.
  • an anti-CD25 antigen-binding domain of the present disclosure bind to mouse CD25 with a K Q of about 420 nM.
  • anti-CD25 antigen-binding domains of the present disclosure may bind to CD25 with an EC50 from about 1 - 10 nM, about 10 - 100 nM, about 100 nM - 1 pM, or above 1 pM.
  • anti- CD25 antigen-binding domains of the present disclosure may bind to CD25 with an ECsofrom about 10 - 100 nM or about 100 nM - 1 pM.
  • anti- CD25 antigen-binding domains of the present disclosure may bind to CD25 with an EC® of about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, about 200 nM, about 225 nM, about 250 nM, about 275 nM, about 300 nM, about 325
  • Table 1-3 sets forth the sequence identifiers of amino acid sequences of the complementarity determining regions (CDR1, CDR2 and CDR3), amino acid and DNA sequences of the full-length camelid anti-CD25 VHH antibodies, as well as amino acid sequences of corresponding humanized anti-CD25 VHH antibodies.
  • Amino acid sequences of additional exemplary anti-CD25 VHH antibodies and corresponding humanized anti-CD25 VHH antibodies are provided in Table 1-4.
  • “Hui” indicates a humanized variant of the parental VHH antibody.
  • “18", “A8", or “18” indicate that the 8 tt; position in the CDR3 of the parental VHH antibody has been mutated to the amino acid residue isoleucine, alanine, or ieucine, respectively.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single- domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position, and indicates an amino acid residue is absent at the particular position): a). GR(K/R/S)FSTLI (SEQ ID NO: 4137); b). GFTFS(N/S)YA (SEQ ID NO: 4140); and c), GRTF(A/S)(S/W/D)(F/N/Y)G (SEQ ID NO: 10309); d). GFTLDYYA (SEQ ID NO: 7342); and e). G(I/M)P(F/-)(A/-)L(P/V/Y)A (SEQ ID NO: 7366).
  • an anti-CD25 antigen-binding domain described herein comprises a CDR1 comprising the sequence GFTFS(N/S)YA (SEQ ID NO: 4140) or GFTLDYYA (SEQ ID NO: 7342).
  • an anti-CD25 antigen-binding domain described herein comprises a CDR1 comprising the sequence GFTFS(N/S)YA (SEQ ID NO: 4140).
  • an anti-CD25 antigen-binding domain described herein comprises a CDR1 comprising the sequence GFTLDYYA (SEQ iD NO: 7342),
  • an anti-CD25 antigen-binding domain described herein may comprise a CDR1 comprising the sequence GR(K/R/S)FSTLI (SEQ iD NO: 4137)
  • the CDR1 may comprise, e.g., the sequence GR(S/K)FSTLI (SEQ ID NO: 4132).
  • an anti-CD25 antigen-binding domain described herein may comprise a CDR1 comprising the sequence GR(K/R/S)FSTL! (SEQ iD NO: 4137), the CDR1 may comprise, e.g., the sequence GRSFSTLI (SEQ ID NO: 4105).
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position, and indicates an amino acid residue is absent at the particular position): a). GRSFSTLI (SEQ ID NO: 4105); b). GR(S/K)FSTLI (SEQ ID NO: 4132); d). GFTFS(N/S)YA (SEQ ID NO: 4140); e). GRTFS(S/W)(F/N/Y)G (SEQ ID NO: 4142); f). GFTLDYYA (SEQ ID NO: 7342); and g). G(I/M)P(F/-)(A/-)L(P/V/Y)A (SEQ ID NO: 7366).
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR2 comprising an amino acid sequence selected from: a). (l/V)(D/E)R(D/G)(D/G)T(A/P/T); b). IYSD(G/S)SGT (SEQ ID NO: 9441); c). IS(Q/R/G)(S/G)GGRT (SEQ ID NO: 10310); d). IS(R/S)(D/S)G(D/G)ST (SEQ ID NO: 7364); e). ISSGGNT (SEQ ID NO: 7346); and f).
  • CDR2 comprising an amino acid sequence selected from: a). (l/V)(D/E)R(D/G)(D/G)T(A/P/T); b). IYSD(G/S)SGT (SEQ ID NO: 9441); c). IS(Q
  • an anti-CD25 antigen-binding domain described herein comprises a CDR2 comprising the sequence IYSD(G/S)SGT (SEQ iD NO: 9441) or iSSTDGRT (SEQ iD NO: 7348).
  • an anti-CD25 antigen-binding domain described herein comprises a CDR2 comprising the sequence IYSD(G/S)SGT (SEQ ID NO: 9441).
  • an anti-CD25 antigen-binding domain described herein comprises a CDR2 comprising the sequence ISSTDGRT (SEQ ID NO: 7348).
  • an anti-CD25 antigen-binding domain described herein may comprise a CDR2 comprising the sequence (i/V)(D/E)R(D/G)(D/G)T(A/P/T)
  • the CDR2 may comprise, e.g., the sequence (i/V)(D/E)R(D/G)GT(A/P/T).
  • an anti-CD25 antigen-binding domain described herein may comprise a CDR2 comprising the sequence (l/V)(D/E)R(D/G)(D/G)T(A/P/T)
  • the CDR2 may comprise, e.g., the sequence l(D/E)RDGT(T/P) (SEQ ID NO: 4135).
  • an anti ⁇ CD25 antigen-binding domain described herein may comprise a CDR2 comprising the sequence (l/V)(D/E)R(D/G)(D/G)T(A/P/T)
  • the CDR2 may comprise, e.g., the sequence l(D/E)R(D/G)(D/G)T(P/T).
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR2 comprising an amino acid sequence selected from: a). (I/V)(D/E)R(D/G)GT(A/P/T); b). I(D/E)RDGT(T/P) (SEQ ID NO: 4135); c). I(D/E)R(D/G)(D/G)T(P/T); d). IYSDGSGT (SEQ ID NO: 4114); e). ISQSGGRT (SEQ ID NO: 4118); f).
  • CDR2 comprising an amino acid sequence selected from: a). (I/V)(D/E)R(D/G)GT(A/P/T); b). I(D/E)RDGT(T/P) (SEQ ID NO: 4135); c). I(D/E)R(D/G)(D/G)T(P/T); d
  • IS(R/S)(D/S)G(D/G)ST (SEQ ID NO: 7364); g). ISSGGNT (SEQ iD NO: 7346); and h). ISSTDGRT (SEQ ID NO: 7348).
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR3 comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) a). NAL(G/L/P/Q/W)Y (SEQ ID NO: 4131); b). NALR(D/H/N/F) (SEQ ID NO: 4134); c). (K/S/T)TLRY (SEQ ID NO: 4136); d).
  • NIYR(P/S)QVP(P/S/T)TRYS (SEQ ID NO: 7365); and h).
  • AAKRLGP(M/I/A/L)VH(Q/R)YSLEVLTPLFLDEYDY (SEQ ID NO: 9423), wherein one or more non-alanine residues in the CDR3 sequence are optionally replaced with an alanine, and/or one or more alanine residues in the CDR3 sequence are optionally replaced with a glycine.
  • an anti-CD25 antigen-binding domain described herein may comprise a CDR3 comprising an amino acid sequence wherein one or more non-alanine residues in the CDR3 sequence are replaced with an alanine, and/or one or more alanine residues in the CDR3 sequence are replaced with a glycine
  • the CDR3 may comprise an amino acid sequence selected from a).(A/V/S)(A/K/T)(A/G)(A/R/K)(A/G/H/N/R)(A/S)(A/G)(A/S/G)(A/Y)(A/P)(A/W/F/L)(A/D/E)(A /D/E)(A/Y/V); and b).
  • an anti-CD25 antigen-binding domain described herein comprises a CDR3 comprising the sequence
  • an anti-CD25 antigen-binding domain described herein comprises a CDR3 comprising the sequence
  • an anti-CD25 antigen-binding domain e.g., antibody such as a singledomain antibody
  • a CDR3 comprising an amino acid sequence selected from: a). NAL(G/L/P/Q/W)Y (SEQ ID NO: 4131); b). NALR(D/H/N/F) (SEQ ID NO: 4134); c). (K/S/T)TLRY (SEQ ID NO: 4136); d). AKGR(H/N)SGSYYPWD(D/E)Y (SEQ ID NO: 4139); e).
  • AAKRLGPMVH(Q/R)YSLEVLTPLFLDEYDY (SEQ ID NO: 7367), wherein one or more non-alanine residues in the CDR3 sequence are optionally replaced with an alanine, and/or one or more alanine residues in the CDR3 sequence are optionally replaced with a glycine.
  • anti-CD25 antigen-binding domains e.g., antibodies such as single-domain antibodies
  • a set of three CDRs i.e., CDR1-CDR2-CDR3
  • amino acid sequences selected from any of the above-described CORI, CDR2, and CDR3 amino acid sequences.
  • a CORI comprising an amino acid sequence of SEQ, ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9441, and a CDR3 comprising an amino acid sequence of (A/V/S)(A/K/T)(A/G)(A/R/K)(A/G/H/N/R)(A/S)(A/G)(A/S/G)(A/Y)(A/Y)(A/P)(A/W/F/L)(A/D/E)(A/D/E)(A/Y/ V); or x).
  • a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of (A/G)(A/G) ⁇ A/K)(A/R)(A/L)(A/G)(A/P)(M/I/A/L)(A/V)(A/H)(A/R/Q)(A/Y)(A/S)(A/L) ⁇ A/E)(A/V)(A/L)(A/T)(A/P )(A/L)(A/F)(A/L)(A/D)(A/E)(A/Y) ⁇ A/D) ⁇ A/Y).
  • a CDR1 comprising an amino acid sequence of SEQ iD NO: 7342
  • a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348
  • a CDR3 comprising an amino acid sequence of (A/G)(A/G)(A/K) ⁇ A/R)(A/L)(A/G)(A/P)(M/I/A/L)(A/V)(A/H) ⁇ A/R/Q)(A/Y)(A/S)(A/L)(A/E)(A/V)(A/L)(A/T)(A/P )(A/L)(A/F)(A/L)(A/D) ⁇ A/E) ⁇ A/Y)(A/D)(A/Y).
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9411; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9412; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9413; iv) a CDR1
  • a CDR1 comprising an amino acid sequence of SEQ ID NO: 4132, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of (IA’ , )(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; ix) a CDR1 comprising an amino acid sequence of SEQ iD NO: 4132, a CDR2 comprising an amino acid sequence of (l/V)( D/E) R(D/G)GT(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO:
  • a CDR1 comprising an amino acid sequence of SEQ !D NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ iD NO: 7367;
  • a CDR1 comprising an amino acid sequence of SEQ iD NO: 4140, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9441, and a CDR3 comprising an amino acid sequence of
  • a CDR1 comprising an amino acid sequence of SEQ iD NO: 7342
  • a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348
  • a CDR3 comprising an amino acid sequence of (A/G)(A/G)(A/K)(A/R)(A/L)(A/G)(A/P)(M/I/A/L)(A/V)(A/H)(A/R/Q)(A/ ⁇ )(A/S)(A/L)(A/E)(A/V)(A/L)(A/T)(A/P )(A/L)(A/F)(A/L)(A/D)(A/E)(A/Y)(A/D)(A/ ⁇ ),
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10504; ii)a CDR1 comprising an amino add sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10505; iii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10506; iv)a CDR1 comprising an amino acid sequence of
  • xxxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10536
  • xxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10537
  • xxxvija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10538
  • xxxvii)a CDR1 comprising an amino acid sequence of SEQ, ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)GT(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO:
  • a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4135, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4142, a CDR2 comprising an amino acid sequence of i(D/E)R(D/G)(D/G)T(P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4136; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4132, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4139; or v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ ID NO:
  • anti-CD25 antigen-binding domains e.g., antibodies such as single-domain antibodies
  • a CDR1 comprising an amino acid sequence selected from any of the CDR1 amino acid sequences listed in Table 1-3, Table 9, Table 10, or Table 12-1, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence selected from SEQ ID Nos: 4101, 4105, 4109, 4113, 4117, 4726-5030, 7342, 7345, 7931-8226, and 9660-9770, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence selected from SEQ ID Nos:
  • anti-CD25 antigen-binding domains comprising a CDR2 comprising an amino acid sequence selected from any of the CDR2 amino acid sequences listed in Table 1-3, Table 9, Table 10, or Table 12-1, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR2 comprising an amino acid sequence selected from SEQ ID Nos:
  • an anti ⁇ CD25 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR2 comprising an amino acid sequence selected from SEQ ID NOs:
  • anti-CD25 antigen-binding domains e.g., antibodies such as single-domain antibodies
  • a CDR3 comprising an amino acid sequence selected from any of the CDR3 amino acid sequences listed in Table 1-3, Table 9, Table 10, or Table 12-1, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR3 comprising an amino acid sequence selected from SEQ ID Nos:
  • an anti-CD25 antigen-binding domain comprises a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4103, 4107, 4111, 4115, 4119, 5337, 5339, 5371, 5375, 5398, 5401, 5431, 5515, 5519, 5521, 5528, 5532, 5542, 5544, 5545, 5547, 5548, 7344, 7347, 7349, 7350, 9411-9416, 9436, 9887, 9966, 9975, 9978, 9979, 9980 and 10504-10544, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino add sequence selected from SEQ ID NO: 4113; a CDR2 comprising an amino add sequence of SEQ ID NO: 9435; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 10504-10518.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino add sequence selected from SEQ ID NO: 7342; a CDR2 comprising an amino add sequence of SEQ ID NO: 7348; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 10519-10544.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino add sequence selected from any one of SEQ ID NOs: 4726-5030, 7931-8226, and 9660-9770; a CDR2 comprising an amino acid sequence selected from any one of SEQ iD NOs: 5031-5335, 8227-8522, and 9771-9880; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ iD Nos: 5336-5640, 8523-8818, and 9881-9991,
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino add sequence selected from any one of SEQ ID NOs: 4726-5030, 7931-8226, and 9660-9770; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5031-5335, 8227-8522, and 9771-9880; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID Nos: 5336-5640, 8523-8818, and 9881-9991.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4726-4758; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5031-5063; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5336-5368.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4726-7458; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5031-5063; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5336-5368.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4759-4785; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5064-0590; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5369-5395.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4759-4785; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5064-5090; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5369-5395,
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4786-4791; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5091-5096; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5396-5401.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4786-4791; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5091-5096; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5396-5401.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence seiected from any one of SEQ iD NOs: 4792-4904 and 9660-9770; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5097-5209 and 9771-9880; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5402-5514 and 9881-9991.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4792-4904 and 9660-9770; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5097-5209 and 9771-9880; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5402-5514 and 9881-9991.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence seiected from any one of SEQ ID NOs: 4905-5030; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5210-5335; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5515-5640.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4905-5030; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5210-5335; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5515-5640.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 7931-8120; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8227-8416; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8523-8712.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 7931-8120; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8227-8416; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8523-8712.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3121-8224; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8417-3520; and/or a CDR3 comprising an amino add sequence selected from any one of SEQ ID NOs: 8713-8816.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence seiected from any one of SEQ ID NOs: 3121-8224; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8417-8520; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs; 8713-8816.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8225-8226; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8521-8522; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8817-8818.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8225-8226; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8521-8522; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8817-8818.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113; a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 10504- 10518.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113; a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 10504- 10518.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342; a CDR2 comprising an amino acid sequence of SEQ ID NQ: 7348; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 9414 and 10519-10544.
  • an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino add sequence of SEQ ID NO: 7342; a CDR2 comprising an amino add sequence of SEQ ID NQ: 7348; and a CDR3 comprising an amino add sequence selected from any one of SEQ ID NOs: 9414 and 10519-10544.
  • anti-CD25 antigen-binding domains e.g., antibodies such as single-domain antibodies
  • anti-CD25 antigen-binding domains comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within any of the exemplary anti-CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 10, or Table 12-1.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises: i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4102, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4103; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4106, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4107; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4109, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4110, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4111; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a
  • Ixiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10520; lxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10521; Ixv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10522;
  • Ixviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10525;
  • Ixixja CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9414; lxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10526; lxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10527;
  • Ixxilja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10528; lxxiii)a CDR1 comprising an amino acid sequence of SEQ ID NQ: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10529;
  • Ixxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10530; lxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10531;
  • Ixxvija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10532;
  • CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10533;
  • Ixxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10534; lxxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10535; lxxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10536; lxxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and
  • Ixxxiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 10539; lxxxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10540;
  • Ixxxvja CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10541;
  • Ixxxvija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10542;
  • Ixxxviija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10543; or
  • an anti-CD25 antigen-binding domain e.g., antibody such as a single-domain antibody
  • an anti-CD25 antigen-binding domain of the present disclosure comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ iD NO: 9436, [0454]ln one embodiment, an anti-CD25 antigen-binding domain (e,g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO:
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10531.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10534.
  • anti-CD25 antigen-binding domains e.g., antibodies such as single-domain antibodies
  • anti-CD25 antigen-binding domains comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within a VHH amino acid sequence as defined by any of the exemplary anti-CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 10 or Table 12-1.
  • antibodies, or antigen-binding fragments thereof comprising the set of CDR1-CDR2-CDR3 amino acid sequences contained within a VHH amino acid sequence selected from SEQ ID Nos: 4104, 4108, 4112, 4116, 4120, 4226-4130, 4143-4725, 5641-5945, 7351-7354, 7359-7362, 7368-2830, 8819-9114, and 9417-9422, 9437, 9439, 9442-9659, 9992-10102, 10214-10276, and 10545-10585.
  • VHH amino acid sequence selected from SEQ ID Nos: 4104, 4108, 4112, 4116, 4120, 4226-4130, 4143-4725, 5641-5945, 7351-7354, 7359-7362, 7368-2830, 8819-9114, and 9417-9422, 9437, 9439, 9442-9659, 9992-10102, 10214-102
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure can include: a), a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4104; b). a variable domain that comprises a CDR1, CDR2, and CDRS contained within a VHH comprising the amino acid sequence of SEQ ID NG: 4108; c). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4112; d).
  • variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7353; i). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7354; or j). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9437.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure can include a), a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4126; b). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4127; c). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4128; d).
  • variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7361; i). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7362; j). a variable domain that comprises a CORI, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9417; k).
  • VHH comprising the amino acid sequence of SEQ ID NO: 10548; u) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10549; v) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10550; w) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10551; x) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10552; y) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10553; z) a variable domain that comprises a CDR1, C
  • variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10566; mm) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9420; nn) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10567; co) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10568; pp) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10569; qq) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10570
  • an anti-CD25 antigen-binding domain e.g., antibody such as a singledomain antibody
  • a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9437 or 9439.
  • an anti-CD25 antigen-binding domain e.g., antibody such as a singledomain antibody
  • a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9422.
  • an anti-CD25 antigen-binding domain e.g., antibody such as a singledomain antibody
  • a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10572.
  • an anti-CD25 antigen-binding domain e.g., antibody such as a singledomain antibody
  • an anti-CD25 antigen-binding domain of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10575.
  • an anti-CD25 antigen-binding domain e.g., antibody such as a single-domain antibody
  • an anti-CD25 antigen-binding domain can include a VHH amino acid sequence selected from SEQ ID Nos: 4104, 4108, 4112, 4116, 4120, 4143-4442, 5641-5945, 7351-7354, 7368-7659, 8819-9114, 9437, 9442-9551, 9992-10102, and 10246-10276, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain e.g., antibody such as a single-domain antibody
  • an anti-CD25 antigen-binding domain of the present disclosure can include a VHH amino acid sequence selected from SEQ ID Nos: 4104, 4108, 4112, 4116, 4120, 7351-7354, 9437, and 10246-10276, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain e.g., antibody such as a single-domain antibody
  • an anti-CD25 antigen-binding domain of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID Nos: 4126-4130, 4443-4725, 7359-7362, 7660-7930, 9417-9422, 9439, 10214- 10245, 9552-9659, and 10545-10585, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity,
  • an anti-CD25 antigen-binding domain e.g., antibody such as a single-domain antibody
  • an anti-CD25 antigen-binding domain of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID NOs: 4126-4130, 7359-7362, 9417-9422, 9439, 10214-10245, and 10545-10585, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain e.g., antibody such as a single-domain antibody
  • an anti-CD25 antigen-binding domain of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID NQs: 9439, 9422, 10572, and 10575, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain e.g., antibody such as a single-domain antibody
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
  • 4129 or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 7359, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 7360, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity,
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
  • an anti-CD25 antigen-binding domain (e.g,, antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
  • 9420 or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 9439, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10214, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10215, or a similar sequence thereof having at least 70%, at least 75%, at least 30%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10216, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ iD NO: 10217, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10218, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10220, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10221, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10222, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10223, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10224, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10225, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at ieast 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ iD NO: 10226, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ iD NO: 10227, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10228, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10229, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10230, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10231, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10232, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10233, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an antl ⁇ CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10234, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10235, or a similar sequence thereof having at least 70%, at least 75%, at least 30%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10236, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ iD NO: 10237, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10238, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10239, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10240, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10241, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10242, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10243, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10244, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at ieast 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10245, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10545, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g,, antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10546, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain e.g., antibody such as a single-domain antibody
  • an anti-CD25 antigen-binding domain of the present disciosure comprises an amino acid sequence of SEQ ID NO: 10547, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10548, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10549, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10550, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10551, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10552, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10553, or a similar sequence thereof having at least 70%, at least 75%, at least 30%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10554, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10555, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10556, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10557, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10558, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10559, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10560, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10561, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10562, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at ieast 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10563, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10564, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g,, antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10565, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10566, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 9420, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10567, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10568, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10569, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10570, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10571, or a similar sequence thereof having at least 70%, at least 75%, at least 30%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10572, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10573, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10574, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10575, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10576, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10577, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10578, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti ⁇ CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10579, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10580, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at ieast 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10581, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10582, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g,, antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10583, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10584, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
  • an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10585, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the present disclosure also includes an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) that competes for binding to CD25 with any one of the exemplary anti-CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 10 or Table 12-1.
  • an anti-CD25 antigen-binding domain e.g., antibody such as a single-domain antibody
  • the present disclosure also includes an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) that binds to the same epitope on CD25 as any one of the exemplary anti ⁇ CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 10 or Table 12-1.
  • the present disclosure also includes an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) that comprises a means for binding an epitope within human CD25 bound by any one of the exemplary anti-CD25 VHH antibodies listed in Table 1-3, Table 1- 4, Table 9, Table 10, or Table 12-1.
  • an anti-CD25 antigen-binding domain e.g., antibody such as a single-domain antibody
  • an anti-CD25 antigen-binding domain e.g., antibody such as a single-domain antibody
  • a single-domain antibody (e.g., VHH) specific for TN FR2 or CD25, as described herein, can be obtained by immunization of dromedaries, camels, llamas, alpacas, or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies.
  • Antigens can be purified from natural sources, or in the course of recombinant production. Immunization and/or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens. By reverse transcription and polymerase chain reaction (PCR), a gene library of single-domain antibodies containing several million clones can be produced.
  • PCR polymerase chain reaction
  • a different method may use gene libraries from animals that have not been previously immunized. Such naive libraries usually contain only antibodies with low affinity to the desired antigen, making it necessary to apply affinity maturation by random mutagenesis as an additional step. See e.g., Saerens, D.; et al. (2008). "Single-domain antibodies as building blocks for novel therapeutics”. Current Opinion in Pharmacology 8 (5): 600-608. [0563]Affinity maturation strategies can be categorized as either targeted/rational approaches or untargeted/random approaches. For targeted approaches information about the VHH of interest is needed, such as hot spots for affinity maturation or structural information on the VHH:antigen complex, whereas for untargeted approaches no prior information is needed.
  • Targeted approaches that may be applied for affinity maturation of VHHs include site-directed in-vitro mutagenesis and in- sr//co/computational approaches.
  • Common untargeted approaches used for affinity maturation of VHHs include random in-vitro mutagenesis, CDR swapping and autonomous hypermutation yeast surface display, with the latter two being novel, emerging and very time efficient techniques.
  • Most of these strategies have in common, that after applying a certain randomization strategy to generate a mutational library, the resulting library can be screened by employing standard display techniques such as yeast, phage or ribosome display to select for the best binders.
  • the choice of the display system is often guided by the library size to be displayed, with yeast display being able to handle library sizes of ⁇ 10 7 - 10 9 , phage display ⁇ 10 8 -10 w and ribosome display ⁇ 10 12 -10 13 (Chan and Groves, 2021).
  • yeast display being able to handle library sizes of ⁇ 10 7 - 10 9
  • phage display ⁇ 10 8 -10 w and ribosome display ⁇ 10 12 -10 13 (Chan and Groves, 2021).
  • the number of highly interactive residues such as aromatic amino acids usually increase in the CDR regions.
  • the selected affinity matured clones may be further evaluated by a developability assessment to test for undesired properties, such as unspecific binding to off-targets or VHH instability.
  • a set of selected residues within the CDRs of a VHH may be mutated (Tiller et al., 2017; Yau et al., 2005). Pre-selection of these residues can be either performed using alanine scanning to identify hot spot residues for mutation or by using structural data of the antigemVHH complex to identify positions to be mutated. These sites can then be either submitted to saturating mutagenesis to substitute a specific site with all possible amino acids or specific amino acid substitutions yielding several smaller libraries. After mutagenesis binders can be displayed to select the best matured candidate. Usually, several rounds of targeted mutagenesis are performed with separate sub-libraries to obtain combinations of individual mutations that cooperatively result in increased binding affinity.
  • silica methods are often used to guide targeted in vitro mutagenesis. Using homology modeling of the target:VHH complex or docking, hotspots for mutations can be identified that are then submitted to in vitro mutagenesis (Bert Schepens et al., 2021; Cheng et al., 2019; Inoue et al., 2013; Mahajan et al., 2018). Further, in silica methods can search all designed variants in a virtual library ( ⁇ 1O 40 members) in a rather short amount of time to identify a feasible number of promising candidates to be tested experimentally. These techniques can be especially valuable if structural data on the drugtarget interaction are available.
  • Untargeted/random affinity maturation strategies that can be applied to affinity mature VHHs include random in vitro mutagenesis, CDR shuffling/swapping and in vivo affinity maturation via yeast display.
  • random in vitro mutagenesis the sequence of either the entire VHH or only the CDRs are mutated randomly (Chen et al., 2021; Ye et al., 2021; Zupancic et al., 2021).
  • the most commonly used technique is error prone PCR employing a DNA polymerase that lacks proof reading activity and PCR conditions that increase the polymerase error rate even further. This technique can be applied without further structural knowledge or information on the importance of residues that contribute to antigemVHH interaction.
  • the resulting mutational library can then be displayed to select the best matured candidate.
  • This technique may also be combined with NGS sequencing of the display elutions to get an in-depth readout of all obtained candidates, enabling the identification of low abundant but still promising clones (Chen et al., 2021).
  • CDR shuffling or swapping is applied for VHH affinity maturation, such as described in Zupancic et al., 2021.
  • enriched libraries can be used as input material for a PCR reaction to individually amplify the CDR of the VHHs.
  • the PCR products can then be mixed and reassembled using overlapping PCR to generate the entire plasmid for further rounds of display to select for the best matured binder.
  • in vivo affinity maturation via yeast display is applied for VHH affinity maturation, such as described in Wellner et al., 2021.
  • the method is based on an autonomous hypermutation yeast surface display (AHEAD), which imitates somatic hypermutation during VHH selection using engineered yeast strains.
  • AHEAD autonomous hypermutation yeast surface display
  • the yeast's error prone orthogonal DNA replication system can generate new variants during plasmid replication by randomly introducing mutations.
  • the new variants can then be displayed and selected using yeast surface display to identify the best binders. This enables the production of high affinity clones in very little time (about 2 weeks), which is significantly faster than classical affinity maturation procedures.
  • the method can be applied using synthetic or immune libraries using unenriched libraries enriched libraries or a subset of preselected clones.
  • binders with medium affinity are required, as it is the case for the V-bodies described herein, and the affinity of the identified candidates need to be decreased, very similar techniques can be applied. For example, mutations that are aiming at lowering the affinity can be introduced using the same targeted or untargeted approaches as described for the affinity maturation. The selection afterwards can be adapted accordingly. If larger libraries are generated that need to be screened via a display technique, the selection strategy can be adapted to enrich medium affinity binders while excluding high affinity candidates. This could, for example be a pre-panning in phage display with low antigen concentration to remove all higher affinity candidates, followed by a selection with high antigen concentration to obtain medium affinity VHHs. For library sizes of up to 1000 candidates a kinetic off- rate characterization can be used to get immediate information about the kinetic behavior of the candidates.
  • DMA sequence can be optimized, for example to improve their stability towards enzymes.
  • Another goal is humanization to prevent immunological reactions of the human organism against the antibody. Humanization can be achieved based on the homology between camelid VHH and human VH fragments, which is described in further detail below.
  • the optimized single-domain antibody can be translated and expressed in suitable organisms such as E. caii or Saccharomyces cerevisiae.
  • Single-domain antibodies can also be derived from conventional antibodies.
  • single-domain antibodies can be made from conventional murine or human IgG with four chains. The process is similar, comprising gene libraries from immunized or naive donors and display techniques for identification of the most specific antigens.
  • the binding region of a conventional IgG consists of two domains (VH and VL), which tend to dimerize or aggregate because of their lipophilicity. Monomerization can be accomplished by replacing lipophilic by hydrophilic amino acids. (See e.g., Borrebaeck, C. A. K.; Ohlin, M. (2002). "Antibody evolution beyond Nature". Nature Biotechnology 20 (12): 1189-90.) If affinity can be retained after monomerization, the single-domain antibodies can likewise be produced in E. coli, S. cerevisiae or other suitable organisms.
  • a "humanized antibody” refers to a chimeric, genetically engineered, antibody in which the amino acid sequences (typically CDRs) from an antibody (donor antibody), e.g., a camelid antibody, are grafted onto a human antibody (acceptor antibody).
  • a humanized antibody typically comprises CDRs from a donor antibody and variable region framework and constant regions, when present, from a human antibody.
  • a “humanized VHH” comprises CDRs that corresponds to the CDRs of a naturally occurring VHH domain (e.g., a camelid VHH), but that has been "humanized”.
  • Humanized VHH may be prepared by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (particularly in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain human antibody.
  • Such humanized VHHs can be obtained in any suitable manner known to a skilled person in the art and thus not strictly limited to methods described herein.
  • a human germline reference that is most similar to the camelid germline sequence of the selected VHH may be identified.
  • Most of the isolated camelid VHHs in literature belong to the camelid IGHV3 subfamily 2 (Nguyen et al., 2000, EMBO J) with DP-47/VH3-23 from the IGHV3 family commonly used as human reference.
  • the framework of the camelid VHH can then be compared to the human reference sequence. Surface exposed residues are substituted to their human counterpart as it is assumed that their contribution to protein stability is rather low. Buried residues however remain of camelid origin, as they likely contribute to the overall VHH stability.
  • residues H37 and H47 are known to interact with the CDR-H3 loop in many VHHs, stabilizing its conformation and thereby contributing to antigen binding affinity.
  • a significant number of VHHs use framework 2 residues H44, H45 and H47 for antigen binding (Zavrtanik et al., 2018, J Mol Biol ⁇ .
  • a full humanization of these residues hence frequently results in reduced solubility or aggregation of the VHHs and a reduced or complete loss of binding affinity for the target antigen (van Faassen et al., 2020, Vincke et al., 2009). in consequence, all or at least some of these hallmark residues in framework 2 remain of camelid origin when humanizing VHHs.
  • CDR grafting is CDR grafting.
  • CDRs of the selected VHHs can be transplanted onto a universal VHH framework that has been partially or fully humanized (Saerens et al., 2009 J Biol Chem, Soler et al., 2021, Vincke et al., 2009 J Biol Chem).
  • CDR grafting has been successfully used in some cases but failed for several others, with VHHs frequently losing their potential to bind to the desired antigen and/or becoming structurally instable with a high tendency to aggregate (van Faassen et al., 2020, FASEB).
  • humanized VHH sequences still retain the residues that are relevant for protein A binding.
  • the engineering activities during humanization may be applied to engineer protein A binding properties into a VHH that did previously not interact with protein A (Graille et al confuse 2000, PNAS).
  • a “cameiized antibody” refers to an antibody having amino acid sequences (typically CD Rs) from a donor antibody, e.g., a human antibody, and variable region framework and constant regions, when present, from a camelid antibody.
  • a “cameiized VH” comprises an amino acid sequence that corresponds to the amino add sequence of a naturally occurring VH domain, but that has been “cameiized”.
  • Cameiized VH may be prepared by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody.
  • the VH sequence that is used as a starting material or starting point for generating or designing the cameiized VH is a VH sequence from a mammal, or the VH sequence of a human antibody.
  • camelized VH can be obtained in any suitable manner known to a skilled person in the art and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.
  • the amino acid residues of a single-domain antibody can be numbered according to the general numbering for VH domains given by Kabat et al.
  • the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering. For example, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering.
  • the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
  • the total number of amino acid residues in a VH domain and a VHH domain is usually in the range of from 110 to 120, often between 112 and 115. However, smaller and longer sequences may also be suitable for the purposes described herein.
  • the boundaries of a given CDR or framework (FR) may vary depending on the scheme used for identification.
  • the Kabat scheme is based on structural alignments
  • the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, "30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • the AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular's AbM antibody modeling software.
  • CDRs can be defined in accordance with any of the Kabat numbering scheme, the Chothia numbering scheme, a combination of Kabat and Chothia, the AbM numbering scheme, and/or the Contact numbering scheme.
  • a VHH typically comprises three CDRs, designated CDR1, CDR2, and CDR3.
  • Table 1-5 below, lists exemplary position boundaries of CDR-Hl, CDR-H2, CORNS as identified by Kabat, Chothia, AbM, and Contact schemes, respectively.
  • residue numbering is listed using both the Kabat and Chothia numbering schemes.
  • FRs are located between CDRs, for example, with FR-H1 located before CDR-Hl, FR-H2 located between CDR-Hl and CDR-H2, FR- H3 located between CDR-H2 and CDR-H3 and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-Hl loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.
  • CDR complementary metal-oxide-semiconductor
  • a particular CDR e.g., a CDR-H3
  • a CDR-H3 contains the amino acid sequence of a corresponding CDR in a given VHH amino acid sequence
  • a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the VHH, as defined by any of the above-mentioned schemes.
  • specific CDR sequences are specified.
  • Exemplary CDR sequences of provided antibodies are described using various numbering schemes (see e.g., Table 1-5), although it is understood that a provided antibody can include CDRs as described according to any of the other above-mentioned numbering schemes or other numbering schemes known to a person of ordinary skill in the art,
  • the framework sequences may be any suitable framework sequences.
  • the framework sequences may be framework sequences derived from a heavy chain variable domain (e.g., a VH sequence or VHH sequence).
  • the framework sequences are either framework sequences that have been derived from a VHH sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences (in which said framework sequences may optionally have been partially or fully camelized).
  • an antigen-binding protein of the present disclosure may comprise naturally occurring sequences (from a suitable species), recombinant sequences, or synthetic or semi-synthetic sequences.
  • nucleotide sequences encoding antigen-binding proteins of the present disclosure may comprise naturally occurring nucleotide sequences, recombinant sequences, or synthetic or semi-synthetic sequences (for example, sequences that are prepared by PCR or isolated from a library).
  • Anti-TNFR2 antigen-binding domains e.g., antibodies such single-domain antibodies
  • anti- CD25 antigen-binding domains of the present disclosure may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy chain variable domains as compared to the exemplary antibody sequences provided herein.
  • Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases.
  • the anti-TNFR2 antigen- binding domains and anti-CD25 antigen-binding domains of the present disclosure may comprise any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations").
  • Germline mutations such sequence changes are referred to herein collectively as "germline mutations"
  • all of the framework and/or CDR residues within the VHH domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3.
  • one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antigen-binding domain was originally derived).
  • the antigen-binding domains of TNFR2 and CD25 may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.
  • antigen-binding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced biological properties (e.g., agonistic effect), reduced immunogenicity, etc.
  • Antigen-binding proteins comprising one or more antigen-binding domains obtained in this general manner are encompassed within the present disclosure.
  • anti-TNFR2 antigen-binding domains and anti-CD25 antigen-binding domains comprising variants of any of the VHH and/or CDR amino acid sequences disclosed herein having one or more amino acid substitutions.
  • the present disclosure includes anti-TNFR2 antigen-binding domains and anti-CD25 antigen-binding domains having VHH and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 3 or fewer, 2, or 1 amino acid substitutions relative to any of the VHH and/or CDR amino acid sequences set forth in Tables 1-3, 1-4, 9, 10, 11 and 12-1. herein.
  • Amino acid substitutions may be introduced into an antigen-binding protein of interest and the resultant variants can screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or reduced ADCC or CDC.
  • amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
  • an amino acid substitution is a conservative substitution, meaning exchanging an amino acid with another amino acid of the same class
  • amino acid substitutions may also include a non-conservative substitution, meaning exchanging an amino acid with an amino acid of a different class.
  • Other exemplary amino acid substitutions are shown in Table 1-6.
  • single-domain antibodies (e.g., VHH) of the present disclosure may comprise one or more mutations to reduce oxidation levels of oxidation-labile residues such as Met (M).
  • Met (M) oxidation-labile residues
  • the single-domain antibodies (e.g., VHH) of the present disclosure may comprise one or more mutations (e.g., substitution mutations) of a Met residue to reduce oxidation.
  • a Met residue may be substituted in any of the single-domain antibodies described herein with, e.g., He (I), Ala (A), or Leu (L), to reduce oxidation.
  • anti-TNFR2 and anti ⁇ CD25 single-domain antibodies (e.g., VHH) of the present disclosure comprise one or more modifications that reduce binding of the single-domain antibodies (e.g., VHH) by pre-existing antibodies found in human blood or serum.
  • single-domain antibodies (e.g,, VHHs) of the present disclosure are modified by mutation of amino acid position 11, for example LeullGlu (L11E), LeullLys (L11K), or LeullVal (L11V).
  • a single-domain antibody (e.g,, VHH) of the present disclosure may comprise a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering).
  • a single-domain antibody (e.g., VHH) of the present disclosure may comprise an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the single-domain antibody (e.g,, VHH).
  • A alanine extension
  • a single-domain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering).
  • a singledomain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering).
  • the C-terminus of a single-domain antibody can be any one of VKVSS (SEQ ID NO: 4032), VQVSS (SEQ ID NO: 4033), VTVKS (SEQ ID NO: 4034), VTVQS (SEQ ID NO: 4035), VKVKS (SEQ ID NO: 4036), VKVQS (SEQ ID NO: 4037), VQVKS (SEQ ID NO: 4038), or VQVQS (SEQ ID NO: 4039).
  • VTVSSA SEQ ID NO: 4040
  • VKVSSA SEQ ID NO: 4041
  • VQVSSA SEQ ID NO: 4042
  • single-domain antibodies e.g., VHH
  • single-domain antibodies are modified by changes in carboxy-terminal region, for example to a terminal sequence having the sequence GQGTLVTVKPGG (SEQ ID NO: 4043) or GQGTLVTVEPGG (SEQ ID NO: 4044) or modification thereof. Additional modification to reduce binding by pre-existing antibodies in human serum can be found in e.g., W02012/175741, WO2015/173325, W02016/150845, WQ2011/003622, WQ2013/024059; US 11,426,468; US 10,526,397, which are incorporated herein by reference in their entities.
  • a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267).
  • a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VAGG (SEQ ID NO: 7266).
  • a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VPAG (SEQ ID NO: 7267).
  • anti-TNFR2 single-domain antibody e.g., VHH
  • VHH single-domain antibody
  • amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 93-640, 2805-3363, 6651-6697, 6931-6992, 7174-7197, 10380, 10381, 10383, 10385, and 10386, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267).
  • anti-TNFR2 single-domain antibody e.g., VHH
  • VHH single-domain antibody
  • amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 10380, 10381, 10383, 10385, and 10386, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267).
  • anti-TNFR2 single-domain antibody e.g., VHH
  • VHH single-domain antibody
  • amino acid sequence selected from any one of SEQ ID NOs: 81-92, 6648-6650, 7092, 7095, 7098, 7101, 7293-7296, 641-1127, 6698-6744, 10375, 10382, 10384, 10394-10408, 10457-10502, 10812, 10720-10727, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ iD NO: 7267).
  • anti-TNFR2 single-domain antibody e.g., VHH
  • VHH single-domain antibody
  • amino acid sequence selected from any one of SEQ ID NOs: 81-92, 6648-6650, 7092, 7095, 7098, 7101, 7293-7296, 10375, 10382, 10384, 10394-10408, 10457-10502, and 10812, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267).
  • anti-TNFR2 single-domain antibody e.g., VHH
  • VHH single-domain antibody
  • amino acid sequence selected from any one of SEQ ID NOs: 10720-10727, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267).
  • anti-CD25 single-domain antibody e.g., VHH
  • VHH single-domain antibody
  • anti-CD25 single-domain antibody of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, 4143-4442, 5641-5945, 7351-7354, 7359-7362, 2268-2830, 8819-9114, 9417-9422, 9437, 9439, 9442- 9659, 9992-10102, 10214-10276, and 10545-10585, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (
  • anti-CD25 single-domain antibody e.g., VHH
  • VHH single-domain antibody
  • amino acid sequence selected from any one of SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, 4126-4130, 7351-7354, 7359-7362, 9417-9422, 9437, 9439, 10214-10276, and 10545-10585, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267).
  • antl-CD25 single-domain antibody of the present disclosure comprises an amino acid sequence seiected from any one of SEQ ID NOs: 4126-4130, 4443-4725, 7359- 7362, 7660-7930, 9417-9422, 9439, 10214-10245, and 10545-10585, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267).
  • anti-CD25 single-domain antibody e.g., VHH
  • VHH single-domain antibody
  • amino acid sequence selected from any one of SEQ ID NOs: 4126-4130, 7359-7362, and 9417-9422, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ, ID NO: 7266) or VPAG (SEQ ID NG: 7267).
  • anti-TNFR2 and anti-CD25 single-domain antibodies are modified to enhance binding to staphylococcal protein A (SpA) or streptococcal protein G (SpG). Binding of SpA and SpG to antibodies or antibody fragments can be useful in the manufacturing process of the antibodies or antibody fragments.
  • SpA staphylococcal protein A
  • SpG streptococcal protein G
  • binding of SpA and SpG to antibodies or antibody fragments can be useful in the manufacturing process of the antibodies or antibody fragments.
  • the high-affinity interaction of the IgG Fc region with SpA and SpG has been extensively exploited and became the gold standard for monoclonal antibody purification (Bjbrck and Kronvall, 1984).
  • Other non-Fc containing antibody fragments, such as VHHs and Fabs do not have the capacity to bind to SpA or SpG via their Fc regions.
  • anti-TNFR2 and anti-CD25 single-domain antibodies e.g., VHH
  • VHH single-domain antibodies
  • the VHH-SpA interface has been mapped to thirteen residues, which duster within the framework at the back side of the V-body, distant to the CDRs (Graille et al, 2000, Henry et al., 2016).
  • superposition of a SpA-Fab crystal structure and a VHH allows for visualizing the binding mode.
  • a SpA-binding motif included in an anti- TNFR2 and anti-CD25 single-domain antibody (e.g., VHH) of the present disclosure may include one or more, or all of the thirteen residues.
  • single-domain antibodies e.g., VHH
  • single-domain antibodies comprise one or more modifications at N-terminus to prevent formation of a pyroglutamate and product heterogeneity.
  • the amino acid residue Glu at the first position of the single-domain antibodies is replaced with Asp (EID).
  • an anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 93-640, 2805-3363, 6651-6697, 6931-6992, 7174-7197, 10380, 10381, 10383, 10385, and 10386, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID).
  • EID Asp
  • an anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NQs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 7090, 10380, 10381, 10383, 10385, and 10386, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID).
  • EID Asp
  • an anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 6643-6650, 7092, 7095, 7098, 7101, 7293-7296, 641-1127, 6698-6744, 10375, 10382, 10384, 10394-10408, 10457- 10502, 10812, 10720-10727, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID).
  • EID Asp
  • an anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID Nos: 81-92, 6648-6650, 7092, 7095, 7098, 7101, 7293-7296, 7101, 10375, 10382, 10384, 10394-10408, 10457-10502, and 10812 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the singledomain antibody (e.g., VHH) is replaced with Asp (EID).
  • EID Asp
  • an anti-TNFR2 single-domain antibody e.g., VHH
  • an anti-TNFR2 single-domain antibody comprises an amino acid sequence selected from any one of SEQ, ID Nos: 10720-10727, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID).
  • EID Asp
  • EID Asp
  • EID Asp
  • EID Asp
  • EID Asp
  • anti-TNFR2 antigen-binding domains and/or anti-CD25 antigen-binding domains and/or multispecific antigen-binding proteins of the present disclosure can adopt an alternative protein scaffold.
  • Such alternative protein scaffold may be a single chain polypeptidic framework, optionally with a reduced size (e.g., less than about 200 amino acids), that contains a highly structured core associated with variable domains of high conformational tolerance allowing insertions, deletions, or other substitutions.
  • antigen-binding proteins may be generated by grafting CDRs or variable regions described herein onto a suitable protein scaffold.
  • the structure of alternative scaffolds may vary, but preferably are of human origin for those developed as therapeutics.
  • an alternative protein scaffold of the present disclosure can be derived from Protein A, e.g., the Z-domain thereof (affibodies), lmmE7 (immunity proteins), BPTI/APPI (Kunitz domains), Ras- binding protein AF-6 (PDZ-domains), charybdotoxin (Scorpion toxin), CTLA-4, Min-23 (knottins), lipocalins (anticalins), neokarzi nostatin, a fibronectin domain (used in "adnectin”), an ankyrin repeat (AR) domain (used in "DARPins”), avidity multimers (also known as "avimers”), or thioredoxin (
  • Antica H ns are a suitable type of non-lg based alternative scaffolds for use in the antigen-binding molecules of the present disclosure
  • Anticalins are a class of engineered ligand-binding proteins that are based on the lipocalin scaffold.
  • Lipocalins are a family of proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. Lipocalins have limited sequence homology, but share a common tertiary structure architecture based on eight antiparallel ⁇ -barrels. Lipocalins contain four exposed loops built on the rigid ⁇ -barrel structure. Exemplary anticalin proteins that are commonly used are about a size of about 180 amino acids and a mass of about 20 kDa.
  • DARPins are another suitable non-lg based alternative scaffold that can be used in the antigenbinding molecules of the present disclosure.
  • DARPins are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. They are derived from natural ankyrin repeat (AR) proteins, which usually contain a 33 amino acid protein motif consisting of two a-helices separated by loops, which repeats mediate protein-protein interactions.
  • AR ankyrin repeat
  • DARPins can be generated using combinatorial AR libraries constructed based on the 33 amino acid AR motif with seven randomized positions.
  • DARPin libraries can be screened using ribosome display, and library members typically are well produced in Escherichia coii, do not aggregate, and display high thermodynamic stability.
  • DARPins contain two to four of these motifs flanked by N- and C- terminal capping motifs to shield hydrophobic regions and allow increased solubility.
  • the avimer structure can also be used as a protein backbone to generate a suitable non-lg based alternative scaffold.
  • Avimers typically consist of two or more peptide sequences of 30 to 35 amino acids each, connected by peptide linker. The individual sequences are derived from A-domains of various membrane receptors and have a rigid structure, stabilized by disulfide bridges and calcium. Each A- domain can bind to a certain epitope of the target protein. The combination of domains binding to different epitopes of the same protein increases affinity to this protein, an effect known as avidity.
  • fibronectin III (FN3) domains can also be used to generate a suitable non-lg based alternative scaffold (also known as "monobody”).
  • FN10 tenth fibronectin type III domain
  • the connecting loops of FN10 each about 15 to 21 amino acids in length, can be randomized and the domains displayed on both phage and yeast to select for a scaffold with the desirable properties.
  • AdnectinsTM Is an exemplary scaffold generated using 10 th FN3 domains randomized and displayed in this way.
  • Another exemplary scaffold comprising FN3 domains is a CentyrinTM.
  • CentryrinsTM contain the consensus sequence of FN3 domains of human Tenascin C (TNC), which is found in the extracellular matrix of various tissues.
  • CentyrinTM scaffolds have loops that have structural homology to antibody variable domains (i.e., CDR1, CDR2 and CDR3), and are small (about 10 kDa), simple, and highly stable single domain proteins that do not contain cysteine, disulfides or glycosylated residues.
  • CentyrinTM possess excellent biophysical properties such as stability to heat, pH, denaturant and organic solvents, reversible unfolding and monodispersity.
  • Another recent exemplary FN3-based scaffold that can be used in the present disclosure is fluctuation-regulated affinity proteins (FLAPs), as described in See et al., 2020. Biotechnology journal 15(12):e2000078, which is incorporated herein by reference in its entirety.
  • multispecific antigen-binding proteins and conjugates comprising one or more anti ⁇ TNFR2 antigen-binding domains (e.g., antibody such as a single-domain antibody) and one or more anti-CD25 antigen-binding domains (e.g., antibody such as a single-domain antibody) that are linked, directly or indirectly, to one or more additional domains or moieties.
  • the one or more anti-TNFR2 antigen-binding domains can specifically bind TNFR2.
  • the one or more anti-CD25 antigen-binding domains can specifically bind CD25.
  • a multispecific antigen-binding protein or conjugate of the present disclosure comprises one or more anti-TNFR2 antigen-binding domains (e.g., antibody such as a singledomain antibody) and one or more anti-CD25 antigen-binding domains (e.g., antibody such as a singledomain antibody) provided herein operably linked to a dimerization domain such as an immunoglobulin Fc region.
  • anti-TNFR2 antigen-binding domains e.g., antibody such as a singledomain antibody
  • anti-CD25 antigen-binding domains e.g., antibody such as a singledomain antibody
  • An immunoglobulin Fc region may be linked indirectly or directly to the one or more anti- TNFR2 antigen-binding domains (e.g., antibody such as a single-domain antibody) and one or more anti- CD25 antigen-binding domains (e.g., antibody such as a single-domain antibody), in some embodiments, a multispecific antigen-binding protein or conjugate of the present disclosure comprises one, two, three, four, five, six or more anti-TNFR2 antigen-binding domains and one, two, three, four, five, six or more anti-CD25 antigen-binding domains provided herein operably linked to an Fc region.
  • a multispecific antigen-binding protein or conjugate of the present disclosure comprises one, two, three, four, five, six or more anti-TNFR2 antigen-binding domains and one, two, three, four, five, six or more anti-CD25 antigen-binding domains provided herein operably linked to an Fc region.
  • the present disclosure provides a multispecific antigen-binding protein or conjugate that comprises (1) one or more anti-TNFR2 antigen-binding domains, such as one or more anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13; (2) one or more anti-CD25 antigen-binding domains, such as one or more anti-CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 10, or Table 12-1; and (3) an Fc region.
  • one or more anti-TNFR2 antigen-binding domains such as one or more anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13
  • one or more anti-CD25 antigen-binding domains such as one or more anti-CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 10, or Table 12-1
  • an Fc region such as one or more anti-CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 10, or Table 12-1
  • the present disclosure provides a muitispecific antigen-binding protein or conjugate that comprises (1) one or more anti-TNFR2 antigen-binding domains, such as one or more anti-TNFR2 VHH antibodies selected from T-001, T-002, T-003, T-004, T-004.VTV, T-005hul, T-006, T- 007, T-008hul, T-008hul.A3, T-007hul with CDR2 G/A, CDR3 T/S, T-009Hul, T-009Hul.NAR, T-OlOHul, T-OlOHul.NAR, T-009Hul_QGR, T-009Hul_SGR, T-009Hul_QGR_.N/C, T-009Hul__NAR_N/C, T- 009Hul_SGR_N/C, T-007Hul.S3.QGR, T-00
  • the present disclosure provides a multispecific antigen-binding protein or conjugate that comprises (1) a means for binding an epitope within human TNFR2 bound by any one of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13; (2) a means for binding an epitope within human CD25 bound by any one of the exemplary anti-CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 19, or Table 12-1; and (3) an Fc region.
  • the present disclosure provides a multispecific antigen-binding protein or conjugate that comprises (1) a means for binding an epitope within human TNFR2 bound by an antibody selected from T-001, T-002, T-003, T-004, T-004.VTV, T-005hul, T-006, T-007, T-008hul, T-008hul.A3, T- 007hul with CDR2 G/A, CDR3 T/S, T-009Hul, T-009HU1.NAR, T-OlOHul, T-OlOHul.NAR, T-009Hul_QGR, T-009HU 1_ SGR, T-009Hul_.QGR_N/C, T-009Hul cognitiveNAR professionN/C, T-009Hul_.SGR__N/C, T-007Hul .S3.QGR, T- 007Hul.S3.EGR, T-005hul, T-006, T
  • T-016Hul.A7 T-016Hul.EQ.SGR, T-007Hul.EQ.S3.NSR, T-011, T-012, T-013, T- 014, T-015, T-016, T-017, T-018, T-019, T-020, T-021, T-016hul.Al, T-016hul.G2, T-016hul.A3, T- 016hul.A4, T-016hul.A5, T-016hul.A6, T-016hul.A7, T-016hul.A9, T-016hul.A10, T-016hul.All, T- 016Hul.A5.A7, T-016Hul.A5.Al, T-016Hul.A5.A3, T-016Hul.A5.A4, T-016Hul.S5.A4, T-016Hul.A5.A6, T-016Hul.A7.Al, T-016Hul.A7.A3, T-016Hul.A7.A4,
  • an Fc region refers to a portion of a heavy chain constant region comprising CH2 and CHS. in some embodiments, an Fc region comprises a hinge, CH2, and CHS. in various embodiments, when an Fc region comprises a hinge, the hinge can mediate dimerization between two Fc-containing polypeptides.
  • An Fc region included in a multispecific antigen-binding protein or conjugate may be an Fc region from any species, or derived from any species, including, but not limited to, human, mouse, rat, monkey (e.g., cyno), camel, llama, shark, goat, rabbit, and/or bovine.
  • an Fc region included in a multispecific antigen-binding protein or conjugate of the present disclosure is a human immunoglobulin Fc region, or is derived from a human immunoglobulin Fc region, in some embodiments, the immunoglobulin Fc region is of IgG, IgE, IgM, IgD, IgA or lg ⁇ isotype. In some embodiments, the immunoglobulin Fc region is an IgG isotype, such as IgGl, lgG2, lgG3, or lgG4 subclass. The immunoglobulin Fc region may comprise a variant or fragment of a native IgG Fc region.
  • an Fc region included in a multispecific antigen-binding protein or conjugate described herein may be a murine (e.g., a mouse or a rat) immunoglobulin Fc region, or derived from a murine immunoglobulin Fc region.
  • an Fc region included in a multispecific antigen-binding protein or conjugate described herein may be a cyno immunoglobulin Fc region, or derived from a cyno immunoglobulin Fc region.
  • a native Fc region typically possesses an effector function, including but not limited to, Fc receptor binding; Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (for example B-cell receptor); and B-cell activation, etc.
  • effector functions generally require the Fc region to be combined with a binding domain (for example, an antibody variable domain) and can be assessed using various assays.
  • a multispecific antigen-binding protein or conjugate of the present disclosure can comprise a dimer of Fc regions.
  • an Fc region mediates dimerization of the anti-TNFR2-binding domains and/or anti-CD25-binding domains at physiological conditions, such as when expressed from a cell, such that a dimer is formed that doubles the number of anti-TNFR2 binding domains and/or anti-CD25-binding domains.
  • a multispecific antigenbinding protein comprising one VHH domain and an Fc region is monovalent as a monomer, but the Fc region can mediate dimerization; as a result, the multispecific antigen-binding protein is bivalent (i.e., having two VHH domains per molecule).
  • two VHH domains (2x) are fused to an IgG Fc region and as a result of dimerization, the multispecific antigen-binding protein is tetravalent (i.e., having four VHH domains per molecule).
  • three VHH domains (3x) are fused to an IgG Fc region and as a result of dimerization, the multispecific antigen-binding protein is hexavalent (i.e., having six VHH domains per molecule), In some embodiments, four VHH domains (4x) are fused to an IgG Fc region and as a result of dimerization, the multispecific antigenbinding protein is octavalent (i.e., having eight VHH domains per molecule).
  • the CH3 domain of the Fc region can be used as homodimerization domain, such that the resulting multispecific antigen-binding protein may be formed from two identical polypeptides.
  • the CH3 dimer interface region of the Fc region can be mutated to enable heterodimerization.
  • a heterodimerization domain can be incorporated into multispecific antigen-binding protein such that the construct is a heterodimeric multispecific antigen-binding protein.
  • the first and second Fc regions may be of the same IgG isotype such as, e.g., IgGl/IgGl, lgG2/lgG2, lgG4/lgG4.
  • the first and second Fc regions may be of different IgG isotypes such as, e.g., lgGl/lgG2, lgGl/lgG4, lgG2/lgG4, etc.
  • the Fc region included in a multispecific antigen-binding protein or conjugate of the present disclosure can be mutated or modified.
  • the mutations include one or more amino acid substitutions to reduce an effector function of the Fc region.
  • mutations to Fc regions to alter, such as reduce, effector function are known, including any as described below, in general, the numbering of the residues in an immunoglobulin heavy chain or portion thereof, such as an Fc region, is according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991),
  • the human IgG Fc region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Non-limiting examples of amino add modifications that can alter ADCC and/or CDC are described in Alegre et al, 1992 J Immunol, 148: 3461-3468; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Shields et al., 2001 JBC, 276(9): 6591-6604; Lazar et al., 2006 PNAS, 103(11): 4005-4010; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Stavenhagen et al., 2008 Advan.
  • an Fc region included in a multispecific antigen-binding protein or conjugate of the present disclosure exhibits reduced effector functions (such as CDC and ADCC).
  • Various in vitro and/or in viva cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A), Pro239Gly (P329G), Asn325Glu (N325E), and/or Ala327Ser (A327S).
  • modifications within the Fc region reduce binding to Fc-receptor-gamma receptors (FcyRs) while have minimal impact on binding to the neonatal Fc receptor (FcRn).
  • the human IgGl Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent glycosylation of the multispecific antigen-binding protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D).
  • the Fc region of the multispecific antigenbinding protein is modified at amino acid Leu235 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E) or Leu235Ala (L235A).
  • the Fc region of the multispecific antigen-binding protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu234Ala (L.234A). In some embodiments, the Fc region of the multispecific antigenbinding protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E).
  • the Fc region of the multispecific antigen-binding protein is altered at both amino acids 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A/L235A) or Leu234Val and Leu235Ala (L234V/L235A). In some embodiments, the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 297, e.g., Leu234Ala, Leu235Ala, Asn297Ala (L234A/L235A/N297A).
  • the Fc region of the multispecific antigenbinding protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro239Ala (L234A/L235A/P329A).
  • the Fc region of the multispecific antigen-binding protein is modified at amino acid Asp265 (Kabat Numbering) to alter Fc receptor interactions, e.g., Asp265Ala (D265A).
  • the Fc region of the multispecific antigen-binding protein is modified at amino acid Pro329 (Kabat Numbering) to alter Fc receptor interactions, e.g., Pro329Ala (P329A) or Pro329Gly (P329G).
  • the Fc region of the multispecific antigen-binding protein is altered at both amino acids 265 and 329, e.g., Asp265Ala and Pro329Ala (D265A/P329A) or Asp265Ala and Pro329Gly (D265A/P329G).
  • the Fc region of the multispecific antigenbinding protein is altered at amino acids at 234, 235, and 265, e.g., Leu234Ala, Leu235Ala, Asp265Ala (L234A/L235A/D265A). In some embodiments, the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro329Gly
  • the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, 265 and 329, e.g., Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A/L235A/D265A/P329G).
  • the Fc region of the multispecific antigen-binding protein is altered at Gly235 to reduce Fc receptor binding. For example, wherein Gly235 is deleted from the multispecific antigen-binding protein.
  • the human IgGl Fc region is modified at amino acid Gly236 to enhance the interaction with CD32A, e.g., Gly236Ala (G236A).
  • the human IgGl Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).
  • the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Gly, Leu235Ser, Gly236Arg (L234G/L235S/G236R). in some embodiments, the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Thr, Giy236Arg (L234S/L235T/G236R).
  • the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Val, Gly236Arg (L234S/L235V/G236R). in some embodiments, the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Gln, Gly236Arg (L234T/L235Q/G236R).
  • the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Thr, Gly236Arg (L234T/L235T/G236R), In some embodiments, the Fc region of the multispecific antigenbinding protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Thr, Leu235Thr, Pro329Gly (L234A/L235A/P329G).
  • the Fc region of the multispecific antigen-binding protein is altered at amino acids at 252, 254, and 256, e.g,, Met252Tyr, Ser254Thr, Thr256Glu (M252Y/S254T/T256E).
  • the Fc region of the multispecific antigen-binding protein is lacking an amino acid at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (1235).
  • the Fc region of the multispecific antigenbinding protein is lacking an amino acid at one or more of the following positions Glu233 (E233), Leu234 (L234), or Leu235 (L235) and is modified at one or more of the Asp265 (D265), Asn297 (N297), or Pro329 (P329) to reduce Fc receptor binding.
  • an Fc region included in a TNFR2 binding polypeptide is derived from a human Fc domain, and comprises a three amino acid deietion in the lower hinge corresponding to IgGl E233, L234, and L235.
  • such Fc polypeptides do not engage FcyRs and thus are referred to as "effector silent" or "effector null.”
  • Fc deletion of these three amino acids reduces the complement protein Clq binding.
  • a polypeptide with an Fc region with Fc deletion of these three amino acids retains binding to FcRn and therefore has extended half-life and transcytosis associated with FcRn mediated recycling.
  • the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino add sequence: IgGl L234A, L235A (also known as "LALA" variant) (mutations bolded in the sequence below)

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Abstract

The present application provides multispecific antigen-binding proteins (e.g., antibodies) that specifically bind tumor necrosis factor receptor 2 (TNFF52) and cluster of differentiation 25 (CD25). The application also provides conjugates comprising the multispecific antigen-binding proteins, polynucleotides and recombinant vectors encoding the multispecific antigen-binding proteins, as well as host cells and methods for preparing the multispecific antigen-binding proteins. The application further provides pharmaceutical compositions comprising the multispecific antigen-binding proteins and methods for treating a disease or disorder using the multispecific antigen-binding proteins.

Description

MULTOPECIFiC MOLECULES BINDING TNFR2 AND CD25 AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
(OOOl)This application claims priority to U.S. Provisional Patent Application No. 63/665,064, filed on June 27, 2024, the disclosure of which is herein incorporated by reference in its entirety.
SEQUENCE LISTING
[0002]The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 26, 2025, is named 260525_.000075__SL.xml and is 10,679,494 bytes in size.
FIELD OF THE INVENTION
[0003]The present invention relates to multispecific antigen-binding proteins, and methods of use thereof. The multispecific antigen-binding proteins, including bispecific molecules, may comprise one or more antigen-binding domains that specifically bind tumor necrosis factor receptor 2 (TNFR2), and one or more antigen-binding domains that specifically bind cluster of differentiation 25 (CD25).
BACKGROUND OF THE INVENTION
[0004]Regulatory T cells (Tregs) are a subset of T ceils that play a crucial role in peripheral self-tolerance and the prevention of autoimmunity. Due to their potent immunosuppressive function, Tregs can be targeted for the treatment of autoimmunity. Current strategies seeking to increase or modulate Tregs in autoimmune patients are based on the ex vivo expansion of Tregs prior to autologous transfer.
However, a major limitation of the current strategies is their inability to stabilize Tregs phenotype to ensure long-lasting immunoregulation.
[0005]While Tregs can support immune homeostasis under normal, healthy conditions, and their activation can be beneficial in the context of autoimmune disease, during proliferative diseases (e.g., cancer), Tregs can accumulate within the tumor microenvironment where they can hamper antitumor responses mounted by infiltrating immune cells, effectively protecting the cancer cells from immune attack. Tregs are capable of suppressing most types of immune cells including CD4+ and CDS-i- T cells, B cells, and antigen-presenting cells (APCs) (e.g., dendritic cells macrophages and monocytes), natural killer (NK) cells, and NKT cells. The number of Tregs is higher in tumors and peripheral blood mononuclear cells (PBMCs) of many cancer patients, and high Treg levels can be associated with poor prognosis, e.g., in solid tumors including breast, cervical, renal, melanomas, ovarian, hepatocellular, gastric, and pancreatic cancers.
[0006]There is a need in the art to develop therapies that can induce a stable immunosuppressive Tregs phenotype for the treatment of diseases such as autoimmune diseases.
SUMMARY OF THE INVENTION
[0007jAs mentioned in the background section above, there is an unmet need in the art to develop therapies that can induce a stable immunosuppressive Tregs phenotype. This application provides compositions and methods to address this and other related needs.
[0008]ln an aspect, provided herein is a multispecific antigen-binding protein comprising one or more binding domains that specifically bind tumor necrosis factor receptor 2 (TNFR2), and one or more binding domains that specifically bind cluster of differentiation 25 (CD25). In some embodiments, the multispecific antigen-binding protein further comprises one or more binding domains that binds to another antigen, e.g., human serum albumin.
[0009]ln some embodiments, the multispecific antigen-binding protein is multivalent, e.g., bivalent, trivalent, tetravalent, pentavalent, hexavalent, septivalent, or octavalent for both antigens.
[OOlOjln some embodiments, the multispecific antigen-binding protein comprises at least one of the anti-TNFR2 antigen-binding domains described herein. In some embodiments, the multispecific antigenbinding protein comprises two of the anti-TNFR2 antigen-binding domains described herein. In some embodiments, the multispecific antigen-binding protein comprises three of the anti-TNFR2 antigenbinding domains described herein. In some embodiments, the multispecific antigen-binding protein comprises four of the anti-TNFR2 antigen-binding domains described herein. In some embodiments, the multispecific antigen-binding protein comprises five of the anti-TNFR2 antigen-binding domains described herein. In some embodiments, the multispecific antigen-binding protein comprises six of the anti-TNFR2 antigen-binding domains described herein.
[OOlljln some embodiments, the multispecific antigen-binding protein comprises at least one of the anti-CD25 antigen-binding domains described herein. In some embodiments, the multispecific antigenbinding protein comprises two of the anti-CD25 antigen-binding domains described herein, in some embodiments, the multispecific antigen-binding protein comprises three of the anti-CD25 antigenbinding domains described herein. In some embodiments, the multispecific antigen-binding protein comprises four of the anti-CD25 antigen-binding domains described herein. In some embodiments, the multispecific antigen-binding protein comprises five of the anti-CD25 antigen-binding domains described herein, in some embodiments, the multispecific antigen-binding protein comprises six of the anti-CD25 antigen-binding domains described herein.
[0012]ln some embodiments of the multispecific antigen-binding protein described herein, the one or more anti-TNFR2 antigen-binding domains and/or the one or more anti-CD25 antigen-binding domains are linked by a flexible linker. In some embodiments of the multispecific antigen-binding protein described herein, the one or more anti-TNFR2 antigen-binding domains and/or the one or more anti- CD25 antigen-binding domains are linked by a rigid linker,
[0013]ln some embodiments of the multispecific antigen-binding protein described herein, the one or more anti-TNFR2 antigen-binding domains may bind to the same epitope on TNFR2. In other embodiments, the one or more anti-TNFR2 antigen-binding domains may bind to different epitopes on TNFR2.
[0014] in some embodiments of the multispecific antigen-binding protein described herein, the one or more anti-CD25 antigen-binding domains may bind to the same epitope on CD25. In other embodiments, the one or more anti-CD25 antigen-binding domains may bind to different epitopes on CD25,
[0015]!n one aspect, the present disclosure provides one or more antigen-binding domains that specifically bind TNFR2, comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from a) (Y/F)¥Q(S/A)LS(T/S)(P/A)N(Y/F)GQ(V/T)F (SEQ ID NO: 60); b) AADSDL(S/R)TV(V/T)(V/T)GPHDY (SEQ ID NO: 61); c) AKDAG(S/G)WG(T/R)GPFG(Y/F)(E/D)¥D¥ (SEQ ID NO: 62); d) A(A/G)(T/A/S)(P/L)(S/T)GKAY(T/S)Y (SEQ ID NO: 10387); e) ATPGPY(T/S/M)YCAPYGSSWSRG¥DY (SEQ ID NO: 64); f) ARV(R/G)G(T/S/A)PY(E/D)Y(N/G)Y (SEQ ID NO: 65); g) (T/A/V)A(S/A)PTGRAF(T/N/A)¥ (SEQ ID NO: 66); h) AGSAFDF (SEQ ID NO: 42); i) S(V/M)(V/L)GRDM(M/V)TY (SEQ ID NO: 67); j) AVGDFEGELVLKGDY (SEQ ID NO: 6635); and k) AAD(L/V)G(F/V/¥)L¥(A/T/V)DYV(P/R)LH(M/T)HHFGS (SEQ ID NO: 7086); wherein one or more non-alanine residues in the CDR3 sequence are optionally replaced with an alanine, and/or one or more alanine residues in the CDR3 sequence are optionally replaced with a glycine.
[0016]ln some embodiments, the CDR3 of the one or more anti-TNFR2 antigen-binding domains comprises an amino acid sequence a). (T/A/V)(A/G)(S/A)(A/P)(A/T/S)(A/G)(A/R) A(A/F)(T/N/A)(A/Y); or b). (A/G)(A/G)(T/A/S)(A/P/L)(A/S/T)(A/G)(A/K)(A/G)(A/Y)(A/T/S)(A/Y).
[0017]ln some embodiments, the CDR3 of the one or more anti~TNFR2 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 3, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 63, 6635, 6639, 6643, 7093, 7099, 7289-7292, 7333, 10374, 10410-10433, 10435-10455, and 10811.
[0018]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains further comprise a CDR1 comprising an amino acid sequence selected from a) GSI(V/F)(R/S)(T/A)(N/D)(S/G/A); b) GFT{F/L)DD(I/Y)A (SEQ ID NO: 69); c) GFTFS(S/R/G)YA (SEQ ID NO: 70); d) GRTFSDYG (SEQ ID NO: 16); e) G(L/F)TLDYYA (SEQ ID NO: 71); f) GF(T/N)FSM¥S (SEQ ID NO: 72); g) GRTF(G/R/S)(N/S)(Y/L)(T/F) (SEQ ID NO: 73); h) GASLSRNA (SEQ ID NO: 40); i) GS(I/T)FRFPP (SEQ ID NO: 74); and j) G(F/V)(S/T)LD(D/Y)(H/Y)T (SEQ ID NO: 7088).
[0019]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains comprise a CORI comprising an amino acid sequence selected from SEQ ID NOs: 1, 5, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 6633, 6637, 6641, 7089, and 7281-7284.
[0020]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains comprise a CDR2 comprising an amino add sequence selected from a) IRSDGF(T/I) (SEQ ID NO: 75); b) l(Y/F)SY(S/G)(S/P)NT (SEQ ID NO: 76); c) l(Y/S)(S/D)DGS(E/D)T (SEQ ID NO: 77); d) INWS(N/Q/E/S)(G/A)RT (SEQ ID NO: 10409); e) l(S/N)(V/T)(S/G)DGST (SEQ ID NO: 78); f) IDT(R/G)GST (SEQ ID NO: 79); g) IR(W/R/Y)(T/P)G(G/L)(S/I)T (SEQ ID NO: 80); h) IYDDGET (SEQ ID NO: 41); i) LTSGGST (SEQ ID NO: 45); and j) l(N/S)SNDG(S/T)(T/V) (SEQ ID NO: 7087).
[0021]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains comprise a CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 2, 9, 13, 17, 21, 25, 23, 33, 37, 41, 45, 6634, 6638, 6642, 7096, 7268, and 7285-7288, and 10388-10393.
[0022]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains comprise i)a CDR1 comprising an amino acid sequence of GSI(V/F)(R/S)(T/A)(N/D)(S/G/A), a CDR2 comprising an amino acid sequence of SEQ iD NO: 75, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 60; ii)a CDR1 comprising an amino acid sequence of SEQ iD NO: 69, a CDR2 comprising an amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 61; iii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 70, a CDR2 comprising an amino acid sequence of SEQ ID NO: 77, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 62; iv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10387; v)a CDR1 comprising an amino acid sequence of SEQ ID NQ: 71, a CDR2 comprising an amino acid sequence of SEQ ID NO: 78, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 64; vi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 72, a CDR2 comprising an amino acid sequence of SEQ ID NO: 79, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 65; vii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 73, a CDR2 comprising an amino acid sequence of SEQ iD NO: 80, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 66; viii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ iD NO: 41, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 42; ix)a CDR1 comprising an amino acid sequence of SEQ ID NQ: 74, a CDR2 comprising an amino acid sequence of SEQ iD NO: 45, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 67; x)a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6635; xi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7088, a CDR2 comprising an amino acid sequence of SEQ !D NO: 7087, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7086; xli)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10409, and a CDR3 comprising an amino acid sequence of (A/G)(A/G)(T/A/S)(A/P/L)(A/S/T)(A/G)(A/K)(A/G){A/Y)(WS)(A/Y); or xiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 73, a CDR2 comprising an amino acid sequence of SEQ iD NO: 80, and a CDR3 comprising an amino acid sequence of
(T/A/V)(A/G)(S/A)(A/P)(A/T/S)(A/G)(A/R)A(A/F)(T/N/A)(A/Y).
[0023)ln some embodiments, the one or more anti-TNFR2 antigen-binding domains comprise a)a CDR1 comprising an amino acid sequence of SEQ ID NO: 69, a CDR2 comprising an amino acid sequence of SEQ iD NO: 76, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 61; b)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10387; c)a CDR1 comprising an amino acid sequence of SEQ ID NO: 73, a CDR2 comprising an amino acid sequence of SEQ iD NO: 80, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 66; d)a CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 63; or e)a CDR1 comprising an amino acid sequence of SEQ ID NG: 16, a CDR2 comprising an amino acid sequence of SEQ !D NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7333; [0024]!n some embodiments, the one or more anti-TNFR2 antigen-binding domains comprise i)a CDR1 comprising an amino acid sequence of GSI(V/F)(R/S)(A/T)(N/D)(G/A), a CDR2 comprising an amino acid sequence of iRSDGFT (SEQ ID NO: 2), and a CDR3 comprising an amino acid sequence of ¥¥Q(S/A)LSSPNYGQ(V/F)F (SEQ ID NO: 7270); ii)a CDR1 comprising an amino acid sequence of GFT(F/L)DD(I/Y)A (SEQ !D NO: 69), a CDR2 comprising an amino acid sequence of l(Y/F)SY(S/G)(S/P) NT (SEQ iD NO: 76), and a CDR3 comprising an amino acid sequence of AADSDLSTVV(V/T)GPHDY (SEQ ID NO: 7271); iii)a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 12), a CDR2 comprising an amino acid sequence of ISDDGSDT (SEQ ID NO: 13), and a CDR3 comprising an amino acid sequence of AKDAGSWGTGPFGYEYDY (SEQ ID NO: 14); iv)a CDR1 comprising an amino acid sequence of GRTFSDYG (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of INWSN(G/A)RT (SEQ ID NO: 7268), and a CDR3 comprising an amino acid sequence of A(A/G)(T/A/S)(P/L)(S/T)GKA¥(T/S)¥ (SEQ ID NO: 10387); v)a CDR1 comprising an amino acid sequence of GLTLDYYA (SEQ ID NO: 20), a CDR2 comprising an amino acid sequence of ISTSDGST (SEQ ID NO: 21), and a CDR3 comprising an amino acid sequence of
ATPGPYTYCAPYGSSWSRGYDY (SEQ ID NO: 22); vi)a CDR1 comprising an amino acid sequence of GF(T/N)FSMYS (SEQ ID NO: 72), a CDR2 comprising an amino acid sequence of IDT(R/G)GST (SEQ ID NO: 79), and a CDR3 comprising an amino acid sequence of ARV(G/R)G(T/A)PYEY(N/G)Y (SEQ ID NO: 7273); vii)a CDR1 comprising an amino acid sequence of GRTF(G/S)S(Y/L)(T/F) (SEQ ID NO: 7274), a CDR2 comprising an amino acid sequence of IR(W/R/Y)(T/P)G(G/L)(S/I)T (SEQ ID NO: 80), and a CDR3 comprising an amino acid sequence of (A/V)A(A/S)PTGRAF(T/N)Y (SEQ ID NO: 7276); viii)a CDR1 comprising an amino acid sequence of GASLSRNA (SEQ ID NO: 40), a CDR2 comprising an amino acid sequence of IYDDGET (SEQ ID NO: 41), and a CDR3 comprising an amino acid sequence of AGSAFDF (SEQ ID NO: 42); ix)a CDR1 comprising an amino acid sequence of GS(T/I)FRFPP (SEQ ID NO: 7277), a CDR2 comprising an amino acid sequence of LTSGGST (SEQ iD NO: 45), and a CDR3 comprising an amino acid sequence of SVLGRDM(M/V)TY (SEQ ID NO: 7275); x)a CDR1 comprising an amino acid sequence of GFTLDDYA (SEQ ID NO: 6633), a CDR2 comprising an amino acid sequence of IFSYSSNT (SEQ ID NO: 6634), and a CDR3 comprising an amino acid sequence of AVGDFEGELVLKGDY (SEQ ID NO: 6635);or xi)a CDR1 comprising an amino acid sequence of GFTLDYYT (SEQ ID NO: 6637), a CDR2 comprising an amino acid sequence of ISSNDGSV (SEQ ID NO: 6638), and a CDR3 comprising an amino acid sequence of AADLGYLYVDYVRLHTHHFGS (SEQ ID NO: 6639).
[0025]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains comprise a)a CDR1 comprising an amino acid sequence of GFT(F/L)DD(I/Y)A (SEQ ID NO: 69), a CDR2 comprising an amino acid sequence of l(¥/F)S¥(S/G)(S/P)NT (SEQ ID NO: 76), and a CDR3 comprising an amino acid sequence of AADSDLSTW(V/T)GPHDY (SEQ ID NO: 7271); b)a CDR1 comprising an amino acid sequence of GRTFSDYG (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of INWSN(G/A)RT (SEQ ID NO: 7268), and a CDR3 comprising an amino acid sequence of A(A/G)(T/A/S)(P/L)(S/T)GKAY(T/S)Y (SEQ ID NO: 10387); or c)a CDR1 comprising an amino acid sequence of GRTF(G/S)S(¥/L)(T/F) (SEQ ID NO: 7274), a CDR2 comprising an amino acid sequence of IR(W/R/Y)(T/P)G(G/L)(S/I)T (SEQ ID NO: 80), and a CDR3 comprising an amino acid sequence of (AAf)A(A/S)PTGRAF(T/N)¥ (SEQ ID NO: 7276).
[0026]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains comprise i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 3; ii) a CDR1 comprising an amino acid sequence of SEQ iD NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6; iii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10; iv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 12, a CDR2 comprising an amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 14; v)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; vi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 20, a CDR2 comprising an amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 22; vii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 24, a CDR2 comprising an amino acid sequence of SEQ ID NO: 25, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 26; viii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 28, a CDR2 comprising an amino acid sequence of SEQ ID NO: 29, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 30; ix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 33, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34; x)a CDR1 comprising an amino acid sequence of SEQ ID NO: 36, a CDR2 comprising an amino acid sequence of SEQ ID NO: 37, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 38; xi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 42; xiija CDR1 comprising an amino acid sequence of SEQ ID NO: 44, a CDR2 comprising an amino add sequence of SEQ ID NO: 45, and a CDR3 comprising an amino add sequence of SEQ ID NO: 46 xiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino add sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino add sequence of SEQ ID NO: 6635; xiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 6637, a CDR2 comprising an amino add sequence of SEQ ID NO: 6638, and a CDR3 comprising an amino add sequence of SEQ ID NO: 6639; xv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino add sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino add sequence of SEQ ID NO: 6643; xvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7089, a CDR2 comprising an amino add sequence of SEQ ID NO: 45, and a CDR3 comprising an amino add sequence of SEQ ID NO: 46; xvii)s CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093; xviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; xix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7099; xx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; xxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; xxi i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7291; xxiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7291; xxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10; xxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093; xxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; xxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: IS, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; xxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10391, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10392, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10393, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; xxxvQa CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; xxxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; xxxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10410; xxxixja CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10411; xl)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10412; xli)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10413; xlii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10414; xliiija CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10415; xlivja CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10416; xlv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10417; xlvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10418; xlvii)a CDRI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10419; xlviiija CDRI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10420; xlix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10421; l)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10422; li)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10423; lii)a CDRI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10424; liii)a CDRI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10425; liv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10426; lv)a CDRI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10427; lvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10428; lvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34;
Iviiija CDRi comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10429; lix)a CDRI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10430; lx)a CDRI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10431; lxi)a CDRI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NQ: 10432; lxii)a CDRI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10433; Ixiii) CDR1 comprising an amino add sequence of SEQ ID NO: 32, a CDR2 comprising an amino add sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10811; lxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10435; lxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10436; lxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10437; lxvii)a CDR1 comprising an amino add sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10438;
Ixvi ii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10439;
Ixvixja CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10440; lxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10441; lxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10442;
Ixxiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10443; lxxiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10444;
Ixxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10445; lxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10446;
Ixxvija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10447;
Ixxviija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino add sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10448; Ixxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10449; ixxvix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10450; lxxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10451;
Ixxxija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10452; lxxxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ !D NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10453;
Ixxxiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10454; or
Ixxxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10455.
[0027]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains comprise a)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; b)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; c)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7099; d)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; e)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino add sequence of SEQ ID NO: 7289; f)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285, and a CDR3 comprising an amino add sequence of SEQ ID NO: 7291; g)a CDR1 comprising an amino acid sequence of SEQ. ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7291; h)a CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10; i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093; j)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6643; k)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10414; l)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10421; m)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10422; n)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10423; o)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10429; p)a CDR1 comprising an amino acid sequence of SEQ ID NQ: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10811; q)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; r)a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10; or s)a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093. [0028]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains comprise a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1128-1686, 6745-6806, and 7102-7125; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1687- 2245, 6807-6868, and 7126-7149; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 2246-2804, 6869-6930, and 7150-7173. [0029]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains is a singie-domain antibody. In some embodiments, the single-domain antibody is a VHH, a VNAR, or a VH domain.
[0030]ln some embodiments, the VHH of the one or more anti-TNFR2 antigen-binding domains is a cameiid VHH. In some embodiments, the VHH of the one or more anti-TNFR2 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 93-640, 2805-3363, 6636, 6640, 6644, 7090, 6651-6697, 6931-6992, 7174-7197, 10380, 10381, 10383, 10385, and 10386, or a sequence having at least 75% identity thereto, in some embodiments, the VHH of the one or more anti-TNFR2 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 10380, 10381, 10383, 10385, and 10386, or a sequence having at least 75% identity thereto.
[0031]ln some embodiments, the VHH of the one or more anti-TNFR2 antigen-binding domains is a humanized VHH. In some embodiments, the VHH of the one or more anti-TNFR2 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 6648, 6649, 6650, 7092, 7095, 7098, 7101,7293-7296, 641-1127, 6698-6744, 10375, 10382, 10384, 10394-10408, 10457-10502, 10720-10727, and 10812, or a sequence having at least 75% identity thereto. In some embodiments, the VHH of the one or more anti-TNFR2 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 6648, 6649, 6650, 7092, 7095, 7098, 7101, 7293-7296, 10375, 10382, 10384, 10394-10408, 10457-10502, 10720-10727, and 10812 or a sequence having at least 75% identity thereto.
[0032]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains of the present disclosure comprise TNF or a variant thereof.
[0033]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains bind to human TNFR2. in some embodiments, the one or more anti-TNFR2 antigen-binding domains bind to human TNFR2 with a KD of less than about 3xl0’6 M. in some embodiments, the one or more anti-TNFR2 antigen-binding domains bind to human TNFR2 with a KD of about IxiO"10 to 5xWs M.
[0034] In some embodiments, the one or more anti-TNFR2 antigen-binding domains bind to cyno TNFR2. In some embodiments, the one or more anti-TNFR2 antigen-binding domains bind to cyno TNFR2 with a KD of less than about 3xl0"6 M. In some embodiments, the one or more anti-TNFR2 antigen-binding domains bind to cyno TNFR2 with a KD of about IxlO’9 to 2xW7 M, [0035)1 n some embodiments, the one or more anti-TNFR2 antigen-binding domains bind to the same epitope(s) on TNFR2 as antibody clone MR2-1. In some embodiments, the one or more anti-TNFR2 antigen-binding domains do not bind to the same epitope(s) on TNFR2 as antibody clone MR2-1. [0036]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains have an agonist effect upon binding to TNFR2. In some embodiments, the one or more anti-TNFR2 antigen-binding domains have an agonist effect upon binding to TNFR2 with an EC50 of about 1-100 nM (e.g., l-10nM, 10-100 nM).
[0037jln some embodiments, the one or more anti-TNFR2 antigen-binding domains increase expression of one or more proteins selected from a protein in the NF-kB pathway, F0XP3, HELIOS, EZH2, HLA-DR, ICAM-1, OX-40, ICOS, and CCR8.
[0038]ln one aspect, the present disclosure provides one or more antigen-binding domains that specifically binds CD25, comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from a) NAL(G/L/P/Q/W)¥ (SEQ ID NO: 4131); b) NALR(D/H/N/F) (SEQ ID NO: 4134); c) (K/S/T)TLRY (SEQ ID NO: 4136); d). (A/V/S)(K/T)G(R/A/K)(G/H/N/R)SG(S/G)YYP(W/F/L)(D/E)(D/E)(Y/V) (SEQ ID NO: 10219); e). AA(S/T)(D/N/Y/K)(F/V)(L/P)(I/L)A(T/I/A)(T/S/A)IS(A/G)(Y/H)DY (SEQ ID NO: 10308); f) AAYVYPDYYCS(D/E)YVLL(K/R)YD¥ (SEQ ID NO: 7363); g) NIYR(P/S)QVP(P/S/T)TR¥S (SEQ ID NO: 7365); and h) AAKRLGP(M/I/A/L)VH(Q/R)YSLEVLTPLFLDEYDY (SEQ ID NO: 9423), wherein one or more non-alanine residues in the CDR3 sequence are optionally replaced with an alanine, and/or one or more alanine residues in the CDR3 sequence are optionally replaced with a glycine.
[0039]ln some embodiments, the CDR3 comprises an amino acid sequence a).(A/V/S)(A/K/T)(A/G)(A/R/K)(A/G/H/N/R)(A/S)(A/G)(A/S/G)(A/Y)(A/Y)(A/P)(A/W/F/L)(A/D/E)(A/D/E)(A/ Y/V); or b). (A/G)(A/G)(A/K)(A/R)(A/L)(A/G)(A/P)(M/I/A/L)(A/V)(A/H)(A/R/Q)(A/Y)(A/S)(A/L)(A/E){A/V) (A/L)(A/T)(A/P)(A/L)(A/F)(A/L)(A/D)(A/E)(A/¥)(A/D)(A/Y).
[0040]ln some embodiments, the CDR3 comprises an amino acid sequence selected from a). NAL(G/L/P/Q/W)Y (SEQ ID NO: 4131); b). NALR(D/H/N/F) (SEQ ID NO: 4134); c). (K/S/T)TLRY (SEQ ID NO: 4136); d). AKGR(H/N)SGSYYPWD(D/E)Y (SEQ ID NO: 4139); e). (A/V)KGR(G/H/N)SGSYYP(W/F)D(D/E)Y (SEQ ID NO: 9440); f). AA(S/T)(D/N/Y)FL(i/L)ATTIS(A/G)YDY (SEQ ID NO: 4141); g). AAYVYPDYYCS(D/E)YVLL(K/R)YDY (SEQ ID NO: 7363); h). NIYR(P/S)QVP(P/S/T)TRYS (SEQ ID NO: 7365); and i). AAKRLGPMVH(Q/R)YSLEVLTPLFLDEYDY (SEQ ID NO: 7367).
(0041]ln some embodiments, the CDR3 of the one or more anti~CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4103, 4107, 4111, 4115, 4119, 4139,
4141, 5337, 5339, 5371, 5375, 5398, 5401, 5431, 5515, 5519, 5521, 5528, 5532, 5542, 5544, 5545, 5547, 5548, 7344, 7347, 7349, 7350, 7367, 9411-9416, 9436, 9440, 9887, 9966, 9975, 9978, 9979, 9980, and 10504-10544.
[0042]ln some embodiments, the CDR3 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4103, 4107, 4111, 4115, 4119, 5337, 5339, 5371, 5375, 5398, 5401, 5431, 5515, 5519, 5521, 5528, 5532, 5542, 5544, 5545, 5547, 5548, 7344, 7347, 7349, 7350, 9411-9416, 9436, 9887, 9966, 9975, 9978, 9979, 9980, and 10504-10544.
[0043]ln some embodiments, the CDR3 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4103, 4107, 4111, 4115, 4119, 7344, 7347, 7349, 7350, 9411-9416, 9436, and 10504-10544.
[0044]ln some embodiments, the one or more anti-CD25 antigen-binding domains described herein may further comprise a CDR1 comprising an amino acid sequence selected from a) GR(K/R/S)FSTLI (SEQ ID NG: 4137); b) GFTFS(N/S)YA (SEQ ID NO: 4140); c) GRTF(A/S)(S/W/D)(F/N/Y)G (SEQ ID NO: 10309); d) GFTLDYYA (SEQ ID NO: 7342); and e) G(I/M)P(F/-)(A/-)L(P/V/Y)A (SEQ ID NO: 7366).
[0045]ln some embodiments, the CDR1 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4101, 4105, 4109, 4113, 4117, 4132,
4142, 4905, 4909, 4918, 7342, and 7345.
[0046]ln some embodiments, the CDR1 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4101, 4105, 4109, 4113, 4117, 4132, 7342, and 7345. [0047] In some embodiments, the one or more anti-CD25 antigen-binding domains described herein may further comprise a CDR2 comprising an amino acid sequence selected from a) (l/V)(D/E)R(D/G)(D/G)T(A/P/T); b) l¥SD(G/S)SGT (SEQ ID NO: 9441); c) IS(Q/R/G)(S/G)GGRT (SEQ ID NO: 10310); d) IS(R/S)(D/S)G(D/G)ST (SEQ ID NO: 7364); e) ISSGGNT {SEQ ID NO: 7346); and f) ISSTDGRT (SEQ ID NO: 7348).
[0048]ln some embodiments, the CDR2 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence seiected from a) (l/V)(D/E)R(D/G)GT(A/P/T); b) l(D/E)RDGT(T/P) (SEQ ID NO: 4135); c) l(D/E)R(D/G)(D/G)T(P/T); d) IYSDGSGT (SEQ ID NO: 4114); e) ISQSGGRT (SEQ ID NO: 4118) f). !S(R/S)(D/S)G(D/G)ST (SEQ ID NO: 7364); g). ISSGGNT (SEQ ID NO: 7346); and h). ISSTDGRT (SEQ ID NO: 7348).
[0049]ln some embodiments, the CDR2 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence seiected from SEQ ID NOs: 4102, 4106, 4110, 4114, 4118, (l/V)(D/E)R(D/G)GT(A/P/T), 4135, l(D/E)R(D/G)(D/G)T(P/T), 5042, 5046, 5059, 5067, 5092, 5214, 5215, 5216, 1117, 7343, 7346, 7348, and 9435.
[0050]ln some embodiments, the CDR2 of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4102, 4106, 4110, 4114, 4118, 7343, 7346, 7348, and 9435.
[0051] In some embodiments, the one or more anti-CD25 antigen-binding domains comprise i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G](D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; in) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V){D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ
ID NO: 4136; iv) a CORI comprising an amino acid sequence of SEQ iD NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)( D/E) R(D/G)GT(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ iD NO: 4131; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4135, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 4134; vi) a CDR1 comprising an amino acid sequence of SEQ iD NO: 4137, a CDR2 comprising an amino acid sequence of l(D/E)R(D/G)(D/G)T(P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4136; vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4132, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; ix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4132, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)GT(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; x) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4135, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; xi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9441, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10219; xii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9441, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9440; xiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4139; xiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 10309, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10310, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10308; xv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4142, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 4141; xvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7364, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7363; xvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7366, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7346, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7365; xviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9423; xix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7367. xx) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9441, and a CDR3 comprising an amino acid sequence of {A/V/S)(A/K/T)(A/G)(A/R/K)(A/G/H/N/RHA/S){A/G)(A/S/G)(A/Y)(A/Y)(A/P)(A/W/F/L){A/D/E)(A/D/E)(A/Y/ V); or xxi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of (A/G)(A/G)(A/K)(A/R)(A/L)(A/G)(A/P)(M/I/A/L)(A/V)(A/H)(A/R/Q)(A/¥)(A/S)(A/L)(A/E)(A/V)(A/L)(A/T){A/P )(A/L)(A/F)(A/L)(A/D)(A/E)(A/Y)(A/D)(A/Y).
[0052]ln some embodiments, the one or more CD25 antigen-binding domains comprise i) a CDR1 comprising an amino add sequence of SEQ ID NO: 4101, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4102, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4103; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4106, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4107; ill) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4109, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4110, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4111; ivj a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4115; v) a CDR1 comprising an amino add sequence of SEQ ID NO: 4117, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 4119; vi) a CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7343, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 7344; vii) a CDR1 comprising an amino acid sequence of SEQ iD NO: 7345, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7346, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 7347; viii) a CDR1 comprising an amino add sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 7349; ix) a CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7350; x) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9411; xi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9412; xii) a CDR1 comprising an amino add sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9413; xiii) a CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9414; xiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9415; xv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ, ID NO: 9416; xvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9975; xvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5431; xviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NO:
9887; xix) a CDR1 comprising an amino acid sequence of SEQ iD NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino add sequence of SEQ ID NO: 9966; xx) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9436; xxi) a CDR1 comprising an amino add sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9978; xxii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ ID NO: 9979; xxiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9980; xxiv) a CDR1 comprising an amino add sequence of SEQ ID NO: 4105, a CDR2 comprising an amino add sequence of SEQ ID NO: 4110, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5339; xxv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5046, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5339; xxvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino add sequence of SEQ ID NO: 5059, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5337; xxvi!) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5046, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5337; xxviii) a CDR1 comprising an amino add sequence of SEQ ID NO: 4101, a CDR2 comprising an amino add sequence of SEQ ID NO: 5067, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5371; xxix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ iD NO: 504S, and a CDR3 comprising an amino acid sequence of SEQ iD NO:
5375; xxx) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4109, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4110, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4111; xxxi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5092, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4111; xxxii) a CDR1 comprising an amino acid sequence of SEQ ID NQ: 4101, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5092, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5398; xxxiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5042, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4111; xxxiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5059, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4111; xxxv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5042, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5401; xxxvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5515; xxxvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4909, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5214, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5519; xxxviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5216, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5521; xxxix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4909, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5217, and a CDR3 comprising an amino acid sequence of SEQ ID NO:
5519; xl) a CDR1 comprising an amino acid sequence of SEQ, ID NO: 4918, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5215, and a CDR3 comprising an amino acid sequence of SEQ, ID NO: 5528; xli) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5532; xlii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5542; xliii) a CDR1 comprising an amino acid sequence of SEQ ID NG: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5544; xliv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5545; xlv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5547; xlvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5548. xlviija CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ ID NG: 10504; xlviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10505; xl ix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10506; l)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10507; li)a CDR1 comprising an amino add sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10508; lii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10509; liii)a CDR1 comprising an amino add sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10510; livja CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10511; lv)a CDR1 comprising an amino add sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10512; lvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10513; lvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 10514;
Iviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10515; lix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10516; lx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10517; lxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10518; lxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10519;
Ixi ii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10520;
Ixivja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10521; lxv)a CDR1 comprising an amino add sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10522;
Ixvija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10523;
Ixvi i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10524; Ixviiija CDR1 comprising an amino acid sequence of SEQ !D NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10525; lxix)a CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 9414; lxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 10526; lxxi)a CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10527; lxxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10528; lxxiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10529;
Ixxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10530;
Ixxvja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10531; lxxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10532;
Ixxviija CDR1 comprising an amino acid sequence of SEQ ID NO; 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10533;
Ixxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10534; lxxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ !D NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10535; ixxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10536; lxxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10537;
Ixxxiija CDR1 comprising an amino add sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10538;
Ixxxiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10539; Ixxxivja CDRI comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10540;
Ixxxvja CDRI comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10541;
Ixxxvija CDRI comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10542;
Ixxxviija CDRI comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10543; or lxxxviii)a CDRi comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10544, [0053]ln some embodiments, the one or more anti-CD25 antigen-binding domains comprise a CDRI comprising an amino acid sequence selected from any one of SEQ ID NOs: 4726-5030, 7931-8226, and 9660-9770; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5031- 5335, 8227-8522, and 9771-9880; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID Nos: 5336-5640, 8523-8818, and 9881-9991.
[0054]ln some embodiments, the one or more anti-CD25 antigen-binding domains include a singledomain antibody. In some embodiments, the single-domain antibody is a VHH, a VNAR, or a VH domain. In some embodiments, the VHH of the one or more anti-CD25 antigen-binding domains is a camelid VHH.
[0055]ln some embodiments, the VHH of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, 4143- 4442, 5641-5945, 7351-7354, 7368-7659, 8819-9114, 9437, 9442-9551, 9992-10102, and 10246-10276, or a sequence having at least 75% identity thereto.
[0056]ln some embodiments, the VHH of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, 7351- 7354, 9437, and 10246-10276, or a sequence having at least 75% identity thereto.
[0057]ln some embodiments, the humanized VHH of the one or more anti-CD25 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ ID NOs: 4126-4130, 4443- 4725, 7359-7362, 7660-7930, 9417-9422, 9439, 9552-9659, 10214-10245, and 10545-10585, or a sequence having at least 75% identity thereto. [0058]ln some embodiments, the humanized VHH of the one or more anti~CD25 antigen-binding domains comprises an amino acid sequence selected from any one of SEQ, ID NOs: 4126-4130, 7359- 7362, 9417-9422, 9439, 10214-10245, and 10545-10585, or a sequence having at least 75% identity thereto.
[0059]ln some embodiments, the one or more anti-CD25 antigen-binding domains of the present disclosure comprise IL-2 or a variant thereof.
[0060)ln some embodiments, the one or more anti-CD25 antigen-binding domains bind to human CD25. in some embodiments, the one or more anti-CD25 antigen-binding domains bind to human CD25 with a KD of less than about 3.5 x!0~7 M. in some embodiments, the one or more anti-CD25 antigen-binding domains bind to human CD25 with a KD of about lxl0“M to about 5xl0~8 M.
[OOSljln some embodiments, the one or more antl-CD25 antigen-binding domains bind to cyno CD25. In some embodiments, the one or more anti-CD25 antigen-binding domains bind to cyno CD25 with a KD of less than about l*10~6 M, In some embodiments, the one or more anti-CD25 antigen-binding domains bind to cyno CD25 with a KD of about lxl0“9 to about 2xl0“7 M.
[0062]ln some embodiments, the one or more anti-CD25 antigen-binding domains bind to the same epitope(s) on CD25 as i L-2, In some embodiments, the one or more anti-CD25 antigen-binding domains compete for binding to CD25 with IL-2. In some embodiments, the one or more anti-CD25 antigenbinding domains have an antagonistic effect upon binding to CD25. In some embodiments, the one or more anti-CD25 antigen-binding domains do not bind to the same epitope(s) as IL-2. In some embodiments, the one or more anti-CD25 antigen-binding domains do not compete for binding to CD25 with IL-2.
[0063]ln some embodiments, the one or more anti-TNFR2 antigen-binding domains and/or the one or more CD25 antigen-binding domains comprise one or more modifications that reduce binding of said antigen-binding protein by pre-existing antibodies found in human blood or serum.
[0064]ln some embodiments, the anti-TNFR2 single-domain antibody and/or the anti-CD25 singledomain antibody comprises one or more modifications at the amino-terminus and/or the carboxyterminus.
[0065]ln some embodiments, the anti-TNFR2 single-domain antibody and/or the anti~CD25 singledomain antibody comprises the amino acid sequence VPAG (SEQ ID NO: 7267) or VAGG (SEQ ID NO: 7266) at the carboxy-terminus starting from position ill according to Chothia. [0066]ln some embodiments, the anti-TNFR2 single-domain antibody and/or the anti-CD25 singledomain antibody comprises a substitution of amino acid residue Glu with Asp (EID) at the first position of the amino-terminus.
[0067pn some embodiments of the muitispecific antigen-binding protein described herein, the one or more anti-TNFR2 antigen-binding domains and/or the one or more CD25 antigen-binding domains are one or more single-domain antibodies. In some embodiments, the one or more single-domain antibodies are one or more VHHs.
[0068]ln some embodiments of the multispecific antigen-binding protein described herein, the multispecific antigen-binding protein further comprises an immunoglobulin Fc region, in some embodiments, the immunoglobulin Fc region is an Fc region of a human immunoglobulin. In some embodiments, the immunoglobulin Fc region is an Fc region of human IgGl, lgG2, lgG3 or lgG4, or a variant thereof.
[0069]ln some embodiments, the immunoglobulin Fc region is an Fc region of human IgGl, or a variant thereof. In some embodiments, the Fc region of human IgGl comprises one or more mutations selected from Leu234Ala (L234A), Leu234Gly (L234G), Leu234Ser (L234S), Leu234Thr (L234T), Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Leu235Ser (L235S), Leu235Thr (L235T), Leu235Val (L235V), Leu235Gln (L235Q), Gly236Arg (G236R), Met252Tyr (M252Y), Ser254Thr (S254T), Thr256Glu (T256E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A), Pro239Gly (P329G), Asn325Glu (N325E), and/or Ala327Ser (A327S) according to EU numbering. In some embodiments, the Fc region of human IgGl comprises a set of mutations selected from:
1JL234A and L235A;
2JL234A, L235A, and P329A;
3JD265A, N297A and P329A;
4)L234A, L235A, and G237A;
5)L234G, L235S, and G236R;
6) L234S, L235T, and G236R;
7)L234S, L235V, and G236R;
8)L234T, L235Q, and G236R;
9)L234T, L235T, and G236R;
10JL234A, L235A, and P329G; and
11)M252Y, S254T, and T25SE. [0070]ln some embodiments, the immunoglobulin Fc region is an Fc region of human IgGl comprising L234A, L235A, and P329A.
[0071]ln some embodiments, the immunoglobulin Fc region is an Fc region of human lgG4, or a variant thereof. In some embodiments, the Fc region of human lgG4 comprises one or more mutations selected from Ser228Pro (S228P), Leu235Glu (L235E), Leu235Ala (L235A), Phe234Ala (F234A), and/or Pro329Gly (P329G) according to EU numbering. In some embodiments, the Fc region of human lgG4 comprises a set of mutations selected from:
1) S228P and L235E;
2JS228P and L235A;
3)S228P, F234A, and L235E;
4JS228P, F234A, and L235A; and
5)P329G, S228P, and L235E.
[0072]ln some embodiments, the immunoglobulin Fc region is an Fc region of human lgG4 comprising S228P and L235E.
[0073]ln some embodiments, the multispecific antigen-binding protein comprises at least one linker, optionally selected from the group consisting of SEQ ID NOs: 3969-4027, and 6261-6362.
[0074]ln some embodiments, the at least one linker is a rigid linker.
[0075]ln some embodiments, the at least one rigid linker is selected from the group consisting of PAPAPAPAPAPAPAPAP (SEQ ID NO: 4009), GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 4012), GGGGSPAPAPAPAPGGGGS (SEQ ID NO: 6361), GGGGSPAPAPAPAPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 6362), and A(EAAAK)nA (SEQ ID NO: 4027), where n is any integer, e.g., 1, 2, 3, 4, 5, 6, or 7, 8, 9 or 10,
[0076]ln some embodiments, the at least one linker is a flexible linker.
[0077]! n some embodiments, the at least one flexible linker is selected from the group consisting of GnS (SEQ ID NO: 4013), SGn (SEQ ID NO: 4014), where n is any integer, e.g., 1, 2, 3, 4, 5, 6, or 7, 8, 9 or 10, and (GGGGS)n (SEQ ID NO: 4015), where n is any integer, e.g., 1, 2, 3, 4, 5, 6, or 7, 8, 9 or 10.
[0078]! n some embodiments, the multispecific antigen-binding protein comprises the amino acid sequence of any one of SEQ ID NOs: 6401-6520 and 10792-10810, or a sequence having at least 70% identity thereto.
[0079]ln another aspect, provided herein is a conjugate comprising the multispecific antigen-binding protein described herein, wherein the multispecific antigen-binding protein is conjugated to a second moiety. In some embodiments, the second moiety is selected from a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytokine, a cytotoxic agent, a chemotherapeutic agent, and a diagnostic agent, or a combination thereof,
[0080]ln another aspect, provided herein is a polynucleotide molecule encoding the muitispecific antigen-binding protein described herein,
[0081]ln some embodiments, the polynucleotide molecule comprises a nucleotide sequence encoding any of the amino acid sequences selected from SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 93-640, 2805-3363, 6636, 6640, 6644, 7090, 6651-6697, 6931-6992, 7174-7197, 10380, 10381, 10383, 10385, and 10386, encoding an anti-TNFR2 VHH, or a sequence having at least 70% identity thereto, in some embodiments, the polynucleotide molecule comprises encoding any of the amino acid sequences selected from SEQ ID NOs: 81-92, 6648, 6649, 6650, 7092, 7095, 7098, 7101,7293-7296, 641-1127, 6698-6744, 10375, 10382, 10384, 10394-10408, 10457-10502, 10720-10727, and 10812, encoding an anti-TNFR2 VHH, or a sequence having at least 70% identity thereto.
[0082]ln some embodiments, the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 48-59, 3364-3922, 6645-6647, 7091, 7094, 7097, 7100, 6993-7054, 7198-7221, and 10611-10661, encoding an anti-TNFR2 VHH, or a sequence having at least 70% identity thereto. In some embodiments, the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 48-59, 6645-6647, 7091, 7094, 7097, and 7100, and 10611-10661, encoding an anti-TNFR2 VHH, or a sequence having at least 70% identity thereto.
[0083]ln some embodiments, the polynucleotide molecule comprises a nucleotide sequence encoding any one of the amino acid sequences selected from SEQ, ID NOs: 4104, 4108, 4112, 4116, 4120, 4143- 4442, 5641-5945, 7351-7354, 7368-7659, 8819-9114, 9437, 9442-9551, 9992-10102, and 10246-10276, encoding an anti-CD25 VHH, or a sequence having at least 70% identity thereto.
[0084]ln some embodiments, the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 4121-4125, 5946-6250, 7355-7358, 9115-9410, 9438, and 10103-10213, encoding an anti-CD25 VHH, or a sequence having at least 70% identity thereto.
[0085]ln an embodiment provided herein, the polynucleotide molecule comprises the nucleotide sequence encoding an amino acid sequence selected from SEQ ID NOs: 4126-4130, 4443-4725, 7359- 7362, 7660-7930, 9417-9422, 9439, 10214-10245, and 9652-9659, encoding a humanized anti-CD25 VHH, or a sequence having at least 70% identity thereto.
[0086]ln an embodiment provided herein, the polynucleotide molecule comprises the nucleotide sequence encoding an amino acid sequence selected from SEQ ID NOs: 9438, 10277-10307, and 10662- 10703 encoding a humanized anti-CD25 VHH, or a sequence having at least 70% identity thereto. [0087] In an embodiment provided herein, the polynucleotide molecule comprises a nucleotide sequence selected from SEQ ID NOs: 6521-6632, or a similar sequence thereof having at least 70% identity thereto.
[0088]ln an embodiment provided herein, the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs- 6521-6632 encoding a multispecific antigen-binding protein. [0089]ln another aspect, provided herein is a recombinant vector comprising the polynucleotide molecule described herein.
[0090] in another aspect, provided herein is a host cell comprising the polynucleotide molecule described herein, or the recombinant vector described herein.
[0091]ln another aspect, provided herein is a kit comprising the multispecific antigen-binding protein, the conjugate, the polynucleotide molecule, the recombinant vector, or the host cell described herein, and optionally, instructions and/or packaging for the same.
[0092]ln another aspect, provided herein is a pharmaceutical composition comprising the multispecific antigen-binding protein, the conjugate, the polynucleotide molecule, or the recombinant vector described herein, and a pharmaceutically acceptable carrier and/or excipient.
[0093]ln another aspect, provided herein is a method for preparing a multispecific antigen-binding protein that specifically binds TNFR2 and CD25, comprising the steps of:
(a) culturing the host cell described herein in a culture medium under conditions suitable for expression of the multispecific antigen-binding protein, and
(bj isolating the multispecific antigen-binding protein from the host cell and/or the culture medium.
[0094]ln another aspect, provided herein is a method for promoting proliferation, activating and/or enhancing suppressive function, and/or stabilizing immunosuppressive phenotype of a population of regulatory T cells (Treg) comprising contacting the population of Treg with the multispecific antigenbinding protein or the conjugate described herein. In some embodiments, said contacting occurs in vitro, in some embodiments, said contacting occurs in vim. In some embodiments wherein the method occurs in vivo, the method further comprises administering the multispecific antigen-binding protein or the conjugate into a subject in need thereof.
[0095]ln another aspect, provided herein is a method of treating or preventing a disease or disorder in a subject in need thereof, said method comprising administering to the subject an effective amount of the multispecific antigen-binding protein or the conjugate described herein. In some embodiments, the disease or disorder is an immunological disease, inflammatory disease, cancer, cardiovascular disease, or an infertility and pregnancy-associated disease.
[0096]ln some embodiments, the immunological disease is selected from an autoimmune disease, a neurological condition, an allergy, asthma, macular degeneration, muscular atrophy, a disease related to miscarriage, atherosclerosis, bone loss, a musculoskeletal disease, obesity, a graft-versus-host disease, and an allograft rejection.
[0097]ln some embodiments, the autoimmune disease is selected from lupus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpastures disease, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lichen sclerosis, lgG4-related disease, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disease, pemphigus vulgaris or related blistering skin disease, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, premature ovarian failure, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, primary ovarian insufficiency, Raynaud’s phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthritis, stiff-man syndrome, type I diabetes, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (Granulomatosis with polyangiitis) or other immune vasculitis.
[0098]ln some embodiments, the lupus is systemic lupus erythematosus (SLE), cutaneous lupus, lupus nephritis, neonatal lupus, or drug-induced lupus. In some embodiments, the cutaneous lupus is acute cutaneous lupus, chronic cutaneous lupus erythematosus, discoid lupus erythematosus (DLE), or subacute cutaneous lupus erythematosus.
[0099jln some embodiments, the autoimmune disease is atopic dermatitis, psoriasis, systemic lupus erythematosus, or arthritis.
[OlOOjln some embodiments, the neurological condition is selected from a brain tumor, a brain metastasis, a spinal cord injury, schizophrenia, epilepsy, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Huntington's disease, Parkinson's disease, and stroke. [OlOlJIn some embodiments, the allergy is selected from food allergy, seasonal allergy, pet allergy, hives, hayfever, allergic conjunctivitis, poison ivy allergy oak allergy, mold allergy, drug allergy, dust allergy, cosmetic allergy, and chemical allergy.
[O1O2J8 n some embodiments, the allograft rejection is selected from skin graft rejection, bone graft rejection, vascular tissue graft rejection, ligament graft rejection, and organ graft rejection, [0103]! n some embodiments, the ligament graft rejection is selected from cricothyroid ligament graft rejection, caudal cruciate ligament graft rejection, periodontal ligament graft rejection, suspensory ligament of the lens graft rejection, palmar radiocarpal ligament graft rejection, dorsal radiocarpal ligament graft rejection, ulnar collateral ligament graft rejection, radial collateral ligament graft rejection, suspensory ligament of the breast graft rejection, anterior sacroiliac ligament graft rejection, posterior sacroiliac ligament graft rejection, sacrotuberous ligament graft rejection, sacrospinous ligament graft rejection, inferior pubic ligament graft rejection, superior pubic ligament graft rejection, anterior cruciate ligament graft rejection, lateral collateral ligament graft rejection, posterior cruciate ligament graft rejection, medial collateral ligament graft rejection, cranial cruciate ligament graft rejection, , and patellar ligament graft rejection.
[0104]ln some embodiments, the organ graft rejection is selected from heart graft rejection, lung graft rejection, kidney graft rejection, liver graft rejection, pancreas graft rejection, intestine graft rejection, and thymus graft rejection.
[0105]ln some embodiments, the graft-versus-host disease arises from a bone marrow transplant or one or more blood cells selected from B-cells, T-cells, basophils, common myeloid progenitor cells, common lymphoid progenitor cells, dendritic cells, eosinophils, hematopoietic stem cells, neutrophils, natural killer cells, megakaryocytes, monocytes, or macrophages.
[OlOSjln some embodiments, the Inflammatory disease is acute or chronic inflammation.
[0107]! n some embodiments, the inflammatory disease is selected from osteoarthritis, atopic dermatitis, endometriosis, polycystic ovarian syndrome, inflammatory bowel disease, fibrotic lung disease, and cardiac inflammation.
[OlOSJin some embodiments, the cancer is selected from adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith- Wiedemann syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dube syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal carcinoma, HIV/AIDS-related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell proiymphocytic leukemia and hairy cell leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CIVIL), chronic T-cell lymphocytic leukemia, eosinophilic leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, hodgkin lymphoma, non-hodgkin lymphoma, lynch syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or M¥H)-associated polyposis, myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian, fallopian tube, and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, soft tissue sarcomas, skin cancer (non-melanoma), small bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Waldenstrom macroglobulinemia (lymphoplasmacytic lymphoma), Werner syndrome, Wilms tumor, or xeroderma pigmentosum, [0109]ln some embodiments, the cardiovascular disease is selected from atherosclerosis, heart failure, left heart failure with reduced ejection fraction, left heart failure with preserved ejection fraction, right ventricular failure, congestive heart failure, restrictive cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, idiopathic cardiomyopathy, and hypertension. [OllOjln some embodiments, the infertility and pregnancy-associated diseases is selected from recurrent pregnancy loss, pre-eclampsia, preterm labor, fetal growth restriction, and intrauterine growth restriction. [OlllJIn another aspect provided herein is a method of regenerating a tissue or organ comprising one or more TNFR2+ and/or CD25+ cells, said method comprising contacting the tissue or organ with an effective amount of the multispecific antigen-binding protein or the conjugate described herein. In another aspect, provided herein is a method of regenerating a tissue or organ comprising one or more TNFR24- cells, said method comprising contacting the tissue or organ with an effective amount of the multispecific antigen-binding protein or the conjugate described herein. In another aspect, provided herein is a method of regenerating a tissue or organ comprising one or more CD25+ cells, said method comprising contacting the tissue or organ with an effective amount of the multispecific antigen-binding protein or the conjugate described herein.
[0112]! n some embodiments, the tissue or organ is selected from pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerve, eye, thymus, tongue, bone, liver, small intestine, large intestine, gastrointestinal, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structures, embryo, and testes tissue. In some embodiments, said contacting occurs m vitro. In some embodiments, said contacting occurs in vivo. In some embodiments wherein the method occurs in vivo, the method further comprises administering the multispecific antigen-binding protein or the conjugate into a subject in need thereof.
[0113]! n various embodiments of the above-described methods, the subject is a mammal. In some embodiments, the mammal is human.
[0114]! n another aspect, provided herein is a method for inducing tolerance to a foreign agent and/or preventing or reducing immune response to a foreign agent in a subject in need thereof, comprising an administering to the subject an effective amount of the multispecific antigen-binding protein, or the conjugate described herein.
[0115]ln some embodiments, the foreign agent is a therapeutic protein, a peptide, a vector, a biochemical vector, a lipid, a carbohydrate, a nucleic acid, a sperm, an oocyte, or an embryo. In some embodiments, the vector is a viral vector, a bacterial vector, or a fungal vector. In some embodiments, the viral vector is a DNA or RNA vector.
BRIEF DESCRIPTION OF DRAWINGS
[OllSjFsgure 1 shows the therapeutic rational of the multispecific antigen-binding proteins of the present disclosure. An exemplary bispecific format is depicted. [0117] Fsgure 2A depicts an exemplary general panning strategy for isolation of tumor necrosis factor (INF) receptor type 2 (TNFR2)-specific variable domain of heavy chain (VHH) antibodies, also referred to herein as V-bodies (Vbs). Binders to human and rodent TNFR2 were enriched from VHH immune libraries by two rounds of phage display. BM, bone marrow.
[0118]Figure 2B shows VHH immune library selection for next-generation sequencing (NGS) across the phage display process. Three initial libraries, 12 samples of the first panning round, and 36 samples of the second panning round, were sequenced with 20 million, 2 million, and 2 million reads, respectively. Comparison of V-body enrichment from the initial library to the first and second round of panning enabled identification of potential V-body candidates.
[O119]F8gure 3 shows a schematic diagram of an exemplary NGS workflow. Following phage display, the VHH region of the phage eluate was amplified via polymerase chain reaction (PCR). Unique and samplespecific barcodes were then fused, and NGS was subsequently performed using the Illumina NovaSeq platform (Genewiz). The raw data were de-multiplexed, and then processed by the NGS analysis pipeline. Forward and reverse sequence pairs were merged via overlapping regions and the VHHs, including complementarity determining regions (CDRs) were annotated. Based on CDRS identity, V-body sequences were clustered, thereby allowing for detailed analysis of, e.g,, V-body enrichment during phage display, sequence diversity, CDR3 length distribution, and cluster abundance. Based on such analyses, more than 600 candidates were selected for DNA synthesis (Twist) and further characterization.
[0120]F8gure§ 4A~4B illustrate human TNFR2 (hTNFR2) V-body binding validation at a fixed concentration of 1 pM V-body. The bar histogram (Figure 4A) and table (Figure 4B) show the percentage of Alexa488-positive cells for all VHHs tested. For the bar histogram, the lower dotted line indicates background staining, and the upper dotted line indicates two times the background level. A V-body having a signal-to-noise ratio greater than 2 is considered as a "binder". Shading within the table indicates binders to hTNFR2.
[0121]Figures 5A-5B illustrate human TNFR2 (hTNFR2) V-body binding validation at a fixed concentration of 100 nM V-body. The bar histogram (Figure 5A) and table (Figure SB) show the percentage of Alexa488-positive cells for all VHHs tested. For the bar histogram, the lower dotted line indicates background staining (~5%), and the upper dotted line indicates two times the background level. A V-body having a signal-to-noise ratio greater than 2 is considered as a "binder". Shading within the table indicates binders to hTNFR2, [0122]Figures SA-6C depict cross-specificity of V-body binding to mouse TNFR2 (mTNFR2) (Figure SA) and cynomolgus TNFR2 (cTNFR2) (Figure SB) at a fixed concentration of WO nM. The bar histograms (Figure 6A-6B) and table (Figure 6C) show the percentage of Aiexa488-positive cells for all VHHs tested. For the bar histogram, the lower dotted line indicates background staining, and the upper dotted line indicates two times the background level.
[0123]Figure 7 shows testing of human TNFR2 V-body binding across a range of concentrations for V- bodies T-002, T-014, T-001, T-006, T-007, T-003. V-bodies were tested at molar concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.12 nM and 1.55 nM.
[0124)Figure 8 shows a schematic diagram of an exemplary experimental setup for determination of binding affinities of the V-bodies for their respective target via surface plasmon resonance (SPR) (left panel) and a corresponding table describing the V-body candidates analyzed (right panel). Figure discloses SEQ. ID NO: 7279.
[0125]Figures 9A-9F depict surface plasmon resonance (SPR) sensorograms of VHH binding to human, cynomolgus, and mouse TNFR2, Fitted binding curves and calculated dissociation constants (KD) are included.
[0126]Fsgure 10 shows a summary of binding affinities of 16 selected anti-TNFR2 V-bodies to human, cynomolgus and mouse TNFR2. Cyno, cynomolgus.
[0127]Fsgure 11 demonstrates that some humanized anti-TNFR2 V-bodies targeted the epitope recognized by a MR2-1 bivalent agonist. T-013hul and T-018hul may recognize the same epitope as MR2-1. MR2-1 binding enhanced binding of T-015hul, T-017hul and T-Ollhul to TNFR2.
[0128]Figures 12A-12E show TNFR2 agonism by multivalent V-body fusion constructs. Agonism of bivalent (Figure 12A), tetravalent (Figures 12B-12C), and IL-2 N88D fusion (Figure 12D) anti-TNFR2 constructs were characterized on NF-KB reporter HEK293 cells stably expressing TNFR2. Dot plots show a dose-dependent response of anti-TNFR2 VHHs compared to a control VHH (Ctrl). Figure 12E shows that activity of T-007_2xVHH-Fc, and an IL-2 mutein, was demonstrated in reporter cell lines specific for each signaling pathway. RLU, relative luminescence unit.
[0129]Figure 13 depicts HEK293 TNFR2 NF-KB (LUC) reporter gene assay controls. Anti-hTNFR2 agonist MR2-1 monoclonal antibodies were tested on NF-xB reporter (Luc) HEK293 reporter cell-line stably expressing TN FR2 (clone 25) versus parental cell line (PCL). RLU, relative luminescence unit.
[O13O]F8gures 14A-14C shows HEK293 TNFR2 NF-KB (LUC) reporter gene assay samples and assay controls. A description of reporter gene assay samples and assay controls is shown in Figure 14A. A bar graph showing protein concentration (mg/mL) for the V-body constructs and respective controls is depicted in Figure MB. A bar graph showing RLUs for V-body constructs and respective controls tested on a PCL control is depicted in Figure 14C.
[0131]Figures 15A-15B depict concentration range curve data generated using MR2-1 (Figure ISA) and TNFa (Figure 15B) controls for four assay plates.
[0132]Flgures 1SA-1SC show exemplary dot plots of RLUs measured across increasing concentrations (mol/L) of control (control 12) and tetravalent V-body fusion constructs comprising four V-bodies mounted onto the fragment crysta lliza ble (Fc) region of a lgG4 variant comprising S228P, L235E and P329G mutations.
[0133]Figures 17A-17F show exemplary dot plots of RLUs measured across increasing concentrations (mol/L) of control (control 10) and an alternative design of tetravalent V-body fusion constructs comprising four V-bodies mounted onto the Fc region of a lgG4 variant comprising S228P, L235E and P329G mutations.
[0134]Figures 18A-18C show exemplary dot plots of RLUs measured across increasing concentrations (mol/L) of control (control 2) and bivalent V-body fusion constructs. Limit of detection, LOD.
[0135]Figures 29A-19C show exemplary dot plots of RLUs measured across increasing concentrations (mol/L) of control (control 13) and IL-2 N88D V-body fusion constructs. Limit of detection, LOD.
[0136]Figure 20 depicts a comparison of RLUs measured across increasing concentrations (mol/L) of monospecific construct 10 (tetravalent Fc) and construct 12 (Vb-Fc-Vb) tested on NF-xB reporter (Luc) HEK293 reporter cell-line stably expressing TNFR2 (clone 8).
[O137]F8gure 21 depicts an exemplary experimental timeline of TNFR2 stimulation by multivalent V-body fusion constructs (e.g., tetravalent Fc, Vb-Fc-Vb, rigid bivalent no Fc) on primary human peripheral blood mononuclear cells (PBMCs) and cluster of differentiation 4 positive (CD4+) CD25* CD127dim regulatory T cells (Tregs).
[0138]Fsgure 22 shows a bar graph of an overview of in-assay concentrations (nM) of multivalent V-body fusion constructs first wave binders. Concentrations (nM) of the VHH constructs is also shown.
[0139]Fjgure 23 illustrates an exemplary gating strategy applied for Treg markers. Treg Donor 1 is shown as an example and an identical strategy was used for Treg Donor 1 and Donor 3. Live cell and CD4 gating were based on Fluorescence Minus One (FMO) FMO control determination of the cut-off point between background fluorescence and positive cell populations. Forkhead box P3 (FoxP3), Human Leukocyte Antigen, DR isotype (HLA-DR), chemokine motif (C-C motif) receptor 8 (CCR8), and OX-40 gating was based on the CD4 subset of IgG control-stained sample from the same donor. For FoxP3, the gate was set at approximately 0.2%. For OX-40, HLA-DR and CCR8, the gate was set at approximately 2%. [0140]Figures 24A-24B demonstrate multivalent anti-TNFR2 V-body fusion constructs increased expression of the Treg suppression marker HLA-DR and CCR8. Histograms displaying expression of lreg suppression marker HLA-DR for specific T-003 binders compared to control formats (Figure 24A). Density plots displaying expression of Treg suppression marker HLA-DR and CCR8 for specific T-003 binders compared to control formats (Figure 24B). Fluorescein isothiocyanate, FITC; Phycoerythrin, PE. [0141]Figure 25A-25B demonstrate tetravalent anti-TNFR2 V-body fusion constructs strongly increased expression of Treg suppression marker HLA-DR on FoxP3+ Tregs. The bar graphs show HLA-DR mean fluorescent intensity (MFI) measured for each of the tetravalent Fc, Vb-Fc-Vb, and rigid bivalent no Fc V- body fusion formats relative to control formats.
[0142]Figure 26 shows dose-response curves based on HLA-DR MFI values of CD4+ FoxP3+ Tregs for construct T-003 and control 10 for Donor 2.
[0143]Fsgure 27 shows dose-dependent induction of Treg suppression marker HLA-DR expression across various concentrations of tetravalent anti-TNFR2 V-body Fc fusion construct 10 for Donor 1 (top panel) and Donor 2 (bottom panel).
[0144]Figure 28 shows dose-dependent induction of Treg suppression marker HLA-DR expression across various concentrations of rigid bivalent anti-TNFR2 V-body fusion construct 2 for Donor 1 (top panel) and Donor 2 (bottom panel),
[0145]Fsgure 29 shows dose-dependent induction of Treg suppression marker HLA-DR expression across various concentrations of tetravalent anti-TNFR2 V-body Vb-Fc-Vb fusion construct 12 for Donor 1 (top panel) and Donor 2 (bottom panel).
[0146]Figure 30 shows exemplary design of multivalent anti-TNFR2 V-body fusion constructs, Anti- TNFR2 V-bodies are shown as ovals, linkers are shown with flexible (e.g., GS linkers) as curved lines, rigid linkers (e.g., proline linker) as straight lines, and Fc domains as dimeric bars. Figure discloses SEQ ID NO: 7280.
[O147]F8gure 31 shows assessment of tetravalent-Fc VHH activity on naive CD4+CD25-rCD45RA+ human Treg. HLA-DR and CCR8 expression on CD4+FQXIP3+ and expansion after 5 day stimulation with anti- CD3/IL-2 plus VHH or MR2-1 are shown.
[0148]Figure 32A shows the ability of TNFR2 VHH to stabilize Treg. Naive CD4+CD25-KD45RA+ human Treg from healthy donors were stimulated with IL-2 and anti-CD3 in the presence of TNFR2 agonist VHH or TNFR2 monoclonal agonist MR2-1 for 5 days. Figure 328 shows the effect of TNFR2 VHH on early markers of Treg stability. Naive CD4*CD25+CD45RA* human Treg from healthy donors were stimulated with IL-2 and anti-CD3 in the presence of TNFR2 agonist VHH or IL-2 mutein for 5 days. [0149]Fjgures 33A-33B show additional results of in vitro treatment of human Treg (CD4+ FQXP3+) with VHH T-007_2xVHH-Fc or IL-2 mutein in the presence of anti-CD3 and IL-2. T-007_2xVHH-Fc induced and expanded a Treg population with high levels of FOXP3, EZH2 (a marker of stability), CCR8, and HLA-DR
(biomarkers of tissue homing and Treg immunosuppressive functionality). T-test: * p < 0.05; ** p < 0.01; n = 3.
[0150]Hgures 34A-34C show the effect of TNFR2 VHH on Treg stability under inflammatory conditions. Human Treg were expanded with IL-2 mutein or TNFR2 VHH in the presence of anti~CD3 and IL-2 for 5 days and then cultured with proinflammatory cytokines (IL-lb, IL-21, and IL-23 +/- TGFb) for 11 to 12 days; IL-17A or IFNy production after PMA/ionomycin stimulation was assessed together with FOXP3 by flow cytometry. The conversion of human Treg in vitro to cells that produce Thl/17 cytokines (IFNy/IL- 17A) triggered by the inflammatory cytokines shown was prevented by co-stimulation with TNFR2 VHH but not with IL-2 mutein. P~vaiues indicate results of a paired T-test; * = p < 0.05; ** = p < 0.01; Fc = human lgG4 mutant Fc.
[OlSljFigure 35 shows assessment of Treg function upon TNFR2 agonism. Naive Treg were stimulated for 7 days with anti-CD3/IL-2 plus TNFR2 VHH (T-007._2xVHH-Fc), control VHH, MR2-1, control IgG, or IL- 2 mutein; after 7 days, stimuli were removed and cells were incubated with cel! tracer-labeled autologous responder cells (na'ive CD4+ T cells); bar graph shows effector CD4 cell proliferation measured as % dividing CD4+FOXP3- cells; FACS histograms show dilution of the cell tracer at different Treg:CD4 (responder) ratios of one of four donors. T-007__2xVHH-Fc induces Treg that are better able to restrain effector CD4 cell proliferation than IL-2 mutein.
[0152]Figure 36A-36B show the effect of TNFR2 agonist VHH on Treg population size in mice. An exemplary design of the experimental procedure is shown. CD4+FOXP3+ Treg expansion in the spleen of mice 5 days after a single injection of 2,5 mg/kg control VHH or TNFR2-specific VHH T-007__2xVHH-Fc is shown.
[0153]Figure 37 shows that TNFR2 agonist VHH activates Treg in vivo. CCR§ is a chemokine receptor expressed on highly suppressive Treg and involved in cell migration (Whiteside et al., Immunol 2021;163:512). ICAM-1 surface adhesion molecule is required for Treg function (Gottrand et al., Immunol 2015;146(4): 657). ICOS costimulatory molecule is upregulated upon Treg activation and maintains FOXP3 expression (Landuyt et al., J Immunol 2019;202(4):1033). Proportion of Treg (CD4+ CD25+ F0XP3+) in spleen expressing activation markers (CCR8, ICAM-1, or ICOS) 5 days after a single injection of control VHH or TNFR2-specific VHH T-007__2xVHH-Fc is shown. 1-way ANOVA was performed for control VHH vs. T-007__2xVHH-Fc; only significant differences are shown; **“ = p < 0.0001. [0154]Figure 38 shows that TNFR2 agonist VHH selectively expands Treg in the spleen. Cell subsets as percentage of CD45+ cells in the spleen 5 days after single injection of TNFR2-specific VHH T- 007__2xVHH-Fc or control VHH are shown.
[0155]Fsgure 39 shows that TNFR2 agonist VHH increases serum level of IL-10. IL-10 is a key antiinflammatory cytokine (Saraiva et al., J Exp Med 2020;217(l):e20190418). Serum cytokine concentration 5 days after single injection of TNFR2 -specific VHH T-007_2xVHH-Fc or control VHH is shown. 1-way ANOVA performed for control VHH vs. T-007_2xVHH~Fc; only significant differences are shown; **** = p < 0.0001.
[0156]Figure 40A-40R show frequency of Treg (CD4+ FOXP3+) among total immune cells (CD45+) in the spleen, blood, colon, and lung in human TNFR2 knock-in mice 5 days after single injection of T-037. T- test: *** p < 0.001; **** p < 0.0001; n = 4.
[0157]Fsgure§ 41A-41E show T-037 selectively increases the Treg population in the spleen 5 days after a single administration compared to IL-2 N88D, a mutein that is active in mice. T-007__2xVHH-Fc is more selective for Treg and induces a higher level of FQXP3 and surface markers (F0XP3, ICAM-1, OX-40, ICOS, and CCR8), consistent with superior function and stability. One-way ANOVA test: **** p < 0.0001; n = 4. [0158]Fsgure§ 42A~42B show that Treg expansion in the spleen as well as increased expression of F0XP3, linked to Treg stability and function, and Treg activation shown by up-regulation of ICAM-1 and ICOS. [0159]Fsgure§ 43A-43C show that reduction of arthritis, measured by paw volume and arthritis score, in a model of collagen-antibody induced arthritis upon treatment with TNFR2 agonists T-037 and T-043. [0160]Figure§ 44A~448 show Treg expansion by TNFR2 agonist without inducing proinflammatory cytokines when compared to CD28 agonist.
[0161]Fsgure 45 depicts an exemplary general panning strategy for isolation of CD25-specific variable domain of heavy chain (VHH) antibodies, also referred to herein as V-bodies (Vbs). Binders to human and rodent CD25 were enriched from VHH immune libraries by two rounds of phage display. BM, bone marrow.
[0162]Figure 46 shows VHH immune library selection for next-generation sequencing (NGS) across the phage display process. Three initial libraries, 12 samples of the first panning round, and 36 samples of the second panning round, were sequenced with 20 million, 2 million, and 2 million reads, respectively. Comparison of V-body enrichment from the initial library to the first and second round of panning enabled identification of potential V-body candidates.
[OlSSjFsgure 47 shows a schematic diagram of an exemplary NGS workflow. Following phage display, the VHH region of the phage eluate was amplified via polymerase chain reaction (PCR). Unique and sample-specific barcodes were then fused, and NGS was subsequently performed using the Illumina NovaSeq platform (Genewiz). The raw data were de-multiplexed, and then processed by the MGS analysis pipeline. Forward and reverse sequence pairs were merged via overlapping regions and the VHHs, including complementarity determining regions (CDRs) were annotated. Based on CDR3 identity, V-body sequences were clustered, thereby allowing for detailed analysis of, e.g., V-body enrichment during phage display, sequence diversity, CDR3 length distribution, and cluster abundance. Based on such analyses, up to ~300 candidates were selected for DNA synthesis (Twist) and further characterization.
[0164]Figure 48 illustrates human CD25 (hCD25) V-body binding validation at a fixed concentration of 100 nM V-body, The bar histogram shows the mean fluorescence intensity (MFI) of Alexa488-positive cells for V-bodies C-004 and C-006 versus an anti-His only control condition.
[0165]Figure 49 illustrates V-body binding to cynomolgus (cCD25) (left panel) and mouse CD25 (mCD25) (right panel) at a fixed concentration of 100 nM V-body. The bar histograms show the mean fluorescence intensity (MFI) of Alexa488-positive cells for tested-bodies C-004 and C-006 versus an anti- His only control condition.
[0166]Fsgure§ 50A-50B show testing of human CD25 V-body binding across a range of concentrations for V-bodies C-004 and C-006. V-bodies were tested at molar concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM, and 0.390625 nM (shown from left to right). The bar histogram in Figure 50A shows the percentage of Alexa488 positive cells for C-004 and C-006. The bar histogram in Figure 503 shows the mean fluorescent intensity (MFI) of Alexa488 positive cells for C-004 and C-006.
[0167]Figure 51 shows a schematic diagram of an exemplary experimental setup for determination of binding affinities of the anti-CD25 V-bodies for their respective target via surface plasmon resonance (SPR). Figure discloses "HHHHHH" as SEQ ID NO: 9425.
[0163]Figures 52A-52C depict surface plasmon resonance (SPR) sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-004 and C-006. Fitted binding curves and calculated dissociation constants (KD) are included. Data corresponding to an anti-CD25 IgG (aCD25 IgG) control condition are also included (Figure 52C). Figure discloses "HHHHHH" as SEQ ID NO: 9425. [0169]Figure S3 shows a summary of binding affinities of two candidate anti~CD25 V-bodies to human, cynomolgus and mouse CD25. Data corresponding to an anti-CD25 IgG (aCD25 IgG) control condition are also included. MB, no binding. [0170]F!gures 54A-54B demonstrate that some humanized anti-CD25 V-bodies targeted the epitope recognized by H--2. Data are shown for a first experiment 1 (Expl) and second experiment 2 (Exp2) performed using V-bodies C-004 (Figure 54A) and C-006 (Figure 548).
[0171]Fsgures 55A-55B demonstrate humanized anti-CD25 V-bodies C-OOlhul, C-002Hul, and C-003 are non-competitive binders. Data are shown for a first experiment 1 (Figure 55A) and second experiment 2 (Figure 55B).
[0172]Figures 56A-56C depict SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-008Hul, C-OlOHul, and C-009Hul. Fitted binding curves and caicuiated dissociation constants (KD) are included.
[0173]Figure§ 57A-57C illustrate ligand (IL-2) competition by SPR. Each panel represents a sensorgram overlay plot for a single V-body captured onto a discrete spot. The sensorgrams display I L-2-Fc competition: association of the human CD25-extracellular domain (CD25-ECD) to the V-body was followed either by additional binding by IL2-Fc, indicating an unoccupied epitope (non-overlapping epitopes), or no i L2-Fc binding, indicating epitope blocking (overlapping epitopes), and a buffer control, association and dissociation of human CD25-ECD in the absence of I L2-Fc.
[0174]Fsgure 58 shows binding of His-tagged anti-CD25 VHHs to Human Embryonic Kidney (HEK) cells transfected with human or cyno CD25 detected by flow cytometry using a fluorescently-labelled secondary anti-His antibody. Binding is expressed as mean fluorescent intensity.
[0175]Figures 59A-59C depict SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-010Hul.A8, C-010Hul.L8, and C-009Hul.L8. Fitted binding curves and calculated dissociation constants (KD) are included.
[017S]Fsgure§ 6QA-60C illustrate exemplary multispecific antibody-binding proteins encompassed by the present invention. Figures 6QB-60C are adapted from C. Spiess et al. Molecular Immunology S7 (2015) 95-106, which is incorporated herein by reference in its entirety.
[0177]Figures S1A-618 show that a TNFR2/CD25 bispecific V-body is able to bind to Treg. Healthy donor PBMC were incubated with mono- (TNFR2 or CD25) or bispecific (TNFR2 and CD25) VHHs and stained for CD4, F0XP3, CD8 and CD14. VHH binding was detected after washing with a secondary fluorescently- labelled anti VHH antibody. Plots show binding to Treg (mean fluorescent intensity) from 2 different donors for molecules that are monospecific for TNFR2 or bispecific for TNFR2 and CD25.
[0178]Hgure§ 62A-62B show that different anti-TNFR2/anti-CD25 multispecific antibodies are able to bind to Treg. Healthy donor PBMC were incubated with mono or bispecific VHHs and stained for CD4, FOXP3, CD8 and CD14. VHH binding was detected after washing with a secondary fluorescently-labelled anti VHH antibody. Histograms show bispecific binding on CD4*FOXP3 Treg, CD4+FOXP3- CD4 effector, CD8 effector and CD.I.4+ monocytes. Insertion of a CD25 VHH at the N- or C-terminus of the Fc portion drastically increases binding to Treg.
[0179]Fsgures 63A-63B shows that a TNFR2/CD25 bispecific V-body is able to activate a reporter cell. Figure 63A shows the results of TNFR2 agonism tested at 3 different dilutions on HEK NF-kB -Luciferase reporter cells expressing TNFR2. Y axis shows maximum agonism/reporter activity in relative luminescence unit (R.L.U.). Antibody formats are described above the respective data points. Figure §38 shows the results of TNFR2 agonism on HEK NF-kB -Luciferase reporter cells expressing TNFR2 only (Clone 18) or both TNFR2 and CD25 (Subcione 48 derived from clone 18). Monospecific and bispecific VHH were tested on TNFR2+ HEK and TNFR2*CD25+ HEK. Plot show equal TNFR2 agonism by TNFR2 VHH on both cells. In contrast, bispecific VHHs induced stronger agonism on reporter expressing CD25 (clone 48) compared to cells not expressing CD25 (clone 18).
[0180]Figures &4A-64B show that Tregs can be activated by mono- and bispecific tetravalent V-bodies. Figure S4A shows the results of total CD4 T cells isolated from PBMC from healthy donors stimulated with anti-CD3/CD28 beads and IL-2 in the presence or absence of mono or bispecific VHHs. Mono and bispecific VHHs led to increased activation as measured by HLA-DR upregulation in Treg after 5 days of stimulation. Figure 648 shows the results of total CD4 T cells isolated from PBMC from healthy donors stimulated with anti-CD3/CD28 beads and IL-2 in the presence or absence of mono or bispecific VHHs, Mono and bispecific VHHs led to increase percentage of FOXP3+ cells among CD4 after 5 days of stimulation.
[0181]Figures 65A-65C depict SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-OllHul, C-012Hul, C-013Hul, C-014Hul, C-015Hul, C-005Hul, C-016Hul, C- 017Hul, and C-018Hul. Fitted binding curves and calculated dissociation constants (KD) are included. [0182]Figures 66A-66C depict SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-031Hul, C-032Hul, C-033Hul, C-034Hul, C-035Hul, C-036Hul, C-037Hul, C- 038Hul, C-039Hul, C~040Hul, and C-041Hul. Fitted binding curves and calculated dissociation constants (KD) are included.
[0183]Figure 67 depicts SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C~019Hul, C-020Hul, and C-022Hul. Fitted binding curves and calculated dissociation constants (KD) are included. [0184]Figure 68 depicts SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-023Hul and C-024Hul. Fitted binding curves and calculated dissociation constants (Ko) are included.
[0185]Fsgures 69A-69B depict SPR sensorgrams of VHH binding to human, cynomolgus, and mouse CD25 for anti-CD25 V-bodies C-025Hul, C-026Hul, C-027Hul, C-028Hul, C-029Hul, and C-030Hul. Fitted binding curves and calculated dissociation constants (KD) are included.
[0186]Rgures 70A-7GF depict SPR sensorograms of VHH binding to human and cynomolgus TNFR2 for anti-TNFR2 V-bodies . Fitted binding curves and calculated dissociation constants (Ko) are included. [0187]Figures 71A-71J depict SPR sensorograms of VHH (obtained by alanine scanning mutagenesis) binding to human and cynomolgus TNFR2 for anti-TNFR2 V-bodies. Fitted binding curves and calculated dissociation constants (KD) are included.
[0188]Fsgure§ 72A-72B depict surface plasmon resonance (SPR) sensorgrams of Fc-tagged bivalent V- bodies binding to human or cynomolgus TNFR2. Fitted binding curves and calculated dissociation constants (Ko) are included.
[0189]Figures 73A-73B depict flow cytometry plots (Figure 73A) and luminescence plots (Figure 73B) upon incubation of TNFR2 HEK reporter cells with the Fc-tagged bivalent V-bodies.
[0190]Figures 74A-74D depict binding of His-tagged VHHs to CD25+ human embryonic kidney (HEK) cells analyzed via flow cytometry. Figures 74A-74B show binding of His-tagged VHH to CD25+ HEK cells detected after washing with secondary anti-His antibody. Figures 74C-74D show binding measured by incubation of pre-incubated VHH/anti-His antibody complex to CD25+ HEK cells.
[0191]Figure 75 depicts Treg cell activation in the presence of a TNFR2/CD25 bispecific V-body compared to a TNFR2/control antibody. Data shows the average of 4 donors +/- SEM.
[O192]F8gure 76 depicts Treg cell expansion upon Injection in human TNFR2/human CD25 double knock- in mice with a TNFR2/CD25 bispecific V-body compared to a TNFR2/control antibody. Data shows the average of 3 replicates +/- STD.
[0193]Figures 77A-77B depict stable TNFR2* HEK cells incubated with a dose range of bispecific TNFR2/CD25 antibodies or control antibodies comprising control VHH with irrelevant specificity in place of the TNFR2 and/or the CD25 VHH.
[0194]Figures 78A-78C depict stable CD25-r/low TNFR2 HEK cells incubated with a dose range of blspecific TNFR2/CD25 antibodies or control antibodies comprising control VHH with irrelevant specificity in place of the TNFR2 and/or the CD25 VHH. [0195] Fsgures 79A-79B depict NF-kB luciferase reporter activity in TNFR2+ HEX cells upon 16-20 hours of incubation with a dose range of bispecific TNFR2/CD25 antibodies or control antibodies comprising control VHH with irrelevant specificity in place of the TNFR2 and/or the CD25 VHH.
[0196]Fsgures 80A-80B depict binding of bispecific TNFR2/CD25 antibodies or control antibodies comprising control VHH with irrelevant specificity in place of the TNFR2 and/or the CD25 VHH to Treg cells (CD4+ FOXP3+) or monocytes (CD14+) from human PBMC.
DETAILED DESCRIPTION OF THE INVENTION
[0197]The present application provides, among other things, multispecific antigen-binding proteins protein comprising: one or more antigen-binding domains that specifically bind to tumor necrosis factor receptor 2 (TNFR2); and one or more antigen-binding domains that specifically bind to cluster of differentiation 25 (CD25).
[0198]TNFR2 signaling has been shown to induce proliferation, sustained suppressive function and FOXP3 promoter demethylation in Tregs (Tseng et al., 2019). TNFR2 signaling also induces the expression of EZH2 (Urbano et al., 2018), a histone methyl transferase involved in the repression of the effector transcriptomic program and stabilization of the Treg phenotype (DuPage et al., 2015). Because of its role in Tregs biology and FOXP3 promoter demethylation, TNFR2 signaling can be leveraged to induce a stable immunosuppressive phenotype and enhance their function to the benefit of autoimmune diseases.
[0199]CD25, also called interleukin-2 receptor subunit alpha (IL-2Ra or IL2RA), is the alpha chain component of the high-affinity heterotrimeric interleukin-2 (IL-2) receptor, a type I transmembrane protein highly expressed on the surface of the majority of Tregs. IL-2 activation of CD25 can facilitate immune tolerance in Tregs. High cell surface expression of CD25 can also occur in malignant cells, e.g., in several lymphomas and leukemias,
[0200]TNFR2 is highly expressed on CD25-f- Tregs and has been shown to induce their expansion, enhance their function and stabilize their suppressive phenotype. While not wishing to be bound by any particular theory, Tregs are believed to be the only cells that express both TNFR2 and CD25 constitutively. In some embodiments, the multispecific antigen-binding proteins described herein can specifically agonize TNFR2 on Tregs using CD25 as an anchor (see Figure 1). Definitions
[02(31] U n less defined otherwise, ail technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shall control.
[0202]As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 5%. For example, as used herein, the expression "about 100“ includes 95 and 105 and all values in between (e.g., 96, 97, 98, 99, etc.).
[0203]The term "antigen" encompasses any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portions thereof, or combinations thereof) that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor. In various embodiments of the present disclosure, the antigens described herein include TNFR2, including human, cynomolgus, and/or mouse TNFR2, and/or CD25, including human CD25.
[0204]The term "epitope" can refer to an antigenic determinant on the surface of an antigen to which an antibody molecule binds. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects (e.g., agnostic or antagonistic effects). Epitopes may be either conformational or linear. A conformational epitope is formed by spatially juxtaposed amino acids from different segments of the linear polypeptide chairs. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include non-peptidic moieties on the antigen, such as saccharides, phosphoryl groups, or sulfonyl groups.
[0205]The term "antigen-binding protein" refers in the broadest sense to a protein that specifically binds an antigen (e.g., TNFR2 and/or CD25). In certain embodiments, an antigen-binding protein comprises an antibody or an antigen-binding fragment of an antibody, such as a human antibody, a humanized antibody; a camelid antibody; a chimeric antibody; a recombinant antibody; a heavy chain antibody; a single-domain antibody (e.g., VHH); a single chain antibody (e.g., single chain fragment variable (scFv)); a diabody; a triabody; a tetrabody; a Fab fragment; a F(ab') 2 fragment; an IgD antibody; an IgE antibody; an igM antibody; an IgGl antibody; an lgG2 antibody; an lgG3 antibody; or an lgG4 antibody, and fragments thereof. The term "antigen-binding protein" also encompasses, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen-binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. In addition, peptide antibody mimetics can be used, as well as scaffolds based on antibody mimetics utilizing fibronectin components (e.g., fibronectin type III domain (FN3)) as a scaffold.
[0206]The term "antigen-binding domain" refers to the portion of the antigen-binding protein that is capable of specifically binding to an antigen or epitope. An antigen-binding protein may comprise more than one antigen-binding domains, for example, two, three, four, five, six, seven, eight or more antigenbinding domains. For example, antigen-binding domains may comprise at least one variable region (either a heavy chain or light chain variable region) or one or more CDRs of an antibody that can bind a particular antigen, Examples of suitable antigen-binding domains include, without limitation, singledomain antibodies (e.g., VHH), single-chain antibodies (e.g., single chain fragment variable (scFv)), Fab fragments, F(ab')2 fragments, and protein scaffolds.
[0207]The term "antibody" and "immunoglobulin" or "Ig" are used interchangeably herein, and is used in the broadest sense and encompasses, for example, individual monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies (e.g., VHH), single chain antibodies, intrabodies, anti-idiotypic (anti-ld) antibodies, and antigen-binding fragments of antibodies, as described below. An antibody can be human, humanized, camellzed, recombinantly produced, chimeric, synthetic, affinity de-matured and/or affinity matured as well as an antibody from other species, for example mouse, camel, llama, rabbit, etc. In specific embodiments, the specific target antigen that can be bound by an antibody provided herein includes a TNFR2 polypeptide, TNFR2 fragment orTNFR2 epitope. In specific embodiments, the specific target antigen that can be bound by an antibody provided herein includes a CD25 polypeptide, CD25 fragment or CD25 epitope. An "antigen-binding fragment" generally refers a portion of an antibody heavy and/or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of antigen-binding fragments include single-domain antibody (e.g., VHH), single-chain Fvs (scFv), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody and minibody, or a chemically modified derivative thereof. In particular, antibodies provided herein include immunoglobulin molecules and molecules that contain immunologically active portion(s) of an immunoglobulin molecule, for example, one or more complementarity determining regions (CDRs) of an antibody that binds to TNFR2 and/or CD25. Such antibody fragments can be found described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec, Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. EnzymoL, 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990), The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl and lgA2), or any subclass (e.g., lgG2a and lgG2b) of immunoglobulin molecule.
[0208]The term "single-domain antibody" or "sdAb" as used herein, refers to an antibody or antibody fragment containing a single antibody variable domain that is able to bind to a specific antigen alone, without the requirement of another antibody variable domain, The complementarity determining regions (CDRs) of a single-domain antibody are part of a single antibody variable domain. Examples of single-domain antibodies include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered antibodies, variable domains derived from the aforementioned antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, shark, goat, rabbit, and/or bovine. In some embodiments, a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. For clarity reasons, the variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH to distinguish it from the conventional VH of four-chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, e.g., camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain, which are also within the scope of the invention. For example, cartilaginous fishes such as sharks can produce immunoglobulin-like structures known as VNAR. In some embodiments, a single-domain antibody may be obtained from a Camelidae VH domain. In some embodiments, a single-domain antibody may be obtained from human VH by camelization. See Saerens et al., Current Opinion in Pharmacology, 2008, 8:600-608, the disclosure of which being incorporated by reference, for review of single-domain antibodies. [0209]The term "specifically binds" as used herein means that an antigen-binding protein forms a complex with a target antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by a dissociation constant (KD) of about Ix.lO'6 M or iess (e.g., less than 1G 5 M, less than 5xl0'7 M, less than 1£T7 M, less than 5xl0's M, less than 10'8 M, less than 5xl0‘9 M, less than 10‘9 M, or less than 1O'W M). Methods for determining the binding affinity of an antigen-binding protein, e.g., an antibody or an antibody fragment, to a target antigen are well known in the art and include, e.g., surface plasmon resonance (e.g., BIACORE® assays), bio-layer interferometry, ligand binding assays (e.g., enzyme-linked immunosorbent assay (ELISA)), equilibrium dialysis, fluorescent-activated cell sorting (FACS), or flow cytometry-based binding assays and the like. Specific binding to a particular target antigen from a certain species does not exclude that the antigen-binding protein can also specifically bind to the analogous target from a different species. For example, specific binding to human TNFR2 does not exclude that the antigen-binding protein can also specifically bind to TNFR2 from cynomolgus monkeys ("cyno") or mouse.
[0210]The term "isolated" when used in the context of antigen-binding proteins (e.g,, antibodies, such as single-domain antibodies), polypeptides, polynucleotides, and vectors, means the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), polypeptides, polynucleotides and vectors are at least partially free of other biological molecules from the cells or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antigen-binding protein may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term "isolated" is not intended to refer to a complete absence of such biological molecules (e.g,, minor or insignificant amounts of impurity may remain) or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies).
[0211]The term "operably linked" as used herein can refer to a functional relationship between two or more regions of a polypeptide chain in which the two or more regions are linked so as to produce a functional polypeptide,
[0212]As used herein, the term "variant", "derivative" or "derived from" in the context of proteins or polypeptides (e.g., antigen-binding proteins or domains thereof) refer to: (a) a polypeptide that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the polypeptide it is a variant or derivative of; (b) a polypeptide encoded by a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a nucleotide sequence encoding the polypeptide it is a variant or derivative of; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions and/or substitutions) relative to the polypeptide it is a variant or derivative of; (d) a polypeptide encoded by nucleic acids can hybridize under high, moderate or typical stringency hybridization conditions to nucleic acids encoding the polypeptide it is a variant or derivative of; (e) a polypeptide encoded by a nucleotide sequence that can hybridize under high, moderate or typical stringency hybridization conditions to a nucleotide sequence encoding a fragment of the polypeptide, it is a variant or derivative of, of at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, or at least 150 contiguous amino acids; or (f) a fragment of the polypeptide it is a variant or derivative of. The terms also encompass a multispecific antigen-binding protein or polypeptide comprising the polypeptide it is a variant or derivative of, [0213]The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.
[0214]As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art, See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur- containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamateaspartate, and asparagine-giutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443- 1445, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
[0215]Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g,, FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each herein incorporated by reference.
[021S]The terms "enhance" or "promote/' or "increase," or "expand," or "improve" refer generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule/composition. A measurable physiological response may include an increase in immune cell expansion, activation, effector function, persistence, and/or an increase in tumor celi death killing ability, among others apparent from the understanding in the art and the description herein, in certain embodiments, an "increased" or "enhanced" amount can be a "statistically significant" amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including ail integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or a control composition.
[0217]The terms "decrease" or "lower," or "lessen," or "reduce," or "abate" refer generally to the ability of composition contemplated herein to produce, elicit, or cause a lesser physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule/composition. In certain embodiments, a "decrease" or "reduced" amount can be a "statistically significant" amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle or a control composition.
[0218]The terms "treat” or "treatment" of a state, disease, disorder or condition include: (1) preventing, delaying, or reducing the incidence and/or likelihood of the appearance of at least one clinical or sub- clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disease, disorder or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disease, disorder or condition; or (2) inhibiting the state, disease, disorder or condition, e.g., arresting, reducing or delaying the development of the state, disease, disorder, or condition or a relapse thereof or at least one clinical or sub-clinical symptom of the state, disease, disorder, or condition; or (3) relieving the state, disease, disorder, or condition, e.g., causing regression of the state, disease, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
[0219]The terms "effective amount" or "therapeutically effective amount" refer to a quantity and/or concentration of a composition containing an active ingredient (e.g., multispecific antigen-binding protein) that when administered into a patient either alone (i.e., as a monotherapy) or in combination with additional therapeutic agents, yields a significant decrease in the progression of the state, disease, disorder, or condition, as, for example, by ameliorating or eliminating symptoms and/or the cause of the state, disease, disorder, or condition. An effective amount may be an amount that relieves, lessens, or alleviates at least one symptom or biological response or effect associated with a state, disease, disorder, or condition, prevents progression of the state, disease, disorder, or condition, or improves physical functioning of the patient. A therapeutically effective amount of a composition containing an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the active agent are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic and/or prophylactic result.
[0220jThe terms “individual", "subject", and "patient" are used interchangeably herein to refer to an animal, for example a mammal. The terms include human and veterinary subjects. In some embodiments, methods of treating mammals, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are provided. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, a subject can be a subject in need of treatment for a disease or disorder. In particular embodiments, the subject is a human.
Muitispecific Antigen-Binding Proteins
[0221pn one aspect, provided herein are multispecific antigen-binding proteins comprising one or more anti-TNFR2 antigen-binding domains and one or more anti CD25 antigen-binding domains that are linked, directly or indirectly, to one another. The multispecific antigen-binding proteins may comprise one or more additional domains or moieties.
[0222]ln some embodiments, the multispecific antigen-binding protein of the present disclosure may be, for example, bispecific, trispecific, tetraspecific, pentaspecific, etc. The terms "bispecific", "trispecific", "tetraspecific", "pentaspecific", etc., all fall under the term "multispecific" and refer to binding to two, three, four, five, etc., different target molecules, respectively. In specific embodiments, the multispecific antigen-binding protein of the present disclosure are bispecific.
[0223]ln some embodiments, the multispecific antigen-binding protein of the present disclosure comprises one or more anti-TNFR2 antigen-binding domains (e.g., single-domain antibodies) and one or more anti CD25 antigen-binding domains (e.g., single-domain antibodies) as described herein. In some embodiments, the multispecific antigen-binding protein is multivalent. For example, the multispecific antigen-binding protein or conjugate of the present disclosure may be at least bivalent, but can also be e.g., trivalent, tetravalent, pentavalent, hexavalent, septivalent, octavalent, etc. The multispecific antigen-binding protein can be bivalent, trivalent, tetravalent, pentavalent, hexavalent, septivalent, or octa va Sent for the different antigens. The terms "bivalent", "trivalent", "tetravaient", "pentavalent", "hexavalent", "septivalent", "octavalent" all fall under the term "multivalent" and indicate the presence of two, three, four, five six, seven, or eight binding domains (e.g., single-domain antibodies), respectively.
[0224]ln some embodiments, the multispecific antigen-binding protein can comprise two or more anti- TNFR2 antigen-binding domains and two or more anti-CD25 binding domains. In some embodiments, the multispecific antigen-binding protein can comprise four or more anti-TNFR2 antigen-binding domains and two or more anti-CD25 binding domains. In some embodiments, the multispecific antigenbinding protein can comprise six or more anti-TNFR2 antigen-binding domains and two or more anti- CD25 binding domains.
[0225]ln some embodiments, the multispecific antigen-binding protein of the present disclosure comprises a single polypeptide. In other embodiments, the multispecific antigen-binding protein or conjugate of the present disclosure comprises more than one polypeptide. In some embodiments, the multispecific antigen-binding protein of the present disclosure comprises two polypeptides.
[0226]ln some embodiments, the multispecific antigen-binding protein can comprise an anti-TNFR2 antigen-binding domain operably connected to an anti-CD25 antigen-binding domain.
[0227]! n certain embodiments, the one or more additional domain or moieties may be one or more additional binding domain that binds to one or more further antigen or protein.
[0228]When two or more anti-TNFR2 antigen-binding domains are included in a multispecific antigenbinding protein, the two or more anti-TNFR2 antigen-binding domains may comprise the same sequence or may comprise different sequences. In such embodiments, the two or more anti-TNFR2 antigenbinding domains may bind to the same epitope on TNFR2 or different epitopes on TNFR2. For example, a multispecific antigen-binding protein or conjugate of the present disclosure may be biparatopic, e.g., if two VHHs bind two different epitopes on TNFR2.
[0229jln various embodiments, multispecific antigen-binding protein of the present disclosure can comprise two or more anti-TNFR2 antigen-binding domains each comprising an amino acid sequence selected from any of the CDR1, CDR2, and/or CDR3 amino acid sequences listed in Table 1-1, Table 5, Table 11, or Table 13, or any combination thereof, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity, or any combination thereof. In various related embodiments, multispecific antigen-binding protein of the present disclosure can comprise two or more anti-TNFR2 antigen-binding domains each comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within any of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13. In some embodiments, the two or more anti-TNFR2 antigen-binding domains can comprise the same set of three CDRs. in some embodiments, the two or more anti-TNFR2 antigenbinding domains can comprise different sets of three CDRs.
[0230]When two or more anti-CD25 antigen-binding domains are included in a multispecific antigenbinding protein or conjugate, the two or more anti-CD25 antigen-binding domains may comprise the same sequence or may comprise different sequences. In such embodiments, the two or more anti-CD25 antigen-binding domains may bind to the same epitope on CD25 or different epitopes on CD25. For example, a multispecific antigen-binding protein or conjugate of the present disclosure may be biparatopic, e.g., if two VHHs bind two different epitopes on CD25.
[0231]ln various embodiments, multispecific antigen-binding protein of the present disclosure can comprise two or more anti-CD25 antigen-binding domains each comprising an amino acid sequence selected from any of the CDR1, CDR2, and/or CDRS amino acid sequences listed in Table 1-3 or Table 9, or any combination thereof, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity, or any combination thereof. In various related embodiments, multispecific antigen-binding protein of the present disclosure can comprise two or more anti-CD25 antigen-binding domains each comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within any of the exemplary anti-CD25 VHH antibodies listed in Table 1-3, Table 1-4, or Table 9. In some embodiments, the two or more anti-CD25 antigen-binding domains can comprise the same set of three CDRs. In some embodiments, the two or more anti-CD25 antigen-binding domains can comprise different sets of three CDRs.
[0232]Exemplary designs of bispecific constructs comprising one or more anti-TNFR2 binding domain (e.g., VHHs) and one or more anti-CD25 binding domain (e.g., VHHs) are shown in Figure SOA.
[O233]8n some embodiments, a multispecific antigen-binding protein of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure from N- terminus to C-terminus: (anti-TNFR2 VHH)n- first linker - Fc region- second linker - (anti-CD25 VHH)m, wherein n and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc.). When n>2, each anti-TNFR2 VHH may be optionally operably linked to another anti-TNFR2 VHH via a linker. When m>2, each anti-TNFR2 VHH may be optionally operably linked to another anti-TNFR2 VHH via a linker. The multiple linkers used in the multispecific antigen-binding protein are not necessarily the same or different.
[0234]ln some embodiments, a multispecific antigen-binding protein or conjugate of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure from N-terminus to C-terminus: (anti-CD25 VHH)n- first linker- Fc - second linker - (anti-TNFR2 VHH)m, wherein n and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc.). When n>2, each anti-CD25 VHH may be optionally operably linked to another anti-CD25 VHH via a linker. When m>2, each anti-TNFR2 VHH may be optionally operably linked to another anti-TNFR2 VHH via a linker. The multiple linkers used in the multispecific antigen-binding protein are not necessarily the same or different.
[0235]ln some embodiments, a multispecific antigen-binding protein of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure from N- terminus to C-terminus: (anti- CD25 VHH)i- (anti~TNFR2 VHH)n- first linker - Fc region- second linker - (anti-TNFR2 VHH)m, wherein I, n, and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc.). The adjacent anti- CD25 VHH and anti-TNFR2 VHH may be optionally operably linked to one another via a linker. When I >2, each anti- CD25 VHH may be optionally operably linked to another anti- CD25 VHH via a linker. When n>2, each anti-TNFR2 VHH may be optionally operably linked to another anti-TNFR2 VHH via a linker. When m>2, each anti-TNFR2 VHH may be optionally operably linked to another anti-TNFR2 VHH via a linker. The multiple linkers used in the multispecific antigen-binding protein are not necessarily the same or different.
[0236]ln some embodiments, a multispecific antigen-binding protein of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure from N- terminus to C-terminus: (anti-TNFR2 VHH)r (anti-CD25 VHH)n- first linker - Fc region- second linker - (anti-TNFR2 VHH)m, wherein I, n, and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc.). The adjacent anti-TNFR2 VHH and anti-CD25 VHH may be optionally operably linked to one another via a linker. When I >2, each anti-TNFR2 VHH may be optionally operably linked to another anti- TNFR2 VHH via a linker. When n>2, each anti-CD25 VHH may be optionally operably linked to another anti-CD25 VHH via a linker. When m>2, each anti-TNFR2 VHH may be optionally operably linked to another anti-TNFR2 VHH via a linker. The multiple linkers used in the multispecific antigen-binding protein are not necessarily the same or different.
[0237]ln a specific embodiment, a multispecific antigen-binding protein of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure from N- terminus to C-terminus: anti-TNFR2 VHH - first linker - Fc region- second linker - anti-CD25 VHH. The multiple linkers used in the multispecific antigen -binding protein are not necessarily the same or different.
[O23S]8n a specific embodiment, a multispecific antigen-binding protein of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure from N- terminus to C-terminus: (anti-TNFR2 VHH)?- first linker - Fc region- second linker - anti-CD25 VHH. The two anti-TNFR2 VHHs may be optionally operably linked to each another via a linker. The multiple linkers used in the multispecific antigen-binding protein are not necessarily the same or different. The two anti- TNFR2 VHHs may bind to the same epitope on TNFR2 or different epitopes on TNFR2.
[0239]ln a specific embodiment, a multispecific antigen-binding protein of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure from N- terminus to C-terminus: anti-CD25 VHH - first linker - Fc region- second linker - (anti-TNFR2 VHH)?.. The two anti-TNFR2 VHHs may be optionally operably linked to each another via a linker. The multiple linkers used in the multispecific antigen-binding protein are not necessarily the same or different, The two anti- TNFR2 VHHs may bind to the same epitope on TNFR2 or different epitopes on TNFR2.
[0240]!n a specific embodiment, a multispecific antigen-binding protein of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure from N- terminus to C-terminus: anti-CD2S VHH - anti-TNFR2 VHH - first linker - Fc region- second linker - anti- TNFR2 VHH. The adjacent anti-TNFR2 VHH and anti-CD25 VHH may be optionally operably linked to one another via a linker. The multiple linkers used in the multispecific antigen-binding protein are not necessarily the same or different. The two anti-TNFR2 VHHs may bind to the same epitope on TNFR2 or different epitopes on TNFR2.
[0241]ln some embodiments, the one or more antl-TNFR2 antigen-binding domains (e.g., VHHs) and one or more anti CD25 antigen-binding domains (e.g., VHHs) may be operably linked to one another in tandem. The adjacent antigen-binding domains (e.g., VHHs) may be optionally operably linked to one another via a linker. The linkers may be a flexible Sinker or a rigid linker.
[0242]ln some embodiments, a multispecific antigen-binding protein of the present disclosure may comprise the following structure from N-terminus to C-terminus: (anti-TNFR2 VHH),,-- linker - (anti- CD25 VHH)m, wherein n and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc.). When n>2, each anti-TNFR2 VHH may be optionally operably linked to another anti-TNFR2 VHH via a linker. When m>2, each anti-CD25 VHH may be optionally operably linked to another anti-CD25 VHH via a linker. The multiple linkers used in the multispecific antigen-binding protein are not necessarily the same or different.
[0243]Sn some embodiments, a multispecific antigen-binding protein of the present disclosure may comprise the following structure from N-terminus to C-terminus: (anti-CD25 VHH)n- linker - (anti-TNFR2 VHH)m, wherein n and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc.). When n>2, each anti-CD25 VHH may be optionally operably linked to another anti-CD25 VHH via a linker. When m>2, each anti-TNFR2 VHH may be optionally operably linked to another anti-TNFR2 VHH via a linker. The multiple linkers used in the multispecific antigen-binding protein are not necessarily the same or different.
[0244]ln some specific embodiments, a multispecific antigen-binding protein of the present disclosure may comprise the following structure from N-terminus to C-terminus: anti-CD25 VHH - linker - (anti- TNFR2 VHH)?.. The two anti-TNFR2 VHHs may bind to the same epitope on TNFR2 or different epitopes on TNFR2. In one embodiment, the linker between anti-CD25 VHH and the adjacent anti-TNFR2 VHH is a flexible linker. In one embodiment, the linker between the two anti-TNFR2 VHH is a rigid linker.
[0245]ln some specific embodiments, a multispecific antigen-binding protein of the present disclosure may comprise the following structure from N-terminus to C-terminus: anti~CD25 VHH - linker - (anti- TNFR2 VHHh. The four anti-TNFR2 VHHs may bind to the same epitope on TNFR2 or different epitopes on TNFR2. In one embodiment, the linker between the anti-CD25 VHH and the adjacent anti-TNFR2 VHH is a flexible linker. In one embodiment, the linker between each of the anti-TNFR2 VHHs is a flexible linker.
[0246]The multispecific antigen-binding protein may also adopt any of the alternative molecular formats shown in Figures 60B-60C and/or described in C. Spiess et al. Molecular Immunology 67 (2015) 95-106, which is Incorporated herein by reference in its entirety.
Antigen-binding Domains
[0247]The antigen-binding domains of the present disclosure can include an antibody or an antigenbinding fragment of an antibody, such as a human antibody, a humanized antibody; a camelid antibody; a chimeric antibody; a recombinant antibody; a heavy chain antibody; a single-domain antibody (e.g., VHH); a single chain antibody (e.g., single chain fragment variable (scFv)); a diabody; a triabody; a tetrabody; a Fab fragment; a F(ab') 2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgGl antibody; an lgG2 antibody; an lgG3 antibody; or an lgG4 antibody, and fragments thereof. [0248]ln some embodiments, an antigen-binding domain that binds to TNFR2 or CD25 is a single-domain antibody (also termed as "sdAb"). The single-domain antibodies of the present disclosure can be derived from numerous sources, including but not limited to VHHs, VNARs, or VH domains (naturally occurring or engineered VH domains), VHHs can be generated from camelid heavy chain only antibodies and libraries thereof. VNARs can be generated from cartilaginous fish heavy chain only antibodies and libraries thereof. Various methods have been implemented to generate monomeric sdAbs from conventionally heterodimeric VH and VL domains, including interface engineering and selection of specific germline families. In some embodiments, sdAbs of the present invention are human or humanized.
[0249]! n some embodiments, a single-domain antibody described herein is a VHH fragment (also known as a nanobody). VHH fragments are also referred to as "V-bodies" in the present disclosure. In some embodiments, the VHH is a camelid VHH, a humanized VHH or a camelized VH. In some embodiments, a single-domain antibody described herein is a VH domain. In some embodiments, a single-domain antibody described herein is a naturally occurring VH domain or engineered VH domain.
[0250]The variable domain of an antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises at least three complementarity determining regions (CDRs) which determine its binding specificity. Preferably, in a variable domain, the CDRs are distributed between framework regions (FRs), The variable domain typically contains 4 framework regions interspaced by 3 CDR regions, resulting in the following typical antibody variable domain structure: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4. CDRs and/or FRs of the single-domain antibody of the present disclosure may be fragments or derivatives from a naturally occurring antibody variable domain or may be synthetic.
[0251]Binding affinity of a molecular Interaction between two molecules, e.g., for an antigen recognized by a multispecific antigen as described herein, can be measured via various techniques, such as surface plasmon resonance (SPR), bio-layer interferometry (BLI ), enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, fluorescent-activated cell sorting (FACS), or flow cytometry binding assays and the like. Surface plasmon resonance is a biosensor technique that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions (see e.g., Ober et al. 2001, Intern. Immunology 13: 1551- 1559). SPR can for example be performed using the BIACORE® system or Carterra ISA system. Another biosensor technique that can be used to determine affinities of biomolecular interactions is bio-layer interferometry (Bid) (see e.g., Abdiche et al, 2008, Anal Biochem. 377; 209-217), Bio-layer interferometry is a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam), A change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Since the interactions can be measured in real-time, association and dissociation rates and affinities can be determined. BLI can for example be performed using the Octet® Systems. Alternatively, affinities can be measured in Kinetic Exclusion Assay (KinExA) (see e.g., Drake et al. 2004, Anal. Biochem., 328: 35-43), which is a solution-based method to measure true equilibrium binding affinity and kinetics of unmodified molecules. Equilibrated solutions of an antibody/antigen complex are passed over a column with beads precoated with antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished with a fluorescently labeled protein binding the antibody (or antigen).
Anti-TNFR2 Antigen-binding Domain
[0252]The present disclosure provides one or more antigen binding domains (e.g., antibodies, such as single-domain antibodies) that bind to tumor necrosis factor receptor 2 (TNFR2).
[0253]TNFR2 is a single pass type-1 membrane protein belonging to the TNFR superfamily. It consists of an extracellular domain with four cysteine rich domains (CRD) and an intracellular domain that is involved in signaling. The cysteine rich domains contain a total of 10 disulfide bonds stabilizing the elongated structure of the protein. Unlike TNFR1 which is widely expressed, the expression of TNFR2 is restricted on immune cells including Tregs, myeloid cells, CD8 and NK cells but also glial cells, endothelial cells and fibroblasts (Medler and Wajant, 2019).
[0254]ln some embodiments, the human TNFR2 protein is encoded by the human TNF receptor superfamily member IB (TNFRSF1B) gene (NCBI Gene ID: 7133) and has the amino acid sequence of MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREY¥DQTAQMCCSKCSPGQHAKVFCTKTSDTVCD SCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL5KQEGCRLCAPLRKCRPGFGVARPG TETSDWCKPCAPGTFSNTTSSTDICRPHQICNWAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPE PSTAPSTSFLLPMGPSPPAEGSTGDFALPVGLIVGVTALGLLIIGWNCVIMTQVKKKPLCLQREAKVPHLPADKARGTQ GPEQQHLLITAPSSSSSSLESSASALDRRAPTRNQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSS DHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS (UniProtKB Accession No. P20333) (SEQ ID NO: 4028) [0255] I n some embodiments, the cyno TNFR2 protein is encoded by the Cyno TNF receptor superfamily member IB (TNFRSF1B) gene (Gene ID: 102144224) and has the amino acid sequence of MVTRRGGDDRRRLKGHRVLGVTLEVLARRCWGGRVGGPAEAGEGRGGGVSKAGWPRPAPPRCLASGPLQRGLSLS VAAGWRAQRSLGRRRCAARARGREGRGNRIPPAPMAPAAVWAALAVGLELWAAGHALPAQVAFTPYAPEPGGTCR LREYYDQTAQMCCSKCPPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRIC TCRPGWYCALSKQEGCRLCAQLRKCRPGFGVARPGTETSDWCKPCAPGTFSNTTSSTDICRPHQICHWAIPGNASM DAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPAPSTAPGTSFLLPVGPSPPAEGSTGDIVLPVGLIVGVTALGLLIIG WNCVIMTQVKKKPLCLQRETKVPHLPADKARGAQGPEQQHLLTTVPSSSSSSLESSASALDRRAPTRNQPQAPGAEK ASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDASPSGSPKDEQVPFSKEECAFRS QLETPETLLGSTEEKPLPLGVPDAGMKPS (UniProtKB Accession No. A0A2K5VET2) (SEQ ID NO: 4029)
[0256]ln some embodiments, the mouse TNFR2 protein is encoded by the mouse TNF receptor superfamily member IB (Tnfrsflb) gene (Gene ID: 21938) and has the amino acid sequence of MAPAALWVALVFELQLWATGHTVPAQWLTPYKPEPGYECQISQEYYDRKAQMCCAKCPPGQYVKHFCNKTSDTVC ADCEASMYTQVWNQFRTCLSCSSSCTTDQVEIRACTKQQNRVCACEAGRYCALKTHSGSCRQCMRLSKCGPGFGVAS SRAPNGNVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDAVCAPESPTLSAIPRTLYVSQPEPTRSQPLDQEPG PSQTPSILTSLGSTPIIEQSTKGGISLPIGLIVGVTSLGLLMLGLVNCIILVQRKKKPSCLQRDAKVPHVPDEKSQDAVGLEQ QHLLTTAPSSSSSSLESSASAGDRRAPPGGHPQARVMAEAQGFQEARASSRISDSSHGSHGTHVNVTCIVNVCSSSDHS SQCSSQASATVGDPDAKPSASPKDEQVPFSQEECPSQSPCETTETLQSHEKPLPLGVPDMGMKPSQAGWFDQIAVKV A (UniProtKB Accession No. P25119) (SEQ ID NO: 4030)
[0257]ln various embodiments, antigen-binding domains of the present disclosure have an agonist effect upon binding to TNFR2. While not wishing to be bound by theory, an agonistic TNFR2 binder can promote or increase activation of TNFR2 and/or potentiate one or more signal transduction pathways mediated by TNFR2. For example, agonistic TNFR2 binders may promote or increase the proliferation of a population of Treg cells. Agonistic TNFR2 binders may promote or increase TNFR2 activation by binding TNFR2, e.g., to induce a conformational change that renders the receptor biologically active. For example, agonistic TNFR2 binders may nucleate the trimerization of TNFR2 in a manner similar to the interaction between TNFR2 and its cognate ligand, tumor necrosis factor (TNF), thus inducing TNFR2- mediated signaling. In some embodiments, agonistic TNFR2 binding domains of the present disclosure may be capable of inducing the proliferation of Treg ceils (e.g., CD4+, CD25+, FOXP3+ Treg cells).
Agonistic TNFR2 binding domains of the present disclosure may also be capable of suppressing the proliferation of cytotoxic T lymphocytes (e.g., CDB-r T-cells), e.g., through activation of immunomodulatory Treg cells or by directly binding TNFR2 on the surface of an autoreactive cytotoxic T- cell and inducing apoptosis.
[025S]ln some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure comprise TNF or a variant thereof. [0259]ln some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure do not impair the binding of its cognate ligand, tumor necrosis factor (TN F), to TNFR2 upon binding to TNFR2. In some embodiments, anti-TNFR2 antigen-binding domain of the present disclosure do not have overlapping epitopes with TNF or do not bind to the same epitope(s) as TNF. In some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure have overlapping epitopes with TNF or bind to the same epitope(s) as TNF. in some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure upon binding to TNFR2 promote or facilitate TNFR2 oligomerization (in the presence or absence of TNF, respectively). In some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure upon binding to TNFR2 multimerize (e.g., dimerize) the TNFR2 trimers to induce intracellular signaling. In some embodiments, the one or more anti-TNFR2 antigen-binding domains compete with binding of TNF to TNFR2, In some embodiments, the one or more anti-TNFR2 antigenbinding domains have an antagonistic effect upon binding to TNFR2. In some embodiments, the one or more anti-TNFR2 antigen-binding domains do not compete with binding of TNF to TNFR2. In some embodiments, the one or more anti-TNFR2 antigen-binding domains have an agonistic effect upon binding to TNFR2.
[0260]ln some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure may have an agonist effect upon binding to TNFR2 with an ECso from about 0.1 - 1 nM, about 1 - 10 nM, about 10 - 100 nM, about 100 nM - 1 pM, or above 1 pM. In some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure may have an agonist effect upon binding to TNFR2 with an ECso from about 1- 100 nM. In some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure may have an agonist effect upon binding to TNFR2 with an ECso from about 1 - 10 nM or about 10 - 100 nM, In some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure may have an agonist effect upon binding to TNFR2 with an ECso of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, or about 100 nM.
[0261]ln some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure bind to human TNFR2. In some embodiments, anti-TNFR2 antigen-binding domains (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human TNFR2 with a KD of less than about IxlO”6 M, for example, less than about 5xl0~7 M, less than about 3xl0’7 M, less than about
1x10 7 M, less than about 3x10 s M, less than about 5x10 8 M, less than about 3x10 8 M, less than about 1x10 8 M, less than about 8x10 9 M, less than about 5x10 9 M, less than about 3x10 9 M, less than about IxlO"3 M, about IxlO’10 to IxlO’9 M, about IxlO’10 to 5xl0~9 M, about IxlO’10 to IxlO"8 IM, about IxlO’10 to 5xl0~s M, about IxlO’9 to IxlO’8 M, about IxlO’9 to 5xl0’s M, about IxlO’9 to lx IQ"7 M, or about IxlO"8 to IxlO"7 M. In certain embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human TNFR2 with a Ko of about IxlO'12 M to about IxlO”6 M. In certain embodiments, antigen-binding proteins (e.g., antibodies such as singledomain antibodies) of the present disclosure may bind to human TNFR2 with a KB of about 1 xiO"8 M to about 2.7 xl0“® M.
[0262]ln some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure bind to cynomolgus monkey ("cyno") TNFR2, In some embodiments, anti-TNFR2 antigen-binding domains (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno TNFR2 with a KB of less than about IxlO"6 M, for example, less than about SxlO"7 M, less than about 3xl0~7 M, less than about IxlO”7 M, less than about 8x10“* M, less than about 5xl0~8 M, less than about 3xl0”8 M, less than about IxlO"8 IM, less than about 8xl0"9 IM, less than about 5xl0"9 IM, less than about 3xl0"9 IM, less than about IxlO"9 M, about IxlO"10 to IxlO"3 IM, about IxlO"1'5 to 5xl0"s M, about IxlO"10 to IxlO"8 M, about IxlO"10 to SxlO”8 M, about IxlO"9 to IxlO"8 M, about 1x10"® to SxlO"8 M, about IxlO"9 to IxlO"7 M, about IxlO"3 to 2x10 7 M, about IxlO"3 to 5xl0’7 M, about 1x10 8 to IxlO"7 M, about IxlO"8 to 2xl0"7 M, about IxlO"8 to 5xl0"7 M, or about IxlO"8 to IxlO"6 M. In certain embodiments, antigenbinding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno TNFR2 with a KB of about 1x10 12 M to about IxlO"6 M. In certain embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno TNFR2 with a Ko of about 3xl0"8 M to about 2.4 xlO"6 IM.
[0263]ln some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure bind to mouse TNFR2. In some embodiments, antl-TNFR2 antigen-binding domains of the present disclosure may bind to mouse TNFR2 with a KD of less than about IxlO"6 IM, far example, less than about 5xl0”7 M, less than about 3xl0"7 IM, less than about IxlO"7 IM, less than about 8x10 s M, less than about 5xl0"8 IM, less than about 3xl0"8 M, less than about IxlO"8 IM, less than about 8xl0"9 M, less than about 5xl0"9 M, less than about 3xl0"9 M, less than about IxlO"9 M, about IxlO"10 to IxlO"9 M, about IxlO"10 to 5xl0"9 IM, about IxlO"10 to IxlO"8 M, about IxlO"10 to 5xl0"8 M, about IxlO"9 to IxlO"8 IM, about IxlO"9 to 5xl0~s IM, about IxlO"9 to IxlO’7 M, about IxlO’9 to 2xl0’7 IM, about IxlO’9 to 5xl0’7 IM, about IxlO’8 to IxlO”7 M, about IxlO’8 to 2xl0"7 M, about IxlO”8 to 5xl0’7 M, or about IxlO’8 to IxlO”6 M. In some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure do not bind to mouse TNFR2.
[0264]ln some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure may bind to TNFR2 with an ECso from about 0.1 - 1 nM, about 1 - 10 nM, about 10 - 100 nM, about 100 nM - 1 pM, or above 1 pM, In some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure may bind to TNFR2 with an ECsofrom about 1- 100 nM. In some embodiments, anti-TNFR2 antigenbinding domains of the present disclosure may bind to TNFR2 with an EC$o from about 1 - 10 nM or about 10 - 100 nM. in some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure may bind to TNFR2 with an ECso of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, or about 100 nM.
[0265]ln some embodiments, anti-TNFR2 antigen-binding domains of the present disclosure may specifically bind TNFR2 without exhibiting specific binding for another receptor of the tumor necrosis factor receptor (TNFR) superfamily,
[0266]The variable domain of an anti-TNFR2 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises at least three complementarity determining regions (CDRs) which determine its binding specificity. Preferably, in a variable domain, the CDRs are distributed between framework regions (FRs). The variable domain typically contains 4 framework regions interspaced by 3 CDR regions, resulting in the following typical antibody variable domain structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. CDRs and/or FRs of the single domain antibody of the invention may be fragments or derivatives from a naturally occurring antibody variable domain or may be synthetic.
[0267]Sequence identifiers corresponding to exemplary anti-TNFR2 VHH antibodies provided herein are listed in Table 1-1. Table 1-1 sets forth the sequence identifiers of amino acid sequences of the complementarity determining regions (CDR1, CDR2 and CDR3), amino acid and DMA sequences of the full-length camelid VHH antibodies, as well as amino acid sequences of corresponding humanized VHH antibodies. In the present disclosure, the suffix "-Hui" in an antibody ID indicates a humanized version of the referenced antibody. Amino acid sequences of additional exemplary anti-TNFR2 VHH antibodies and corresponding humanized VHH antibodies are provided in Table 1-2. Table 1-1. Sequence Identifiers for exemplary anti-TNFR2 VHH antibodies
Table 1-2. Sequence Identifiers for additional exemplary anti-TNFR2 VHH antibodies and humanized anti-THFR2 VHH antibodies
[0268]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) a) GSI(V/F)(R/S)(T/A)(N/D)(S/G/A); b) GFT(F/L)DD(I/Y)A (SEQ ID NO: 69); c) GFTFS(S/R/G)YA (SEQ ID NO: 70); d) GRTFSDYG (SEQ ID NO: 16); e) G(L/F)TLDYYA (SEQ ID NO: 71); f) GF(T/N)FSMYS (SEQ ID NO: 72); g) GRTF(G/R/S)(N/S)(Y/L)(T/F) (SEQ ID NO: 73); h) GASLSRNA (SEQ ID NO: 40); i) GS(I/T)FRFPP (SEQ ID NO: 74); j) GFTLDDYA (SEQ ID NO: 6633); and k) G(F/V)(S/T)LD(D/Y)(H/Y)T (SEQ ID NO: 7088)
[0269]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g,, antibody such as a singledomain antibody) of the present disclosure comprises a CDR2 comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) a) IRSDGF(T/i) (SEQ ID NO: 75); b) l(Y/F)SY(S/G)(S/P)NT (SEQ ID NO: 76); c) l(Y/S)(S/D)DGS(E/D)T (SEQ ID NO: 77); d) INWSN(G/A)RT (SEQ ID NO: 7268); e) l(S/N)(V/T)(S/G)DGST (SEQ ID NO: 78); f) IDT(R/G)GST (SEQ ID NO: 79); g) IR(W/R/Y)(T/P)G(G/L)(S/I)T (SEQ ID NO: 80); h) IYDDGET (SEQ ID NO: 41); i) LTSGGST (SEQ ID NO: 45); j) IFSYSSNT (SEQ ID NO: 6634); k) l(N/S)SNDG(S/T)(T/V) (SEQ ID NO: 7087); and l) INWS(N/Q/E/S)(G/A)RT (SEQ ID NQ:10409).
[0270]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR3 comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) a) (Y/F)YQ(S/A)LS(T/S)(P/A)N(Y/F)GQ(V/T)F (SEQ ID NO: 60); b) AADSDL(S/R)TV(V/T)(V/T)GPHDY (SEQ ID NO: 61); c) AKDAG(S/G)WG(T/R)GPFG(Y/F)(E/D)YDY (SEQ ID NO: 62); d) AA(T/A)PSGKAY(T/S)Y (SEQ ID NO: 63); e) ATPGPY(T/S/M)YCAPYGSSWSRGYDY (SEQ ID NO: 64); f) ARV(R/G)G(T/S/A)PY(E/D)Y(N/G)Y (SEQ ID NO: 65); g) (T/A/V)A(S/A)PTGRAF(T/N/A)Y (SEQ ID NO: 66); h) AGSAFDF (SEQ ID NO: 42); i) S(V/M)(V/L)GRDM(M/V)TY (SEQ ID NO: 67); j) AVGDFEGELVLKGDY (SEQ ID NO: 6635); k) AAD(L/V)G(F/V/Y)LY(A/T/V)DYV(P/R)LH(M/T)HHFGS (SEQ ID NO: 7086); l) A(A/G)(T/A)(P/L)(S/T)GKAY(T/S)Y (SEQ ID NQ: 7333); and m) A(A/G)(T/A/S)(P/L)(5/T)GKAY(T/S)Y (SEQ ID NO: 10387), wherein one or more non-alanine residues in the CDR3 sequence are optionally replaced with an alanine, and/or one or more alanine residues in the CDR3 sequence are optionally replaced with a glycine.
[0271]! n certain embodiments, the CDR3 sequence of an anti-TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises the amino acid sequence: a). (WV)(A/G)(S/A)(A/P)(A/T/S)(A/G)(A/R)A(A/F)(T/N/A)(A/Y); or b). (A/G)(A/G)(WS)(A/P/L){A/S/T)(A/G)(A/K)(A/G)(A/Y)(A/T/S)(A/Y).
[0272]in certain embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i)a CDR1 comprising an amino acid sequence of GSI(V/F)(R/S)(T/A)(N/D)(S/G/A), a CDR2 comprising an amino acid sequence of SEQ iD NO: 75, and a CDR3 comprising an amino acid sequence of
SEQ ID NO: 60; ii)a CDR1 comprising an amino acid sequence of SEQ iD NO: 69, a CDR2 comprising an amino acid sequence of SEQ iD NO: 76, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 61; iii)a CDR1 comprising an amino acid sequence of SEQ iD NO: 70, a CDR2 comprising an amino acid sequence of SEQ iD NO: 77, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 62; iv)a CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 63; v)a CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 7333; vi)a CDR1 comprising an amino acid sequence of SEQ iD NO: 71, a CDR2 comprising an amino acid sequence of SEQ iD NO: 78, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 64; vii)a CDR1 comprising an amino acid sequence of SEQ iD NO: 72, a CDR2 comprising an amino acid sequence of SEQ iD NO: 79, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 65; viii)a CDR1 comprising an amino acid sequence of SEQ ID NG: 73, a CDR2 comprising an amino acid sequence of SEQ !D NO: 80, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 66; ix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 42; x)a CDR1 comprising an amino acid sequence of SEQ ID NO: 74, a CDR2 comprising an amino acid sequence of SEQ iD NO: 45, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 67; xi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6635; xii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7088, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7087, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7086; xiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10387; xivja CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10409, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10387; xv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10409, and a CDR3 comprising an amino acid sequence of (A/G)(A/G)(T/A/S)(A/P/L)(A/S/T)(A/G)(A/K)(A/G){A/Y)(WS)(A/Y); or xvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 73, a CDR2 comprising an amino acid sequence of SEQ ID NO: 80, and a CDR3 comprising an amino acid sequence of (T/A/V)(A/6)(S/A)(A/P)(An/S)(A/G)(A/R)A(A/F)(T/N/A)(A/Y).
[0273]ln certain embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a)a CDR1 comprising an amino acid sequence of SEQ ID NO: 69, a CDR2 comprising an amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 61; b)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 63; c)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7333; - d)a CDR1 comprising an amino acid sequence of SEQ ID NO: 73, a CDR2 comprising an amino acid sequence of SEQ ID NO: 80, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 66; e)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7268, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10387; f) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10409, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10387; g) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10409, and a CDR3 comprising an amino acid sequence of (A/G)(A/G)(T/A/S)(A/P/L)(A/S/T)(A/G)(A/K)(A/G)(A/Y)(A/T/S)(A/Y); or h) a CDR1 comprising an amino acid sequence of SEQ ID NO: 73, a CDR2 comprising an amino acid sequence of SEQ ID NO: 80, and a CDR3 comprising an amino acid sequence of (T/A/V)(A/G)(S/A){A/P){A/T/S)(A/G)(A/R)A(A/F){T/N/A)(A/¥).
[0274]ln certain embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i)a CDR1 comprising an amino acid sequence of GSI(V/F)(R/S)(A/T)(N/D)(G/A), a CDR2 comprising an amino acid sequence of IRSDGFT (SEQ ID NO: 2), and a CDR3 comprising an amino acid sequence of YYQ(S/A)LSSPNYGQ(V/T)F (SEQ ID NO: 7270); ii)a CDR1 comprising an amino acid sequence of GFTFDDIA (SEQ ID NO: 8), a CDR2 comprising an amino acid sequence of IYSYGPNT (SEQ ID NO: 9), and a CDR3 comprising an amino acid sequence of AADSDLSTW(V/T)GPHDY (SEQ ID NO: 7271); iii)a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 12), a CDR2 comprising an amino acid sequence of ISDDGSDT (SEQ iD NO: 13), and a CDR3 comprising an amino acid sequence of AKDAGSWGTGPFGYEYDY (SEQ ID NO: 14); iv)a CDR1 comprising an amino acid sequence of GRTFSDYG (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of INWSN(G/A)RT (SEQ ID NO: 7268), and a CDR3 comprising an amino acid sequence of AA(T/A)PSGKAYSY (SEQ ID NO: 7272); v)a CDR1 comprising an amino acid sequence of GRTFSDYG (SEQ ID NQ: 16), a CDR2 comprising an amino acid sequence of INWSN(G/A)RT (SEQ ID NO: 7268), and a CDR3 comprising an amino acid sequence of A(A/G)(T/A)(P/L)(S/T)GKAY(T/S)Y (SEQ ID NO: 7333); vi)a CDR1 comprising an amino acid sequence of GLTLDYYA (SEQ ID NO: 20), a CDR2 comprising an amino acid sequence of ISTSDGST (SEQ ID NO: 21), and a CDR3 comprising an amino acid sequence of ATPGPYTYCAPYGSSWSRGYDY (SEQ ID NO: 22); vii)a CDR1 comprising an amino acid sequence of GF(T/N)FSMYS (SEQ ID NO: 72), a CDR2 comprising an amino acid sequence of IDT(R/G)GST (SEQ ID NO: 79), and a CDR3 comprising an amino acid sequence of ARV(G/R)G(T/A)PYEY(N/G)Y (SEQ ID NO: 7273); viii)a CDR1 comprising an amino acid sequence of GRTF(G/S)S(Y/L)(T/F) (SEQ ID NO: 7274), a CDR2 comprising an amino acid sequence of IR(W/R/Y)(T/P)G(G/L)(S/I)T (SEQ ID NO: 80), and a CDR3 comprising an amino acid sequence of (A/V)A(A/S)PTGRAF(T/N)Y (SEQ ID NO: 7276); ix)a CDR1 comprising an amino acid sequence of GASLSRNA (SEQ ID NO: 40), a CDR2 comprising an amino acid sequence of IYDDGET (SEQ ID NO: 41), and a CDR3 comprising an amino acid sequence of AGSAFDF (SEQ ID NO: 42); x)a CDR1 comprising an amino acid sequence of GS(T/i)FRFPP (SEQ ID NO: 7277), a CDR2 comprising an amino add sequence of LTSGGST (SEQ ID NO: 45), and a CDR3 comprising an amino acid sequence of SVLGRDM(M/V)TY (SEQ ID NO: 7275); xi)a CDR1 comprising an amino acid sequence of GFTLDDYA (SEQ ID NO: 6633), a CDR2 comprising an amino acid sequence of IFSYSSNT (SEQ ID NO: 6634), and a CDR3 comprising an amino add sequence of AVGDFEGELVLKGDY (SEQ ID NO: 6635); xii)a CDR1 comprising an amino acid sequence of GFTLDYYT (SEQ ID NO: 6637), a CDR2 comprising an amino acid sequence of ISSNDGSV (SEQ, iD NO: 6638), and a CDR3 comprising an amino acid sequence of AADLGYLYVDYVRLHTHHFGS (SEQ ID NO: 6639); xiii)a CDR1 comprising an amino acid sequence of GRTFSDYG (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of INWSN(G/A)RT (SEQ ID NO: 7268), and a CDR3 comprising an amino acid sequence of A(A/G)(T/A/S)(P/L)(S/T)GKAY(T/S)Y (SEQ ID NO: 10387); or xiv)a CDR1 comprising an amino acid sequence of GFT(F/L)DD(I/Y)A (SEQ ID NO: 69), a CDR2 comprising an amino acid sequence of i(Y/F)SY(S/G)(S/P)NT (SEQ ID NO: 76), and a CDR3 comprising an amino acid sequence of AADSDLSTW(V/T)GPHDY (SEQ ID NO: 7271).
[0275]! n certain embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a)a CDR1 comprising an amino acid sequence of GFTFDDIA (SEQ ID NO: 8), a CDR2 comprising an amino acid sequence of IYSYGPNT (SEQ ID NO: 9), and a CDR3 comprising an amino acid sequence of AADSDLSTW(V/T)GPHDY (SEQ ID NO: 7271); b)a CDR1 comprising an amino acid sequence of GRTFSDYG (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of INWSN(G/A)RT (SEQ ID NO: 7268), and a CDR3 comprising an amino acid sequence of AA(T/A)PSGKAYSY (SEQ ID NO: 7272); c)a CDR1 comprising an amino acid sequence of GRTFSDYG (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of INWSN(G/A)RT (SEQ ID NO: 7268), and a CDR3 comprising an amino acid sequence of A(A/G)(T/A)(P/L)(S/T)GKAY(T/S)Y (SEQ ID NO: 7333); d)a CDR1 comprising an amino acid sequence of GRTF(G/S)S(Y/L)(T/F) (SEQ ID NO: 7274), a CDR2 comprising an amino acid sequence of IR(W/R/Y)(T/P)G(G/L)(S/I)T (SEQ ID NO: 80), and a CDR3 comprising an amino acid sequence of (A/V)A(A/S)PTGRAF(T/N)Y (SEQ ID NO: 7276); e)a CDR1 comprising an amino acid sequence of GRTFSDYG (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of INWSN(G/A)RT (SEQ ID NO: 7268), and a CDR3 comprising an amino acid sequence of A(A/G)(T/A/S)(P/L)(S/T)GKAY(T/S)Y (SEQ ID NO: 10387); or f)a CDR1 comprising an amino acid sequence of GFT(F/L)DD(I/Y)A (SEQ ID NO: 69), a CDR2 comprising an amino add sequence of l(Y/F)SY(S/G)(S/P)NT (SEQ ID NO: 76), and a CDR3 comprising an amino acid sequence of AADSDLSTW(V/T)GPHDY (SEQ ID NO: 7271).
[0276]lncluded herein are anti-TNFR2 antigen-binding domains (e.g., antibodies such as single-domain antibodies) comprising a CDR1 (CDR1) comprising an amino add sequence selected from any of the CDR1 ammo acid sequences listed in Table 1-1, Table 5, Table 11, or Table IB, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0277]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence selected from SEQ ID Nos: 1, 5, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 6633, 6637, 6641, 7089, 7281-7284, 1128-1686, 6745-6806, and 7102-7125, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0278]lncluded herein are anti~TNFR2 antigen-binding domains (e.g., antibodies such as single-domain antibodies) comprising a CDR2 (CDR2) comprising an amino acid sequence selected from any of the CDR2 amino acid sequences listed in Table 1-1, Table 5, Table 11, or Table 13, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0279]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g,, antibody such as singledomain antibody) comprises a CDR2 comprising an amino acid sequence selected from SEQ ID Nos: 2, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 6634, 6638, 6642, 7096, 7285-7288, 1687-2245, 6807-6868, 7126-7149, and 10388-10393, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0280]lncluded herein are anti-TNFR2 antigen-binding domains (e.g., antibodies such as single-domain antibodies) comprising a CDR3 (CDR3) comprising an amino acid sequence selected from any of the CDR3 amino acid sequences listed in Table 1-1, Table 5, Table 11, or Table 13, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0281]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR3 comprising an amino acid sequence selected from SEQ ID Nos: 3, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 6635, 6639, 6643, 7093, 7099, 2246-2804, 6869-6930, 7150-7173, 7289-7292, 7333, 10374, 10410-10433, 10435-10455, and 10811, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0282]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR3 comprising an amino acid sequence selected from SEQ ID Nos: 34, 7099, 10410-10433, 10435-10455, and 10811, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0283]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR3 comprising an amino acid sequence selected from SEQ ID Nos: 10414, 10421, 10422, 10423, 10429, and 10811, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0284]lnciuded herein are anti-TNFR2 antigen-binding domains (e.g., antibodies such as single-domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within any of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 21, or Table 13. In certain embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 3; ii) a CDR1 comprising an amino acid sequence of SEQ !D NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6; iii)a CDR1 comprising an amino acid sequence of SEQ !D NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10; iv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 12, a CDR2 comprising an amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 14; v)a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; vi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 20, a CDR2 comprising an amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 22; vii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 24, a CDR2 comprising an amino acid sequence of SEQ ID NO: 25, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 26; viii)a CDR1 comprising an amino acid sequence of SEQ, ID NO: 28, a CDR2 comprising an amino acid sequence of SEQ ID NO: 29, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 30; ix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 33, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34; x) a CDR1 comprising an amino acid sequence of SEQ ID NO: 36, a CDR2 comprising an amino acid sequence of SEQ ID NO: 37, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 38; xi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 41, and a CDRS comprising an amino acid sequence of SEQ ID NO: 42; xii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 44, a CDR2 comprising an amino acid sequence of SEQ ID NO: 45, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 46; xiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6635; xiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 6637, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6638, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6639; xv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6643; xvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7089, a CDR2 comprising an amino acid sequence of SEQ ID NO: 45, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 46; xvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093; xvii i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; xix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7099; xx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; xxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; xxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7291; xxiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7291; xxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10; xxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093; xxviija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; xxviiija CDR1 comprising an amino add sequence of SEQ ID NO: 16, a CDR2 comprising an amino add sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; xxix)a CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10374; xxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10391, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10392, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10393, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxvja CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10338, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; xxxvija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; xxxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; xxxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10410; xxxixja CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10411; xl)a CDR1 comprising an amino acid sequence of SEQ. ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10412; xli)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10413; xlii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10414; xliiija CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10415; xilv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10416; xlv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10417; xlvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10418; xlvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10419; xlviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10420; xlix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10421; l)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10422; li)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10423; lii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10424; liii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10425; livja CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10426; lv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10427;
Ivija CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10428; lvii)a CDR1 comprising an amino add sequence of SEQ ID NO: 32, a CDR2 comprising an amino add sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino add sequence of SEQ ID NO: 34;
Ivii i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10429; lixja CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10430; lx)a CDR1 comprising an amino add sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10431; lxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10432; bcii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10433;
IxliiJCDRl comprising an amino acid sequence of SEQ iD NO: 32, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10811; lxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10435; lxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10436; lxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10437; lxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10438;
Ixviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10439; lxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10440; lxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10441; lxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10442; ixxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10443; ixxiiija CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10444; ixxivja CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10445; lxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10446; Ixxvija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10447;
Ixxviija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10448; lxxviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ !D NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10449;
Ixxixja CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10450; lxxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10451; lxxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10452;
Ixxxiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10453;
Ixxxiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10454; or lxxxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10455.
[0285]! n some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; b)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; c)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7099; d)a CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 7289; e)a CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 7289; f)a CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 7291; g)a CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 7291; h)a CDR1 comprising an amino acid sequence of SEQ iD NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10; i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093; j)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6643; k)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 10414; l) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10421; m) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10422; n) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10423; o) a CDR1 comprising art amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10429; p) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10811; q) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino add sequence of SEQ iD NO: 7096, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10374; r) a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino add sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10; or s) a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising art amino acid sequence of SEQ !D NO: 6634, and a CDR3 comprising an amino add sequence of SEQ ID NO: 7093. [0286]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 34, 7099, 10410-10433, 10435-10455, and 10811.
[0287]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 10414, 10421, 10422, 10423, 10429, and 10811.
[0288]in a related embodiment, included herein are anti-TNFR2 antigen-binding domainsje.g., antibodies such as single-domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within a VHH amino acid sequence as defined by any of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13. For example, provided herein are antibodies, or antigen-binding fragments thereof, comprising the set of CDR1-CDR2-CDR3 amino acid sequences contained within a VHH amino acid sequence selected from SEQ ID Nos: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, S640, 6644, 7090, 10380, 10381, 10383, 10385, 10386, 81-92, 6648, 6649, 6650, 7095, 7098, 7101, 7092, 93-640, 641-1127, 2805-3363, 3364-3922, 6651-6697, 6698-6744, 6931-6992, 7174-7197, 10375, 10382, 10384, 10394-10408, 10457-10502, 10720-10727, and 10812. [0289]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure can include a)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4; b)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7; c)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 11; d)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 15; e)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 19; f)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 23; g)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 27; h)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 31; i)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 35; j)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 39; k)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 43; l)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 47; m)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 6636; n)a variable domain that comprises a CDR1; CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 6640; o)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 6644; p)a variable domain that comprises a CDR1; CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7090; q)a variable domain that comprises a CORI, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7095; r)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7098; s)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7101; t)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 6650; u)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7293; v)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7294; w)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7295; x)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7296; y) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10375; z) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10382; aa) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10384; bb) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10380; cc) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10381; dd) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NG: 10383; ee) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10385; ff) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NG: 10386; gg) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10394; hh) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NG: 10395; ii) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10396; jj) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10397; kk) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NG: 10398;
II) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10399; mm) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10400; nn) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10401; oo) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10402; pp) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10403; qq) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10404; rr) a variable domain that comprises a CDR1,. CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10405; ss) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10406; tt) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10407; uu) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10408; w) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10457; ww) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10458; xx) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10459; yy) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10460; zz) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10461; aaa) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10462; bbb) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10463; ccc) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10464; ddd) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10465; eee) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10466; fff) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10467; ggg) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10468; hhh) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10469; iii) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10470; jjj) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10471; kkk) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10472;
III) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10473; mmm) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10474; nnn) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino add sequence of SEQ ID NO: 10475; ooo) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10476; ppp) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10477; qqq) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10478; nr) a variable domain that comprises a CDRl, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10479; sss) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10480; ttt) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10481; uuu) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10482; vw) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10483; www) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10484; xxx) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10485; yyy) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10486; zzz) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10487; aaaa) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10488; bbbb) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10489; cccc) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10490; dddd) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10491; eeee) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10492; ffff) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10493; gggg) 3 variable domain that comprises a CDR1; CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10494; hhhh) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10495; iiii) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10496; jjjj) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10497; kkkk) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10498;
Illi) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10499; mmmm) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10500; nnnn) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10501; oooo) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10502; pppp) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10720; qqqq) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10721; rrrr) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10722; ssss) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10723; tttt) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10724; uuuu) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10725; vwv) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10726; or wwww) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10727.
[0290]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure can include a)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 19; b)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 6644; c)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7095; d)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7098; e)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7101; f)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7293; g)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7294; h)a variable domain that comprises a CDR1; CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7295; i)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7296; j)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10375; k)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10382; l)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10384; m)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10380; n)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10381; o)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10383; p)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10385; or q)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10386; r)a variable domain that comprises a CDR1; CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10394; s)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10395; t)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10396; u)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10397; v)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10398; w)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10399; x)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10400; y)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10401; z)a variable domain that comprises a CDR1? CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10402; aa)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10403; bb)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10404; cc)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10405; dd)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10406; ee)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10407; ff)a variable domain that comprises a CDR1, CDR2? and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10408; ggja variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10457; hh)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10458; ii)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10459; jj)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10460; kk)a variable domain that comprises a CDR1, CDR2? and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10461; ll)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10462; mm)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10463; nn)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10464; oo)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10465; pp)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10466; qq)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10467; rr)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10468; ss)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10469; tt)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10470; uu)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NQ: 10471; vv)a variable domain that comprises a CORI, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10472; ww)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10473; xx)a variable domain that comprises a CDR1; CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10474; yy)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10475; zz)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10476; aaa)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10477; bbb)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10478; ccc)a variable domain that comprises a CDR1; CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10479; ddd)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10480; eee)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10481; fff)a variable domain that comprises a CDR1; CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10482; ggg)a variable domain that comprises a CDR1, CDR2? and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10483; hhh)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10484; iii)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10485; jjj)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10486; kkk)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NQ: 10487; lil)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10488; mmm)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10489; nnn)a variable domain that comprises a CDR1; CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10490; oooja variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10491; ppp)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10492; qqq)a variable domain that comprises a CDR1; CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10493; rrr)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10494; sss)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10495; ttt)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10496; utiuja variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10497; vw)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10498; www)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10499; xxx)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino add sequence of SEQ ID NO: 10500; yyy)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10501; m)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10502; aaaa)a variable domain that comprises a CDR1, COR2,. and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10720; bbbb)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10721; cccc)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10722; dddd)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10723; eeee)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10724; ffff)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10725; gggg)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10726; hhhh)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10727; or iiii)a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10812.
[0291]ln an embodiment provided herein, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure can include a VHH amino acid sequence selected from SEQ ID Nos: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 93-640, 2805-3363, 6651-6697, 6931-6992, 7174-7197, 7222-7254, 10380, 10381, 10383, 10385, and 10386, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0292]ln an embodiment provided herein, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure can Include a VHH amino acid sequence selected from SEQ ID Nos: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 7222-7254, 10380, 10381, 10383, 10385, and 10386, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0293]ln an embodiment provided herein, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID Nos: 81-92, 6648, 6649, 6650, 7092, 7095, 7098, 7101, 7293-7296, 641-1127, 6698-6744, 10375, 10382, 10384, 10394-10408, 10457-10502, 10720-10727, 10812, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0294]ln an embodiment provided herein, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID Nos: 10461, 10468, 10469, 10470, 10477, and 10812, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0295]! n an embodiment provided herein, an anti-TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure can include a VHH amino acid sequence selected from SEQ ID NOs: 19, 6644, 6650, 7095, 7098, 7101, 7293-7296, 7222-7254, 10375, 10382, 10384, 10380, 10381, 10383, 10385, and 10386, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0296]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ, ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 6643. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 6650 or 10720. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino add sequence of SEQ ID NO: 6643. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) comprises the amino add sequence of SEQ ID NO: 6650 or 10720, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0297]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some
Ill embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289. In some embodiments, an anti~TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10394. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10394, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0298]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 7294. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 7294, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0299]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289. In some embodiments, an anti~TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10395. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10395, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0300]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10396. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10396, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0301]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289. In some embodiments, an anti~TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10397. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 7289. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10397, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0302]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10398. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10398, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0303]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10399. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10399, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0304]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10400. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10400, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0305]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10401. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10401, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0306]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10391. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10402. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10391, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10402, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0307]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10392. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10403. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10392, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10403, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0308]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10393. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10404. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10393, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10404, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0309]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 18. in some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10405. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10405, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0310]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 18. in some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10406. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10406, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0311]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 18. in some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10407. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10407, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0312]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10391. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10408. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10391, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10408, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0313]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10410. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10457. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10410. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10457, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0314]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10411. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10458. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10411. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10458, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0315]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10412. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10459. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10412. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10459, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0316]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10413. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10460. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10413. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10460, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0317]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10414. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10461. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10414. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10461, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0318]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10415. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10462. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10415. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10462, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0319]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10416. In some embodiments, an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10463. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10416. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10463, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0320]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10417. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10464. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10417. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10464, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0321pn some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10418. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10465. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10418. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10465, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0322]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10419. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10466. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10419. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10466, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0323]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10420. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10467 or 10721, In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10420. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10467 or 10721, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0324]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10421. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10468 or 10722. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10421. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10468 or 10722, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0325]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10422. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10469 or 10723. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10422. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10469 or 10723, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0326]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10423. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10470 or 10724. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10423. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10470 or 10724, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0327]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10424. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10471 or 10725. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10424. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10471 or 10725, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0323]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10425. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10472. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10425. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10472, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0329]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10426. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10473. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10426. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10473, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0330]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10427. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10474 or 10726. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10427. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10474 or 10726, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0331]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10428. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10475. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10428. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10475, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0332]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 34. in some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10476. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10476, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0333]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10429. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10477 or 10727. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10429. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10477 or 10727, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0334]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10430. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10478. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10430. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10478, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0335]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10431. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10479. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10431. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10479, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0336]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10432. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10480. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10432. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10480, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0337]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10433. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10481. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10433. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10481, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0338]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 32. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10811. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10812. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10811. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10812, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0339]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10435. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10482. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10435. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10482, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0340]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10436. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10483. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10436. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10483, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0341]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7099. In some embodiments, an anti~TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 7101. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 7099. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 7101, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0342]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10437. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10484. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10437. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10484, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0343]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10438. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10485. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10438. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10485, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0344]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10439. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10486. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10439. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10486, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0345]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10440. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10487. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10440. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10487, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0346]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10441. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10488. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10441. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10488, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0347]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10442. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10489. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10442. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10489, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0348]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10443. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10490. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10443. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10490, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0349]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10444. In some embodiments, an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10491. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDRS comprising an amino acid sequence of SEQ ID NO: 10444. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10491, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0350]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10445. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10492. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10445. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10492, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0351]ln some embodiments, an anti-TNFR2 antigen- domain protein (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10446. In some embodiments, an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10493. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10446. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10493, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0352]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10447. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10494. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10447. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10494, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0353]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10448. In some embodiments, an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10495. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10448. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10495, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0354]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10449. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10496. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10449. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10496, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0355]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10450. In some embodiments, an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10497. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10450. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10497, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0356]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10451. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10498. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10451. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10498, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0357]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10452. In some embodiments, an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10499. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10452. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10499, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0358]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10453. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10500. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10453. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10500, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0359]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an antl-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10454. In some embodiments, an antl-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10501. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10454. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10501, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0360]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10455. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10502. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10455. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ, ID NO: 10502, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0361]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-TNFR2 antigembinding domain (e,g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10375. In some embodiments, an anti- TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO 10374. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10375, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0362]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 8. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 9. In some embodiments, an anti- TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 7095 or 10380. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a CDRS comprising an amino acid sequence of SEQ ID NO: 7093. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 7095 or 10380, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0363]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singie-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: S634. in some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10. in some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 10381 or 10382, In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10381 or 10382, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0364]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 10383 or 10384. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 10383 or 10384, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0365]ln some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7281. In some embodiments, an antl-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289. In some embodiments, an anti~TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ !D NO: 7293 or 10385. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7281, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 7289. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 7293 or 10385, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0366]ln some embodiments, an anti~TNFR2 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7283. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR2 comprising an amino acid sequence of SEQ ID NO: 7287. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 7291. In some embodiments, an anti-TNFR2 antigenbinding domain (e.g., antibody such as single-domain antibody) described herein comprises a set of three CDRs (I.e., CDR1-CDR2-CDR3) contained within an anti-TNFR2 VHH antibody comprising the amino acid sequence of SEQ ID NO: 7295 or 10386. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises a CORI comprising an amino acid sequence of SEQ ID NO: 7283, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7287, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7291. In some embodiments, an anti-TNFR2 antigen-binding domain (e.g., antibody such as single-domain antibody) comprises the amino acid sequence of SEQ ID NO: 7295 or 10386, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0367)1 n some embodiments, the present disclosure also includes an anti~TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) that competes for binding to TNFR2 with any one of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13.
[0368]! n some embodiments, the present disclosure also includes an anti-TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) that binds to the same epitope on TNFR2 as any one of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13.
[0369]ln some embodiments, the present disclosure also includes an anti-TNFR2 antigen-binding domain (e.g., antibody such as a single-domain antibody) that comprises a means for binding an epitope within human TNFR2 bound by any one of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13.
Anti-CD25 Antigen-binding Domnins
[0370]The present disclosure provides one or more anti-CD25 antigen-binding domains (e.g., antibodies, such as single-domain antibodies) that bind to CD25.
[0371]Ciuster of differentiation 25 (CD25), also called interleukin-2 receptor subunit alpha (IL-2Ra or IL2RA) is the alpha chain component of the heterotrimeric interleukin-2 receptor complex. IL-2Ro is a single pass type-1 transmembrane protein with a total length of 251 amino acids. The receptor subunit consists of two sushi or elbow domains that are connected via an unordered loop region (Wang et al., Science 310, 1159--1163. 2005). The C-terminal domain of the protein is a long, disordered region that is needed to allow CD25 forming a cap like structure in the IL-2 receptor complex but still being anchored in the membrane. The actual structure and positioning of the loop has not been resolved in any of the available crystal structures. The sushi domains of CD25 form five stranded beta sheet sandwiches that are related to each other in a pseudo-2-fold symmetry. Sushi domain 1 accounts for most of the interactions with IL-2 (82%) while Sushi domain 2 contributes significantly less (Stauber et al., Proc Natl Acad Sci U S A 103, 2788-2793. 2006). The structure of CD25 is stabilized by several intradomain and two interdomain disulfide bonds, in addition, CD25 carries several glycans with one N-glycosylation located at the C-terminus of Sushi domain 2 and four O-glycans located in the C-terminal unordered region.
[0372JCD25 interacts with IL-2 in a tight manner. It is postulated that the IL-2 receptor complex forms in a stepwise manner starting with IL-2 binding to CD25/IL-2Ro, then engaging subunit p and finally interacting with the y receptor subunit (Stauber et al., Proc Natl Acad Sci U S A 103, 2788-2793. 2006). Interestingly, it has been reported that CD25 can present IL-2 in cis and in trans (Liao et aL, Immunity 38, 13-25. 2013; Wuest et aL, Nat Med 17, 604-609. 2011), both resulting in IL-2 receptor complex assembly. The CD25/IL-2Ra has the largest interface with 11-2 within the complex, which is reflected in the very high affinity between IL-2 and CD25 (Liao et al., 2013). However, in the complex itself CD25/IL- 2Ra makes no direct contact with the other two subunits P or y. Deglycosylation experiments of the individual subunits were found to impact the complex formation with the y subunit aggregating, while the subunits a and P were still able to bind to IL-2 (Stauber et al., Proc Natl Acad Sci U S A 103, 2788- 2793. 2006). Hence, the glycosylation on CD25/IL-2Ra is not essential for the interaction with IL-2. IL-2- Ry and I L-2Rf are also part of other interleukin receptor complexes while CD25/IL-2Ra is exclusively found in the IL-2 receptor complex (Liao et aL, Immunity 38, 13-25. 2013).
[0373)6esides the membrane anchored version of CD25, it has been reported that soluble CD25 can be found in the human serum (Pedersen and Lauritsen, Scand J Immunol 70, 40-43. 2009). This soluble form of CD25 can result from a shedding event of the membrane anchored protein, producing a truncated CD25 with a molecular weight (MW) of ~20kDa.
[0374)1 n some embodiments, antigen-binding domains (e.g., antibodies, such as single-domain antibodies) described herein bind to human CD25. In some embodiments, the human CD25 protein is encoded by the human interleukin-2 receptor subunit alpha (IL2RA) gene (NCBI Gene ID: 3559) and has the amino acid sequence of MDSYLLMWGLLTFIMVPGCQAELCDDDPPEiPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWD NQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFWGQMVYYQC VQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAAT METSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI (UniProtKB Accession No. P01589) (SEQ ID NO:
6310)
[0375)1 n some embodiments, antigen-binding domains (e.g., antibodies, such as single-domain antibodies) described herein bind to cynomolgus monkey ("cyno") CD25. In some embodiments, the cyno CD25 protein is encoded by the cyno interleukin-2 receptor subunit alpha (IL2RA) gene (NCBI Gene ID: 102123605) and has the amino acid sequence of MDPYLLMWGLLTFITVPGCQAELCDDDPPKITHATFKAVAYKEGTMLNCECKRGFRRIKSGSPYMLCTGNSSHSSWDN QCQCTSSAARNTTKQVTPQPEEQKERKTTEMQSQMQLADQVSLPGHCREPPPWENEATERIYHFVVGQTVYYQCVQ GYRALHRGPAESICKMTHGKTRWTQPQLICTGETEPSQFPGEEEPQASPDGLPESETSRLVTTTDFRIQTEVAATMETFI FTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKNRRTI (GenBank Accession No. EHH64536.1) (SEQ ID NO:
6311)
[0376)1 n some embodiments, antigen-binding domains (e.g., antibodies, such as single-domain antibodies) described herein bind to mouse CD25. In some embodiments, the mouse CD25 protein is encoded by the mouse interleukin 2 receptor subunit alpha (Il2ra) gene (NCBI Gene ID: 16184) and has the amino acid sequence of MEPRLLMLGFLSLTIVPSCRAELCLYDPPEVPNATFKALSYKNGTILNCECKRGFRRLKELVYMRCLGNSWSSNCQCTSN SHDKSRKQVTAQLEHQKEQQTTTDMQKPTQSMHQENLTGHCREPPPWKHEDSKRIYHFVEGQSVHYECIPGYKALQ RG PAISI CKM KCG KTG WTQPQLTCVDEREH H RFLASEESQGSRNSSPESETSCPITTTDFPQPTETTAMTETFVLTM EYK VAVASCLFLLISILLLSGLTWQHRWRKSRRTI (UniProtKB Accession No. P01530) (SEQ ID NO: 6312)
[0377]ln some embodiments, the one or more anti-CD25 antigen-binding domains of the present disclosure comprise IL-2 or a variant thereof.
[0378]ln some embodiments, anti-CD25 antigen-binding domains of the present disclosure upon binding to CD25 do not impair the binding of its cognate ligand, interleukin-2 (IL-2), to CD25. In some embodiments, anti-CD25 antigen-binding domains of the present disclosure do not have overlapping epitopes with IL-2.
[0379]ln some embodiments, anti-CD25 antigen-binding domains of the present disclosure upon binding to CD25 may impair the binding of IL-2 to CD25. In some embodiments, anti-CD25 antigen-binding domains of the present disclosure may have overlapping epitopes with IL-2. In some embodiments, when the anti-CD25 antigen-binding domains have overlapping epitopes with IL-2, the anti-CD25 antigen-binding domains may impair IL-2 binding to CD25. In some embodiments, when the anti-CD25 antigen-binding domains have overlapping epitopes with IL-2, the anti-CD25 antigen-binding domains may compete for binding to CD25 with IL-2.
[0380]ln various embodiments, anti~CD25 antigen-binding domains of the present disclosure may have an antagonistic effect (e.g., a blocking effect) upon binding to CD25. An antagonistic CD25 binder can block or decrease activation of CD25 and/or attenuate one or more signal transduction pathways mediated by CD25. Antagonistic CD25 binders may block or decrease CD2S activation by binding CD25, e.g., to induce a conformational change that renders the receptor biologically inactive. For example, antagonistic CD25 binders may prevent the trimerization of an IL-2 receptor complex as can occur due to the interaction between CD25 and its cognate ligand, IL-2, thus impairing CD25-mediated signaling. [0381]ln some embodiments, when the anti-CD25 antigen-binding domains of the present disclosure have overlapping epitopes with IL-2, such anti-CD25 antigen-binding domains may have an antagonistic effect upon binding to CD25.
[0382]ln various embodiments, anti-CD25 antigen-binding domains of the present disclosure may have an agonistic effect (e.g., a stimulatory effect) upon binding to CD25. An agonistic CD25 binder can stimulate or enhance activation of CD25 and/or strengthen one or more signal transduction pathways mediated by CD25. Agonistic CD25 binders may stimulate or enhance CD25 activation by binding CD25, e.g., to induce a conformational change that renders the receptor biologically active. For example, agonistic CD25 binders may promote the trimerization of an IL-2 receptor complex as can occur due to the interaction between CD25 and its cognate ligand, IL-2, thus promoting CD25-mediated signaling. [0383]in some embodiments, when the anti-CD25 antigen-binding domains of the present disclosure have overlapping epitopes with IL-2, such anti~CD25 antigen-binding domains may have an agonistic effect upon binding to CD25.
[0384]ln some embodiments, anti-CD25 antigen-binding domains of the present disclosure bind to human CD25. in some embodiments, anti-CD25 antigen-binding domains (e.g., antibodies such as singledomain antibodies) of the present disclosure may bind to human CD25 with a Ko of less than about IxlO"6 M, for example, less than about 5xlO~7 M, less than about 3xl0"7 M, less than about IxlO"7 M, less than about 3xl0"8 M, less than about 5xl0"8 M, less than about 3xl0"8 M, less than about IxlO"8 M, less than about 8xl0"9 M, less than about 5xl0“9 M, less than about 3xl0~9 M, less than about IxlO"9 M, about IxlO"10 to IxlO"9 M, about lxlQ~w to 5xl0"9 M, about IxlO"10 to IxlO'8 M, about IxlO"10 to 5xl0"8 M, about IxlO"9 to 1x10"* M, about IxlO"9 to 5x10"* M, about IxlO"9 to IxlO"7 M, or about IxlO"8 to lxlO"7 M.
[0385]ln one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 1.6 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 7.6 nM. In one embodiment, an anti- CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KB of about 9.4 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 10 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 11 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 12 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 13 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 14 nM. In one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KB of about 17 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about IS nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 19 nM. In one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KB of about 20 nM. In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 21 nM. In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 22 nM. In one embodiment an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KD of about 26 nM. in one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 31 nM. In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 35 nM. in one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KD of about 49 nM. In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 50 nM. In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 58 nM. In one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a of about 61 nM. In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 62 nM, In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 66 nM. In one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KD of about 73 nM. In one embodiment an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 76 nM, In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a Ko of about 97 nM. In one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KD of about 102 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 107 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 149 nM. In one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to human CD25 with a KD of about 241 nM. in one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to human CD25 with a KD of about 348 nM.
[0386]!n some embodiments, anti-CD25 antigen-binding domains of the present disclosure bind to cynomolgus monkey ("cyno") CD25. In some embodiments, anti-CD25 antigen-binding domains (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno CD25 with a KD of less than about IxlO-6 M, for example, less than about 5xlO-7 M, less than about 3xl£T7 M, less than about IxlO”7 M, less than about 8xl0”8 M, less than about 5xl0”8 M, less than about 3xl0’8 M, less than about IxlO"8 M, less than about 8xl0”9 M, less than about 5xl0~9 M, less than about 3xl0~9 M, less than about IxlO’9 M, about IxlO’10 to IxlO”3 M, about IxlO”10 to 5xl0”s M, about IxlO”10 to IxlO”8 M, about IxlO”10 to 5xl0”s M, about IxlO’9 to IxlO’8 M, about IxlO”9 to 5xl0’8 M, about IxlO’9 to IxlO’7 M, about IxlO’9 to 2xl0’7 M, about IxlO”9 to 5xl0”7 M, about IxlO”8 to IxlO’7 M, about IxlO”8 to 2xl0”7 M, about IxlO”8 to 5xl0”7 M, or about IxlO”8 to IxlO”6 M.
[0387]in one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 706 pM, in one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 793 pM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 1.5 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 73 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 34 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 48 nM. In one embodiment, an anti- CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 49 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 52 nM, In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 57 nM. in one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to cyno CD25 with a KD of about 70 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 79 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 97 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ka of about 107 nM. In one embodiment, an anti- CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 112 nM. in one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KB of about 115 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 117 nM. In one embodiment, an anti-CD25 antigenbinding domain of the present disclosure binds to cyno CD25 with a KD of about 119 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 121 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ka of about 131 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 136 nM. in one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KB of about 142 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 146 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 148 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 149 nM, In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 162 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KB of about 163 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 186 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 191 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 211 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 235 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KES of about 283 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 339 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a Ko of about 380 nM, In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KD of about 411 nM. In one embodiment, an anti-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KES of about 956 nM. In one embodiment, an antl-CD25 antigen-binding domain of the present disclosure binds to cyno CD25 with a KB of about 2.1 pM.
[0388]ln some embodiments, anti-CD25 antigen-binding domains of the present disclosure bind to mouse CD25. In some embodiments, anti-CD25 antigen-binding domains of the present disclosure may bind to mouse CD25 with a KD of less than about IxlO”6 M, for example, less than about 5xl0"7 M, less than about 3xl0’7 M, less than about IxlO’7 M, less than about 8x10 8 M, less than about 5xlO’’8 M, less than about 3xl0’8 M, less than about IxlO’8 M, less than about 8xl0’3 M, less than about 5xl0’s M, less than about 3xl0"9 M, less than about IxlO"9 M, about IxlO"10 to IxlO’3 M, about IxlO"10 to 5xl0’9 M, about IxlO’10 to IxlO"8 M, about 1x10 ”10 to 5xl0"8 M, about IxlO"9 to IxlO"8 M, about IxlO"9 to 5xl0’8 M, about IxlO’3 to IxlO”7 M, about IxlO’3 to 2xl0"7 M, about IxlO’9 to 5x10 ”7 M, about IxlO’8 to IxlO’7 M, about IxlO’8 to 2xl0’7 M, about IxlO’8 to 5x10 ”7 M, or about IxlO"8 to IxlO’6 M. In some embodiments, anti-CD25 antigen-binding domains of the present disclosure do not bind to mouse CD25. [0389]ln one embodiment, an anti-CD25 antigen-binding domain of the present disclosure bind to mouse CD25 with a KQ of about 420 nM. [0390]ln some embodiments, anti-CD25 antigen-binding domains of the present disclosure may bind to CD25 with an EC50 from about 1 - 10 nM, about 10 - 100 nM, about 100 nM - 1 pM, or above 1 pM. in some embodiments, anti- CD25 antigen-binding domains of the present disclosure may bind to CD25 with an ECsofrom about 10 - 100 nM or about 100 nM - 1 pM. In some embodiments, anti- CD25 antigen-binding domains of the present disclosure may bind to CD25 with an EC® of about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, about 100 nM, about 125 nM, about 150 nM, about 175 nM, about 200 nM, about 225 nM, about 250 nM, about 275 nM, about 300 nM, about 325 nM, about 350 nM, about 375 nM, about 400 nM, about 425 nM, about 450 nM, about 475 nM, about 500 nM, about 525 nM, about 550 nM, about 575 nM, about 600 nM, about 625 nM, about 650 nM, about 675 nM, about 700 nM, about 725 nM, about 750 nM, about 775 nM, about 800 nM, about 825 nM, about 850 nM, about 875 nM, about 900 nM, about 925 nM, about 950 nM, about 975 nM, or about 1 pM.
[0391]Sequence identifiers corresponding to exemplary anti-CD25 VHH antibodies provided herein are listed in Table 1-3. Table 1-3 sets forth the sequence identifiers of amino acid sequences of the complementarity determining regions (CDR1, CDR2 and CDR3), amino acid and DNA sequences of the full-length camelid anti-CD25 VHH antibodies, as well as amino acid sequences of corresponding humanized anti-CD25 VHH antibodies. Amino acid sequences of additional exemplary anti-CD25 VHH antibodies and corresponding humanized anti-CD25 VHH antibodies are provided in Table 1-4. In the present disclosure, "Hui" indicates a humanized variant of the parental VHH antibody. "18", "A8", or "18" indicate that the 8tt; position in the CDR3 of the parental VHH antibody has been mutated to the amino acid residue isoleucine, alanine, or ieucine, respectively.
Table 1-3, Sequence identifiers for exemplary anti-CMS VHH antibodies
Table 1-4. Sequence identifiers for additional exemplary anti-CD25 VHH antibodies and humanized anti-CMS VHH antibodies
[0392]in some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a single- domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position, and indicates an amino acid residue is absent at the particular position): a). GR(K/R/S)FSTLI (SEQ ID NO: 4137); b). GFTFS(N/S)YA (SEQ ID NO: 4140); and c), GRTF(A/S)(S/W/D)(F/N/Y)G (SEQ ID NO: 10309); d). GFTLDYYA (SEQ ID NO: 7342); and e). G(I/M)P(F/-)(A/-)L(P/V/Y)A (SEQ ID NO: 7366).
[0393]ln some embodiments, an anti-CD25 antigen-binding domain described herein comprises a CDR1 comprising the sequence GFTFS(N/S)YA (SEQ ID NO: 4140) or GFTLDYYA (SEQ ID NO: 7342).
[0394]ln some embodiments, an anti-CD25 antigen-binding domain described herein comprises a CDR1 comprising the sequence GFTFS(N/S)YA (SEQ ID NO: 4140).
[0395]in some embodiments, an anti-CD25 antigen-binding domain described herein comprises a CDR1 comprising the sequence GFTLDYYA (SEQ iD NO: 7342),
[0396]ln some embodiments, when an anti-CD25 antigen-binding domain described herein may comprise a CDR1 comprising the sequence GR(K/R/S)FSTLI (SEQ iD NO: 4137), the CDR1 may comprise, e.g., the sequence GR(S/K)FSTLI (SEQ ID NO: 4132).
[0397]ln some embodiments, when an anti-CD25 antigen-binding domain described herein may comprise a CDR1 comprising the sequence GR(K/R/S)FSTL! (SEQ iD NO: 4137), the CDR1 may comprise, e.g., the sequence GRSFSTLI (SEQ ID NO: 4105).
[0398]ln some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position, and indicates an amino acid residue is absent at the particular position): a). GRSFSTLI (SEQ ID NO: 4105); b). GR(S/K)FSTLI (SEQ ID NO: 4132); d). GFTFS(N/S)YA (SEQ ID NO: 4140); e). GRTFS(S/W)(F/N/Y)G (SEQ ID NO: 4142); f). GFTLDYYA (SEQ ID NO: 7342); and g). G(I/M)P(F/-)(A/-)L(P/V/Y)A (SEQ ID NO: 7366).
[0399]! n some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR2 comprising an amino acid sequence selected from: a). (l/V)(D/E)R(D/G)(D/G)T(A/P/T); b). IYSD(G/S)SGT (SEQ ID NO: 9441); c). IS(Q/R/G)(S/G)GGRT (SEQ ID NO: 10310); d). IS(R/S)(D/S)G(D/G)ST (SEQ ID NO: 7364); e). ISSGGNT (SEQ ID NO: 7346); and f). ISSTDGRT (SEQ ID NO: 7348). [0400]ln some embodiments, an anti-CD25 antigen-binding domain described herein comprises a CDR2 comprising the sequence IYSD(G/S)SGT (SEQ iD NO: 9441) or iSSTDGRT (SEQ iD NO: 7348).
[0401]ln some embodiments, an anti-CD25 antigen-binding domain described herein comprises a CDR2 comprising the sequence IYSD(G/S)SGT (SEQ ID NO: 9441).
[0402]ln some embodiments, an anti-CD25 antigen-binding domain described herein comprises a CDR2 comprising the sequence ISSTDGRT (SEQ ID NO: 7348).
(0403]ln some embodiments, when an anti-CD25 antigen-binding domain described herein may comprise a CDR2 comprising the sequence (i/V)(D/E)R(D/G)(D/G)T(A/P/T), the CDR2 may comprise, e.g., the sequence (i/V)(D/E)R(D/G)GT(A/P/T).
[0404]ln some embodiments, when an anti-CD25 antigen-binding domain described herein may comprise a CDR2 comprising the sequence (l/V)(D/E)R(D/G)(D/G)T(A/P/T), the CDR2 may comprise, e.g., the sequence l(D/E)RDGT(T/P) (SEQ ID NO: 4135).
[0405]ln some embodiments, when an anti~CD25 antigen-binding domain described herein may comprise a CDR2 comprising the sequence (l/V)(D/E)R(D/G)(D/G)T(A/P/T), the CDR2 may comprise, e.g., the sequence l(D/E)R(D/G)(D/G)T(P/T).
[0406]!n some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR2 comprising an amino acid sequence selected from: a). (I/V)(D/E)R(D/G)GT(A/P/T); b). I(D/E)RDGT(T/P) (SEQ ID NO: 4135); c). I(D/E)R(D/G)(D/G)T(P/T); d). IYSDGSGT (SEQ ID NO: 4114); e). ISQSGGRT (SEQ ID NO: 4118); f). IS(R/S)(D/S)G(D/G)ST (SEQ ID NO: 7364); g). ISSGGNT (SEQ iD NO: 7346); and h). ISSTDGRT (SEQ ID NO: 7348).
[0407]! n some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR3 comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) a). NAL(G/L/P/Q/W)Y (SEQ ID NO: 4131); b). NALR(D/H/N/F) (SEQ ID NO: 4134); c). (K/S/T)TLRY (SEQ ID NO: 4136); d). (A/V/S)(K/T)G(R/A/K)(G/H/N/R)SG(S/G)YYP(W/F/L)(D/E)(D/E)(Y/V) (SEQ ID NO: 10219); and e). AA(S/T)(D/N/Y/K)(F/V)(L/P)(I/L)A(T/I/A)(T/S/A)IS(A/G)(Y/H)DY (SEQ ID NO: 10308); f). AAYVYPDYYCS(D/E)YVLL(K/R)YDY (SEQ ID NO: 7363); g). NIYR(P/S)QVP(P/S/T)TRYS (SEQ ID NO: 7365); and h). AAKRLGP(M/I/A/L)VH(Q/R)YSLEVLTPLFLDEYDY (SEQ ID NO: 9423), wherein one or more non-alanine residues in the CDR3 sequence are optionally replaced with an alanine, and/or one or more alanine residues in the CDR3 sequence are optionally replaced with a glycine.
[0408]ln certain embodiments, when an anti-CD25 antigen-binding domain described herein may comprise a CDR3 comprising an amino acid sequence wherein one or more non-alanine residues in the CDR3 sequence are replaced with an alanine, and/or one or more alanine residues in the CDR3 sequence are replaced with a glycine, the CDR3 may comprise an amino acid sequence selected from a).(A/V/S)(A/K/T)(A/G)(A/R/K)(A/G/H/N/R)(A/S)(A/G)(A/S/G)(A/Y)(A/Y)(A/P)(A/W/F/L)(A/D/E)(A /D/E)(A/Y/V); and b). (A/G)(A/G)(A/K)(A/R)(A/L)(A/G)(A/P)(M/I/A/L)(A/V)(A/H)(A/R/Q)(A/Y)(A/S)(A/L)(A/E)(A/V) (A/L)(A/T)(A/P)(A/L)(A/F)(A/L)(A/D)(A/E)(A/Y)(A/D)(A/Y).
[0409]ln some embodiments, an anti-CD25 antigen-binding domain described herein comprises a CDR3 comprising the sequence
(A/V/S)(A/K/T)(A/G)(A/R/K)(A/G/H/N/R)(A/S)(A/G)(A/S/G)(A/Y)(A/Y)(A/P)(A/W/F/L)(A/D/E)(A/D/E)(A/Y/ V).
[0410]ln some embodiments, an anti-CD25 antigen-binding domain described herein comprises a CDR3 comprising the sequence
(A/G)(A/G)(A/K)(A/R)(A/L)(A/G)(A/P)(M/I/A/L)(A/V)(A/H)(A/R/Q)(A/Y)(A/S)(A/L)(A/E)(A/V) (A/L)(A/T)(A/P)(A/L)(A/F)(A/L)(A/D)(A/E)(A/Y)(A/D)(A/Y).
[0411]ln some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a CDR3 comprising an amino acid sequence selected from: a). NAL(G/L/P/Q/W)Y (SEQ ID NO: 4131); b). NALR(D/H/N/F) (SEQ ID NO: 4134); c). (K/S/T)TLRY (SEQ ID NO: 4136); d). AKGR(H/N)SGSYYPWD(D/E)Y (SEQ ID NO: 4139); e). (A/V)KGR(G/H/N)SGSYYP(W/F)D(D/E)Y (SEQ ID NO: 9440); f). AA(S/T)(D/N/Y)FL(I/L)ATTIS(A/G)YDY (SEQ ID NO: 4141); g). AAYVYPDYYCS(D/E)YVLL(K/R)YDY (SEQ ID NO: 7363); h). NIYR(P/S)QVP(P/S/T)TRYS (SEQ ID NO: 7365); and i). AAKRLGPMVH(Q/R)YSLEVLTPLFLDEYDY (SEQ ID NO: 7367), wherein one or more non-alanine residues in the CDR3 sequence are optionally replaced with an alanine, and/or one or more alanine residues in the CDR3 sequence are optionally replaced with a glycine.
[0412]ln some embodiments, anti-CD25 antigen-binding domains (e.g., antibodies such as single-domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) comprising amino acid sequences selected from any of the above-described CORI, CDR2, and CDR3 amino acid sequences. In certain embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino add sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino add sequence of SEQ ID NO:4131; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino add sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino add sequence of SEQ ID NO: 4136; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NG: 4139; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4142, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4141; vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7364, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7363; vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7366, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7346, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7365; viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO:
7367; ix) a CORI comprising an amino acid sequence of SEQ, ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9441, and a CDR3 comprising an amino acid sequence of (A/V/S)(A/K/T)(A/G)(A/R/K)(A/G/H/N/R)(A/S)(A/G)(A/S/G)(A/Y)(A/Y)(A/P)(A/W/F/L)(A/D/E)(A/D/E)(A/Y/ V); or x). a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of (A/G)(A/G){A/K)(A/R)(A/L)(A/G)(A/P)(M/I/A/L)(A/V)(A/H)(A/R/Q)(A/Y)(A/S)(A/L){A/E)(A/V)(A/L)(A/T)(A/P )(A/L)(A/F)(A/L)(A/D)(A/E)(A/Y){A/D){A/Y).
[0413]ln certain embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ iD NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)( D/E) R(D/G)GT(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4135, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of i(D/E)R(D/G)(D/G)T(P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4136; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4139; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4142, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4141; vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7364, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7363; vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7366, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7346, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7365; viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7367; ix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9441, and a CDR3 comprising an amino acid sequence of (A/V/S)(A/K/T)(A/G)(A/R/K)(A/G/H/N/RHA/S){A/G)(A/S/G)(A/Y)(A/Y)(A/P)(A/W/F/L)(A/D/E)(A/D/E)(A/Y/ V); or
>:), a CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of (A/G)(A/G)(A/K){A/R)(A/L)(A/G)(A/P)(M/I/A/L)(A/V)(A/H){A/R/Q)(A/Y)(A/S)(A/L)(A/E)(A/V)(A/L)(A/T)(A/P )(A/L)(A/F)(A/L)(A/D){A/E){A/Y)(A/D)(A/Y).
[0414]!n certain embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ iD NO: 4132, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ iD NO: 4131; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E]R(D/G){D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ iD NO: 4136; iv) a CDR1 comprising an amino acid sequence of SEQ iD NO: 4140, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 4139; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4142, a CDR2 comprising an amino acid sequence of SEQ !D NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NG: 4141; vi) a CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ !D NO: 7364, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 7363; vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7366, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7346, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7365; or viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7367. [0415)1 n certain embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9411; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9412; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9413; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9414; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9415; or vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9416. [0416]ln certain embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V){D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4136; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino add sequence of (!/V)(D/E)R(D/G)GT(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4135, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of l(D/E)R(D/G)(D/G)T(P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO:
4136; vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4132, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of (IA’,)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; ix) a CDR1 comprising an amino acid sequence of SEQ iD NO: 4132, a CDR2 comprising an amino acid sequence of (l/V)( D/E) R(D/G)GT(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; x) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4135, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; xi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9441, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10219; xii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9441, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9440; xiii) a CORI comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4139; xiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 10309, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10310, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10308; xv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4142, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4141; xvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7364, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7363; xvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7366, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7346, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7365; xviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO:
9423; xix) a CDR1 comprising an amino acid sequence of SEQ !D NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ iD NO: 7367; xx) a CDR1 comprising an amino acid sequence of SEQ iD NO: 4140, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9441, and a CDR3 comprising an amino acid sequence of
(A/V/S)(A/K/T)(A/G)(A/R/K)(A/G/H/N/R)(A/S)(A/G)(A/S/G)(A/Y)(A/Y)(A/P)(A/W/F/L)(WE)(A/D/E)(A/Y/ V); or xxi). a CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of (A/G)(A/G)(A/K)(A/R)(A/L)(A/G)(A/P)(M/I/A/L)(A/V)(A/H)(A/R/Q)(A/¥)(A/S)(A/L)(A/E)(A/V)(A/L)(A/T)(A/P )(A/L)(A/F)(A/L)(A/D)(A/E)(A/Y)(A/D)(A/¥),
[0417]ln certain embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10504; ii)a CDR1 comprising an amino add sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10505; iii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10506; iv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ !D NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10507; v)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10508; vi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10509; vii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10510; viii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10511; ix)a CDR1 comprising an amino add sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10512; x)a CORI comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10513; xi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 10514; xiija CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10515; xiii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10516; xiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10517; xv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10518; xvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10519; xvii)a CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10520; xviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10521; xix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10522; xx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10523; xxi)a CDR1 comprising an amino add sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10524; xxii)a CDR1 comprising an amino add sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NQ: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10525; xxiiija CDR1 comprising an amino add sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO:
9414; xxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10526; xxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10527; xxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10528; xxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10529; xxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10530; xxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10531; xxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10532; xxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10533; xxxiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10534; xxxm)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO:
10535; xxxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10536; xxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10537; xxxvija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10538; xxxvii)a CDR1 comprising an amino acid sequence of SEQ, ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10539; xxxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ, ID NO: 10540; xxxixja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10541; xl)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10542; xli)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10543; or xlii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10544. [041S]ln some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)GT(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO:
4131; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4135, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4142, a CDR2 comprising an amino acid sequence of i(D/E)R(D/G)(D/G)T(P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4136; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4132, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4139; or v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4141. [0419]Provided herein are anti-CD25 antigen-binding domains (e.g., antibodies such as single-domain antibodies) comprising a CDR1 comprising an amino acid sequence selected from any of the CDR1 amino acid sequences listed in Table 1-3, Table 9, Table 10, or Table 12-1, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0420]ln some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence selected from SEQ ID Nos: 4101, 4105, 4109, 4113, 4117, 4726-5030, 7342, 7345, 7931-8226, and 9660-9770, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0421]ln some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR1 comprising an amino acid sequence selected from SEQ ID Nos:
4101, 4105, 4109, 4113, 4117, 4132, 4142, 4905, 4909, 4918, 7342, and 7345, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0422]Provided herein are anti-CD25 antigen-binding domains (e.g., antibodies such as single-domain antibodies) comprising a CDR2 comprising an amino acid sequence selected from any of the CDR2 amino acid sequences listed in Table 1-3, Table 9, Table 10, or Table 12-1, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0423]ln some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR2 comprising an amino acid sequence selected from SEQ ID Nos:
4102, 4106, 4110, 4114, 4118, 5031-5335, 7343, 7346, 7348, 8227-8522, 9435, and 9771-9880, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0424]ln some embodiments, an anti~CD25 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR2 comprising an amino acid sequence selected from SEQ ID NOs:
4102, 4106, 4110, 4114, 4118, 5042, 5046, 5059, 5067, 5092, 5214, 5215, 5216, 5217, 7343, 7346, 7348, and 9435, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0425]Provided herein are anti-CD25 antigen-binding domains (e.g., antibodies such as single-domain antibodies) comprising a CDR3 comprising an amino acid sequence selected from any of the CDR3 amino acid sequences listed in Table 1-3, Table 9, Table 10, or Table 12-1, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0426]! n some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR3 comprising an amino acid sequence selected from SEQ ID Nos:
4103, 4107, 4111, 4115, 4119, 5336-5640, 7344, 7347, 7349, 7350, 8523-8818, 9411-9416, 9436, 9881- 9991, and 10504-10544, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0427]ln some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as singledomain antibody) comprises a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 4103, 4107, 4111, 4115, 4119, 5337, 5339, 5371, 5375, 5398, 5401, 5431, 5515, 5519, 5521, 5528, 5532, 5542, 5544, 5545, 5547, 5548, 7344, 7347, 7349, 7350, 9411-9416, 9436, 9887, 9966, 9975, 9978, 9979, 9980 and 10504-10544, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.
[0428]ln some embodiments, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino add sequence selected from SEQ ID NO: 4113; a CDR2 comprising an amino add sequence of SEQ ID NO: 9435; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 10504-10518.
[0429]ln some embodiments, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino add sequence selected from SEQ ID NO: 7342; a CDR2 comprising an amino add sequence of SEQ ID NO: 7348; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 10519-10544.
[0430]ln some embodiments, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino add sequence selected from any one of SEQ ID NOs: 4726-5030, 7931-8226, and 9660-9770; a CDR2 comprising an amino acid sequence selected from any one of SEQ iD NOs: 5031-5335, 8227-8522, and 9771-9880; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ iD Nos: 5336-5640, 8523-8818, and 9881-9991,
[0431]in some embodiments, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino add sequence selected from any one of SEQ ID NOs: 4726-5030, 7931-8226, and 9660-9770; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5031-5335, 8227-8522, and 9771-9880; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID Nos: 5336-5640, 8523-8818, and 9881-9991.
[0432]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4726-4758; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5031-5063; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5336-5368.
[0433]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4726-7458; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5031-5063; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5336-5368.
[0434]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4759-4785; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5064-0590; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5369-5395.
[0435]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4759-4785; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5064-5090; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5369-5395,
[0436]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4786-4791; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5091-5096; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5396-5401.
[0437]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4786-4791; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5091-5096; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5396-5401. [0438]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence seiected from any one of SEQ iD NOs: 4792-4904 and 9660-9770; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5097-5209 and 9771-9880; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5402-5514 and 9881-9991.
[0439]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4792-4904 and 9660-9770; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5097-5209 and 9771-9880; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5402-5514 and 9881-9991.
[0440]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence seiected from any one of SEQ ID NOs: 4905-5030; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5210-5335; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5515-5640.
[0441]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4905-5030; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5210-5335; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5515-5640.
[0442]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 7931-8120; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8227-8416; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8523-8712.
[0443]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 7931-8120; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8227-8416; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8523-8712.
[0444] In one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 3121-8224; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8417-3520; and/or a CDR3 comprising an amino add sequence selected from any one of SEQ ID NOs: 8713-8816.
[0445]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence seiected from any one of SEQ ID NOs: 3121-8224; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8417-8520; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs; 8713-8816.
[0446]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8225-8226; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8521-8522; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8817-8818.
[0447pn one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8225-8226; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8521-8522; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 8817-8818.
[0448]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113; a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 10504- 10518.
[0449]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113; a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435; and a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 10504- 10518.
[0450]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342; a CDR2 comprising an amino acid sequence of SEQ ID NQ: 7348; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 9414 and 10519-10544.
[0451]ln one embodiment, an anti-CD25 antigen-binding domain comprises a CDR1 comprising an amino add sequence of SEQ ID NO: 7342; a CDR2 comprising an amino add sequence of SEQ ID NQ: 7348; and a CDR3 comprising an amino add sequence selected from any one of SEQ ID NOs: 9414 and 10519-10544.
[0452]Provided herein are anti-CD25 antigen-binding domains (e.g., antibodies such as single-domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within any of the exemplary anti-CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 10, or Table 12-1. In certain embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises: i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4102, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4103; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4106, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4107; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4109, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4110, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4111; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4115; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4117, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4119; vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7343, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7344; vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7345, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7346, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7347; viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7349; ix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7350; x) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9411; xi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9412; xii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 9413; xiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9414; xiv) a CDR1 comprising an amino add sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 9415; xv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 9416; xvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino add sequence of SEQ ID NO: 9975; xvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 5431; xviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 9887; xix) a CDR1 comprising an amino acid sequence of SEQ iD NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 9966; xx) a CDR1 comprising an amino acid sequence of SEQ ID NQ: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 9436; xxi) a CDR1 comprising an amino acid sequence of SEQ iD NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 9978; xxii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 9979; xxi si) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9980; xxiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4110, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5339; xxv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ iD NO: 5046, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5339; xxvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ iD NO: 5059, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 5337; xxvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ iD NO: 5046, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 5337; xxvlii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino add sequence of SEQ ID NO: 5067, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5371; xxix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino add sequence of SEQ ID NO: 5046, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5375; xxx) a CDR1 comprising an amino add sequence of SEQ ID NO: 4109, a CDR2 comprising an amino add sequence of SEQ iD NO: 4110, and a CDR3 comprising an amino add sequence of SEQ ID NO: 4111; xxxi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino add sequence of SEQ ID NO: 5092, and a CDR3 comprising an amino add sequence of SEQ ID NO: 4111; xxxii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino add sequence of SEQ ID NO: 5092, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5398; xxxiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino acid sequence of SEQ iD NO: 5042, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 4111; xxxiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5059, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4111; xxxv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5042, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5401; xxxvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5515; xxxvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4909, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5214, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5519; xxxviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5216, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5521; xxxix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4909, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5217, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5519; xl) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4918, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5215, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5528; xli) a CDR1 comprising an amino acid sequence of SEQ ID NG: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5532; xlii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5542; xliii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5544; xliv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino add sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5545; xlv) a CDR1 comprising an amino acid sequence of SEQ ID NO:49, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5547; xl vs) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino add sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5548; xlviija CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10504; xlviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10505; xlix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10506; l)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10507; li)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10508; lii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10509; liii)a CDR1 comprising an amino add sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10510; liv)a CDR1 comprising an amino add sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10511; lv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10512; lvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10513; lvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10514; lviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10515; lix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10516; lx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10517; lxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10518; lxii)a CDR1 comprising an amino acid sequence of SEQ ID NG: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10519;
Ixiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10520; lxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10521; Ixv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10522;
Ixvijs CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10523; lxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10524;
Ixviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10525;
Ixixja CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9414; lxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10526; lxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10527;
Ixxilja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10528; lxxiii)a CDR1 comprising an amino acid sequence of SEQ ID NQ: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10529;
Ixxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10530; lxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10531;
Ixxvija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10532;
Ixxviija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10533;
Ixxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10534; lxxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10535; lxxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10536; lxxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10537; lxxxii)a CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10538;
Ixxxiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 10539; lxxxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10540;
Ixxxvja CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10541;
Ixxxvija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10542;
Ixxxviija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10543; or
Ixxxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ !D NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10544, [0453]ln one embodiment, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ iD NO: 9436, [0454]ln one embodiment, an anti-CD25 antigen-binding domain (e,g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 9416.
[0455]ln one embodiment, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10531.
[0456]ln one embodiment, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10534.
[0457]ln a related embodiment, provided herein are anti-CD25 antigen-binding domains (e.g., antibodies such as single-domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within a VHH amino acid sequence as defined by any of the exemplary anti-CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 10 or Table 12-1. For example, provided herein are antibodies, or antigen-binding fragments thereof, comprising the set of CDR1-CDR2-CDR3 amino acid sequences contained within a VHH amino acid sequence selected from SEQ ID Nos: 4104, 4108, 4112, 4116, 4120, 4226-4130, 4143-4725, 5641-5945, 7351-7354, 7359-7362, 7368-2830, 8819-9114, and 9417-9422, 9437, 9439, 9442-9659, 9992-10102, 10214-10276, and 10545-10585.
[0458]ln some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure can include: a), a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4104; b). a variable domain that comprises a CDR1, CDR2, and CDRS contained within a VHH comprising the amino acid sequence of SEQ ID NG: 4108; c). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4112; d). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NG: 4116; e). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4120; f). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7351; g). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7352; h). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7353; i). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7354; or j). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9437. [0459]ln some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure can include a), a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4126; b). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4127; c). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4128; d). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4129; e). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 4130; f). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7359; g). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7360; h). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7361; i). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 7362; j). a variable domain that comprises a CORI, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9417; k). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino add sequence of SEQ ID NO: 9418; l), a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9419; m). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9420; n). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9421; o). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9422; p). a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9439; q) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10545; r) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10546; s) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10547; t) a variable domain that comprises a CDR1, CDR2? and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10548; u) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10549; v) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10550; w) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10551; x) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10552; y) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10553; z) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10554; aa) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10555; bb) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10556; cc) a variable domain that comprises a CORI, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10557; dd) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10558; ee) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10559; ff) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10560; gg) a variable domain that comprises a CDR1, CDR2; and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10561; hh) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10562; ii) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10563; jj) a variabie domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10564; kk) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10565;
II) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10566; mm) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9420; nn) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10567; co) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10568; pp) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10569; qq) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10570; rr) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10571; ss) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10572; tt) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10573; uu) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10574; w) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10575; ww) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10576; xx) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10577; yy) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10578; zz) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10579; aaa) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10580; bbb) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10581; ccc) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10582; ddd) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10583; eee) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10584; or fff) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10585.
[0460]ln some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9437 or 9439. [0461]ln some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 9422.
[0462]ln some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10572. [0463]ln some embodiments, an anti-CD25 antigen-binding domain (e.g., antibody such as a singledomain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 10575.
[0464]in an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure can include a VHH amino acid sequence selected from SEQ ID Nos: 4104, 4108, 4112, 4116, 4120, 4143-4442, 5641-5945, 7351-7354, 7368-7659, 8819-9114, 9437, 9442-9551, 9992-10102, and 10246-10276, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0465]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure can include a VHH amino acid sequence selected from SEQ ID Nos: 4104, 4108, 4112, 4116, 4120, 7351-7354, 9437, and 10246-10276, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0466]!n an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID Nos: 4126-4130, 4443-4725, 7359-7362, 7660-7930, 9417-9422, 9439, 10214- 10245, 9552-9659, and 10545-10585, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity,
[0467]!n an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID NOs: 4126-4130, 7359-7362, 9417-9422, 9439, 10214-10245, and 10545-10585, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0468]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID NQs: 9439, 9422, 10572, and 10575, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0469]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
4126, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at ieast 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0470]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
4127, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0471]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
4128, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0472]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
4129, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0473]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
4130, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0474]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 7359, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0475]ln an embodiment provided herein, an anti~CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 7360, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity,
[0476]in an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
7361, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0477]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
7362, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity,
[0478]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
9417, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0479]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g,, antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
9418, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0480]! n an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
9419, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0481]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
9420, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0482]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
9421, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0483]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO:
9422, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0484]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 9439, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0485]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10214, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0486] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10215, or a similar sequence thereof having at least 70%, at least 75%, at least 30%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0487] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10216, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0488] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ iD NO: 10217, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0489] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10218, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0490] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10220, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0491] In an embodiment provided herein, an anti~CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10221, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0492] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10222, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0493] in an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10223, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0494] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10224, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0495] in an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10225, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at ieast 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0496] in an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ iD NO: 10226, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0497] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ iD NO: 10227, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0498] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10228, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0499] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10229, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0500] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10230, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0501] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10231, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0502] in an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10232, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0503] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10233, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0504] In an embodiment provided herein, an antl~CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10234, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0505] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10235, or a similar sequence thereof having at least 70%, at least 75%, at least 30%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0506] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10236, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0507] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ iD NO: 10237, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0508] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10238, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0509] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10239, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0510] In an embodiment provided herein, an anti~CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10240, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0511] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10241, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0512] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10242, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0513] In an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10243, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0514] in an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10244, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at ieast 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0515] in an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10245, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0516]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10545, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0517]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g,, antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10546, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0518]! n an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disciosure comprises an amino acid sequence of SEQ ID NO: 10547, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0519]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10548, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0520]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10549, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0521]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10550, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0522]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10551, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0523]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10552, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0524]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10553, or a similar sequence thereof having at least 70%, at least 75%, at least 30%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0525]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10554, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0526]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10555, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0527]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10556, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0528]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10557, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[OS29J8 n an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10558, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0530]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10559, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0531]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10560, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0532]ln an embodiment provided herein, an anti~CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10561, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0533]in an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10562, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at ieast 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0534]in an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10563, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0535]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10564, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0536]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g,, antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10565, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0537]! n an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10566, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0538]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 9420, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0539]! n an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10567, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0540]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10568, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0541]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10569, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0542]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10570, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0543]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10571, or a similar sequence thereof having at least 70%, at least 75%, at least 30%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0544]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10572, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0545)1 n an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10573, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0546]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10574, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0547]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10575, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0548]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10576, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0549]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10577, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0550]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10578, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0551]ln an embodiment provided herein, an anti~CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10579, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0552]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10580, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at ieast 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0553]in an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10581, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0554]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10582, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at ieast 90%, at ieast 91%, at ieast 92%, at ieast 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0555]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g,, antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10583, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0556]! n an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10584, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at ieast 98%, or at least 99% sequence identity.
[0557]ln an embodiment provided herein, an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) of the present disclosure comprises an amino acid sequence of SEQ ID NO: 10585, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0558]! n some embodiments, the present disclosure also includes an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) that competes for binding to CD25 with any one of the exemplary anti-CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 10 or Table 12-1. [0559]i n some embodiments, the present disclosure also includes an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) that binds to the same epitope on CD25 as any one of the exemplary anti~CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 10 or Table 12-1. [0560]ln some embodiments, the present disclosure also includes an anti-CD25 antigen-binding domain (e.g., antibody such as a single-domain antibody) that comprises a means for binding an epitope within human CD25 bound by any one of the exemplary anti-CD25 VHH antibodies listed in Table 1-3, Table 1- 4, Table 9, Table 10, or Table 12-1.
Smcsie-Domain Antibodies
[0561]A single-domain antibody (e.g., VHH) specific for TN FR2 or CD25, as described herein, can be obtained by immunization of dromedaries, camels, llamas, alpacas, or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies. Antigens can be purified from natural sources, or in the course of recombinant production. Immunization and/or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens. By reverse transcription and polymerase chain reaction (PCR), a gene library of single-domain antibodies containing several million clones can be produced. Screening techniques such as phage display, yeast display, and ribosome display help to identify the clones binding the antigen. Methods generation of heavy-chain antibody fragments are described in e.g., WO 94/04678; Hamers-Casterman et al. 1993; Muyldermans et al. 2001; and Arbabi Ghahroudi, M. et al. (1997). FEBS Letters 414 (3): 521-526, each of which is incorporated herein by reference in its entirety.
[0562]A different method may use gene libraries from animals that have not been previously immunized. Such naive libraries usually contain only antibodies with low affinity to the desired antigen, making it necessary to apply affinity maturation by random mutagenesis as an additional step. See e.g., Saerens, D.; et al. (2008). "Single-domain antibodies as building blocks for novel therapeutics". Current Opinion in Pharmacology 8 (5): 600-608. [0563]Affinity maturation strategies can be categorized as either targeted/rational approaches or untargeted/random approaches. For targeted approaches information about the VHH of interest is needed, such as hot spots for affinity maturation or structural information on the VHH:antigen complex, whereas for untargeted approaches no prior information is needed. Targeted approaches that may be applied for affinity maturation of VHHs include site-directed in-vitro mutagenesis and in- sr//co/computational approaches. Common untargeted approaches used for affinity maturation of VHHs include random in-vitro mutagenesis, CDR swapping and autonomous hypermutation yeast surface display, with the latter two being novel, emerging and very time efficient techniques. Most of these strategies have in common, that after applying a certain randomization strategy to generate a mutational library, the resulting library can be screened by employing standard display techniques such as yeast, phage or ribosome display to select for the best binders. The choice of the display system is often guided by the library size to be displayed, with yeast display being able to handle library sizes of ~107 - 109, phage display ~108-10w and ribosome display ~1012-1013 (Chan and Groves, 2021). Notably, during affinity maturation the number of highly interactive residues such as aromatic amino acids usually increase in the CDR regions. The selected affinity matured clones may be further evaluated by a developability assessment to test for undesired properties, such as unspecific binding to off-targets or VHH instability.
[0564]For targeted in vitro mutagenesis, a set of selected residues within the CDRs of a VHH may be mutated (Tiller et al., 2017; Yau et al., 2005). Pre-selection of these residues can be either performed using alanine scanning to identify hot spot residues for mutation or by using structural data of the antigemVHH complex to identify positions to be mutated. These sites can then be either submitted to saturating mutagenesis to substitute a specific site with all possible amino acids or specific amino acid substitutions yielding several smaller libraries. After mutagenesis binders can be displayed to select the best matured candidate. Usually, several rounds of targeted mutagenesis are performed with separate sub-libraries to obtain combinations of individual mutations that cooperatively result in increased binding affinity.
[0565]Computer-aided//n silica methods are often used to guide targeted in vitro mutagenesis. Using homology modeling of the target:VHH complex or docking, hotspots for mutations can be identified that are then submitted to in vitro mutagenesis (Bert Schepens et al., 2021; Cheng et al., 2019; Inoue et al., 2013; Mahajan et al., 2018). Further, in silica methods can search all designed variants in a virtual library (~1O40 members) in a rather short amount of time to identify a feasible number of promising candidates to be tested experimentally. These techniques can be especially valuable if structural data on the drugtarget interaction are available.
[0566]Untargeted/random affinity maturation strategies that can be applied to affinity mature VHHs include random in vitro mutagenesis, CDR shuffling/swapping and in vivo affinity maturation via yeast display. For random in vitro mutagenesis the sequence of either the entire VHH or only the CDRs are mutated randomly (Chen et al., 2021; Ye et al., 2021; Zupancic et al., 2021). The most commonly used technique is error prone PCR employing a DNA polymerase that lacks proof reading activity and PCR conditions that increase the polymerase error rate even further. This technique can be applied without further structural knowledge or information on the importance of residues that contribute to antigemVHH interaction. The resulting mutational library can then be displayed to select the best matured candidate. This technique may also be combined with NGS sequencing of the display elutions to get an in-depth readout of all obtained candidates, enabling the identification of low abundant but still promising clones (Chen et al., 2021).
[0567]ln some embodiments, CDR shuffling or swapping is applied for VHH affinity maturation, such as described in Zupancic et al., 2021. For CDR swapping, enriched libraries can be used as input material for a PCR reaction to individually amplify the CDR of the VHHs. The PCR products can then be mixed and reassembled using overlapping PCR to generate the entire plasmid for further rounds of display to select for the best matured binder. One limitation of this approach is that it can only be used for VHHs comprising the same framework as it is the case for synthetic libraries, [0568]!n some embodiments, in vivo affinity maturation via yeast display is applied for VHH affinity maturation, such as described in Wellner et al., 2021. The method is based on an autonomous hypermutation yeast surface display (AHEAD), which imitates somatic hypermutation during VHH selection using engineered yeast strains. The yeast's error prone orthogonal DNA replication system can generate new variants during plasmid replication by randomly introducing mutations. The new variants can then be displayed and selected using yeast surface display to identify the best binders. This enables the production of high affinity clones in very little time (about 2 weeks), which is significantly faster than classical affinity maturation procedures. The method can be applied using synthetic or immune libraries using unenriched libraries enriched libraries or a subset of preselected clones.
[0569]ln case binders with medium affinity are required, as it is the case for the V-bodies described herein, and the affinity of the identified candidates need to be decreased, very similar techniques can be applied. For example, mutations that are aiming at lowering the affinity can be introduced using the same targeted or untargeted approaches as described for the affinity maturation. The selection afterwards can be adapted accordingly. If larger libraries are generated that need to be screened via a display technique, the selection strategy can be adapted to enrich medium affinity binders while excluding high affinity candidates. This could, for example be a pre-panning in phage display with low antigen concentration to remove all higher affinity candidates, followed by a selection with high antigen concentration to obtain medium affinity VHHs. For library sizes of up to 1000 candidates a kinetic off- rate characterization can be used to get immediate information about the kinetic behavior of the candidates.
[0570jWhen the most potent clones have been identified, their DMA sequence can be optimized, for example to improve their stability towards enzymes. Another goal is humanization to prevent immunological reactions of the human organism against the antibody. Humanization can be achieved based on the homology between camelid VHH and human VH fragments, which is described in further detail below. Finally, the optimized single-domain antibody can be translated and expressed in suitable organisms such as E. caii or Saccharomyces cerevisiae.
[0571]Single-domain antibodies can also be derived from conventional antibodies. In some embodiments, single-domain antibodies can be made from conventional murine or human IgG with four chains. The process is similar, comprising gene libraries from immunized or naive donors and display techniques for identification of the most specific antigens. However, the binding region of a conventional IgG consists of two domains (VH and VL), which tend to dimerize or aggregate because of their lipophilicity. Monomerization can be accomplished by replacing lipophilic by hydrophilic amino acids. (See e.g., Borrebaeck, C. A. K.; Ohlin, M. (2002). "Antibody evolution beyond Nature". Nature Biotechnology 20 (12): 1189-90.) If affinity can be retained after monomerization, the single-domain antibodies can likewise be produced in E. coli, S. cerevisiae or other suitable organisms.
[0572]A "humanized antibody" refers to a chimeric, genetically engineered, antibody in which the amino acid sequences (typically CDRs) from an antibody (donor antibody), e.g., a camelid antibody, are grafted onto a human antibody (acceptor antibody). Thus, a humanized antibody typically comprises CDRs from a donor antibody and variable region framework and constant regions, when present, from a human antibody. Accordingly, a "humanized VHH" comprises CDRs that corresponds to the CDRs of a naturally occurring VHH domain (e.g., a camelid VHH), but that has been "humanized". Humanized VHH may be prepared by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (particularly in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain human antibody. Such humanized VHHs can be obtained in any suitable manner known to a skilled person in the art and thus not strictly limited to methods described herein.
[0573]Humanization of VHHs can achieved using resurfacing or CDR grafting. Resurfacing strategies have been described in e.g., Conrath et al., 2005 J Mol Biol; Kazemi-Lomedasht et al., 2018; Vincke et al., 2009 J Biol Chem, and CDR grafting strategies have been described in e.g., ben Abderrazek et al., 2011; van Faassen et al., 2020 FASEB; Li et al., 2018; Vaneycken et al., 2010; Vincke et al., 2009 J Biol Chem; and Yu et al., 2017, each of which is incorporated herein by reference in its entirety.
[0574]To humanize a camelid VHH using a resurfacing approach, a human germline reference that is most similar to the camelid germline sequence of the selected VHH may be identified. Most of the isolated camelid VHHs in literature belong to the camelid IGHV3 subfamily 2 (Nguyen et al., 2000, EMBO J) with DP-47/VH3-23 from the IGHV3 family commonly used as human reference. The framework of the camelid VHH can then be compared to the human reference sequence. Surface exposed residues are substituted to their human counterpart as it is assumed that their contribution to protein stability is rather low. Buried residues however remain of camelid origin, as they likely contribute to the overall VHH stability. Humanization of framework regions 1, 3 and 4 usually does not impact the physicochemical properties of the VHHs, whereas a general humanization of framework 2 would significantly increase local hydrophobicity. Residues H37, H44, H45 and H47 (Chothia numbering) in framework 2, the so called tetrade or hallmark residues, have a rather hydrophobic nature in human VHs (VGLW) as they are partially buried and involved in VH/VL paring, while in camelid VHHs these residues are partially charged (FERG), which significantly increases VHH solubility and inhibits paring of camelid VL (Soler et al., 2021, Biomolecules, Conrath et al., 2005 J Mol Biol). Further, residues H37 and H47 are known to interact with the CDR-H3 loop in many VHHs, stabilizing its conformation and thereby contributing to antigen binding affinity. In addition, a significant number of VHHs use framework 2 residues H44, H45 and H47 for antigen binding (Zavrtanik et al., 2018, J Mol Biol}. A full humanization of these residues hence frequently results in reduced solubility or aggregation of the VHHs and a reduced or complete loss of binding affinity for the target antigen (van Faassen et al., 2020, Vincke et al., 2009). in consequence, all or at least some of these hallmark residues in framework 2 remain of camelid origin when humanizing VHHs.
[0575]Another approach that may be applied to humanize VHHs is CDR grafting. CDRs of the selected VHHs can be transplanted onto a universal VHH framework that has been partially or fully humanized (Saerens et al., 2009 J Biol Chem, Soler et al., 2021, Vincke et al., 2009 J Biol Chem). CDR grafting has been successfully used in some cases but failed for several others, with VHHs frequently losing their potential to bind to the desired antigen and/or becoming structurally instable with a high tendency to aggregate (van Faassen et al., 2020, FASEB). This is mostly attributed to interactions of CDR3 with specific residues in framework 2 that are important for CDR3 conformation, general VHH stability and overall hydrophobicity, which are impaired by this approach. Sometimes camelid backmutations are introduced into the framework to compensate for these effects (van Faassen et al., 2020, FASEB). [0576]An alternative strategy to mitigate the need of humanizing the selected VHH sequences is to use fully or partially humanized synthetic VHH libraries instead of camelid immune libraries for VHH discovery (Moutel et al. 2016, eLife; McMahon, 2018, NSMB; Zimmermann et al., 2018, eLife). In many of these libraries the hallmark residues are still of camelid origin for reasons discussed above. [0577]Other suitable humanizing substitutions are described in WO 09/138519 and WO 08/020079, as well as Tables A-3 to A-8 from WO 08/020079 (which are lists showing possible humanizing substitutions), each of which is incorporated herein by reference in its entirety. Non-limiting examples of such humanizing substitutions include Q108L and A14P, Such humanizing substitutions may also be suitably combined with one or more other mutations as described herein (such as with one or more mutations that reduce binding by pre-existing antibodies).
[0578]ln some embodiments, humanized VHH sequences still retain the residues that are relevant for protein A binding. In some embodiments, the engineering activities during humanization may be applied to engineer protein A binding properties into a VHH that did previously not interact with protein A (Graille et al„ 2000, PNAS).
[0579] Li ke a "humanized antibody", a "cameiized antibody" refers to an antibody having amino acid sequences (typically CD Rs) from a donor antibody, e.g., a human antibody, and variable region framework and constant regions, when present, from a camelid antibody. Accordingly, a "cameiized VH" comprises an amino acid sequence that corresponds to the amino add sequence of a naturally occurring VH domain, but that has been "cameiized". Cameiized VH may be prepared by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner, for example as described in WO 2008/020079. Such "carnalizing" substitutions are usually inserted at amino acid positions that form and/or are present at the VH—-VL interface, and/or at the so-called Camelidae hallmark residues, e.g., F37, E44, R45 and F47 (see for example WO 94/04678 and Davies and
Riechmann (1994 and 1996)). in one embodiment, the VH sequence that is used as a starting material or starting point for generating or designing the cameiized VH is a VH sequence from a mammal, or the VH sequence of a human antibody. However, such camelized VH can be obtained in any suitable manner known to a skilled person in the art and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material. [0580]The amino acid residues of a single-domain antibody can be numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelids described in Riechmann and Muyidermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see for example FIG. 2 of this publication). The total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering. For example, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering. As a result, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain is usually in the range of from 110 to 120, often between 112 and 115. However, smaller and longer sequences may also be suitable for the purposes described herein.
[0581]Determination of CDR regions in a single-domain antibody may be accomplished using different methods, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol, Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745," ("Contact" numbering scheme); Lefranc M P et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 January; 27(l):55-77 ("IMGT" numbering scheme); Honegger A and Piuckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun. 8; 309(3):657-70, ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272, ("AbM" numbering scheme), each reference cited herein is incorporated by reference in its entirety.
[0582]The boundaries of a given CDR or framework (FR) may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, "30a," and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular's AbM antibody modeling software.
[0583]ln some embodiments, CDRs can be defined in accordance with any of the Kabat numbering scheme, the Chothia numbering scheme, a combination of Kabat and Chothia, the AbM numbering scheme, and/or the Contact numbering scheme. A VHH typically comprises three CDRs, designated CDR1, CDR2, and CDR3. Table 1-5, below, lists exemplary position boundaries of CDR-Hl, CDR-H2, CORNS as identified by Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-Hl, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-H1 located before CDR-Hl, FR-H2 located between CDR-Hl and CDR-H2, FR- H3 located between CDR-H2 and CDR-H3 and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-Hl loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.
Table 1-5. CDRs definitions according to various numbering schemes.
1Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5sh Ed. Public Health Service, National Institutes of Health, Bethesda, MD;
2AI-Lazikani et al., (1997) JMB 273, 927-94S
[0584]Thus, unless otherwise specified, a "CDR" or "complementarity determining region," or individual specified CDRs (e.g., CDR-Hl, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) CDR as defined by any of the above-mentioned schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VHH amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the VHH, as defined by any of the above-mentioned schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes (see e.g., Table 1-5), although it is understood that a provided antibody can include CDRs as described according to any of the other above-mentioned numbering schemes or other numbering schemes known to a person of ordinary skill in the art,
[0585)1 n a single-domain antibody sequence of the present disclosure, the framework sequences may be any suitable framework sequences. For example, the framework sequences may be framework sequences derived from a heavy chain variable domain (e.g., a VH sequence or VHH sequence). In some embodiments, the framework sequences are either framework sequences that have been derived from a VHH sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences (in which said framework sequences may optionally have been partially or fully camelized).
[058S]Antigen-binding fragments (or combinations of fragments) of any of single-domain antibodies described herein, such as fragments that contain one or more CDR sequences, suitably flanked by and/or linked via one or more framework sequences, are also encompassed within the present disclosure.
[0587]lt should be noted, however, that the present disclosure is not limited to the origin of the singledomain antibody (or of the nucleotide sequence used to express it), nor to the way that the singledomain antibody or nucleotide sequence is generated or obtained. Thus, an antigen-binding protein of the present disclosure may comprise naturally occurring sequences (from a suitable species), recombinant sequences, or synthetic or semi-synthetic sequences. Similarly, nucleotide sequences encoding antigen-binding proteins of the present disclosure may comprise naturally occurring nucleotide sequences, recombinant sequences, or synthetic or semi-synthetic sequences (for example, sequences that are prepared by PCR or isolated from a library).
[0588]Anti-TNFR2 antigen-binding domains (e.g., antibodies such single-domain antibodies) and anti- CD25 antigen-binding domains of the present disclosure may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy chain variable domains as compared to the exemplary antibody sequences provided herein. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The anti-TNFR2 antigen- binding domains and anti-CD25 antigen-binding domains of the present disclosure may comprise any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the VHH domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antigen-binding domain was originally derived).
[0589]Furthermore, the antigen-binding domains of TNFR2 and CD25 may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antigen-binding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced biological properties (e.g., agonistic effect), reduced immunogenicity, etc. Antigen-binding proteins comprising one or more antigen-binding domains obtained in this general manner are encompassed within the present disclosure.
[0590]Provided herein are anti-TNFR2 antigen-binding domains and anti-CD25 antigen-binding domains comprising variants of any of the VHH and/or CDR amino acid sequences disclosed herein having one or more amino acid substitutions. For example, the present disclosure includes anti-TNFR2 antigen-binding domains and anti-CD25 antigen-binding domains having VHH and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 3 or fewer, 2, or 1 amino acid substitutions relative to any of the VHH and/or CDR amino acid sequences set forth in Tables 1-3, 1-4, 9, 10, 11 and 12-1. herein. Amino acid substitutions may be introduced into an antigen-binding protein of interest and the resultant variants can screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or reduced ADCC or CDC.
[0591]Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. in some embodiments, an amino acid substitution is a conservative substitution, meaning exchanging an amino acid with another amino acid of the same class, in some embodiments, amino acid substitutions may also include a non-conservative substitution, meaning exchanging an amino acid with an amino acid of a different class. Other exemplary amino acid substitutions are shown in Table 1-6.
Table 1-6. Exemplary amino acid substitutions
[0592]ln some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure may comprise one or more mutations to reduce oxidation levels of oxidation-labile residues such as Met (M). In certain embodiments, it may be desirable to address Met (M) oxidation liability by mutation of a Met (M) residue, in some embodiments, the single-domain antibodies (e.g., VHH) of the present disclosure may comprise one or more mutations (e.g., substitution mutations) of a Met residue to reduce oxidation. As a non-limiting example, a Met residue may be substituted in any of the single-domain antibodies described herein with, e.g., He (I), Ala (A), or Leu (L), to reduce oxidation.
[0593]in some embodiments, anti-TNFR2 and anti~CD25 single-domain antibodies (e.g., VHH) of the present disclosure comprise one or more modifications that reduce binding of the single-domain antibodies (e.g., VHH) by pre-existing antibodies found in human blood or serum. In some embodiments, single-domain antibodies (e.g,, VHHs) of the present disclosure are modified by mutation of amino acid position 11, for example LeullGlu (L11E), LeullLys (L11K), or LeullVal (L11V). In one embodiment, a single-domain antibody (e.g,, VHH) of the present disclosure may comprise a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering). As another example, a single-domain antibody (e.g., VHH) of the present disclosure may comprise an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the single-domain antibody (e.g,, VHH). The C-terminus of a VHH is normally VTVSS (SEQ ID NO: 4031). in one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering). In another embodiment, a singledomain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering). Accordingly, the C-terminus of a single-domain antibody (e.g., VHH) can be any one of VKVSS (SEQ ID NO: 4032), VQVSS (SEQ ID NO: 4033), VTVKS (SEQ ID NO: 4034), VTVQS (SEQ ID NO: 4035), VKVKS (SEQ ID NO: 4036), VKVQS (SEQ ID NO: 4037), VQVKS (SEQ ID NO: 4038), or VQVQS (SEQ ID NO: 4039). In another embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering), optionally a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) and an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension at the C-terminus of the single-domain antibody (e.g., VHH) (such that the C-terminus of the single-domain antibody (e.g., VHH) for example has the sequence VTVSSA (SEQ ID NO: 4040), VKVSSA (SEQ ID NO: 4041) or VQVSSA (SEQ ID NO: 4042)). In further embodiments, single-domain antibodies (e.g., VHH) of the present disclosure are modified by changes in carboxy-terminal region, for example to a terminal sequence having the sequence GQGTLVTVKPGG (SEQ ID NO: 4043) or GQGTLVTVEPGG (SEQ ID NO: 4044) or modification thereof. Additional modification to reduce binding by pre-existing antibodies in human serum can be found in e.g., W02012/175741, WO2015/173325, W02016/150845, WQ2011/003622, WQ2013/024059; US 11,426,468; US 10,526,397, which are incorporated herein by reference in their entities.
[0594]ln one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267). in one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VAGG (SEQ ID NO: 7266). In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VPAG (SEQ ID NO: 7267).
[0595]Additional carboxy-terminus modifications to introduce to a single-domain antibody (e.g., VHH) of the present disclosure include those described in in e.g., WO2024/238790, which is incorporated herein by reference in its entirety.
[0596]ln some embodiments, anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 93-640, 2805-3363, 6651-6697, 6931-6992, 7174-7197, 10380, 10381, 10383, 10385, and 10386, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267).
[0597]ln some embodiments, anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 10380, 10381, 10383, 10385, and 10386, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267).
[0598]ln some embodiments, anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 6648-6650, 7092, 7095, 7098, 7101, 7293-7296, 641-1127, 6698-6744, 10375, 10382, 10384, 10394-10408, 10457-10502, 10812, 10720-10727, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ iD NO: 7267).
[O599]8n some embodiments, anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 6648-6650, 7092, 7095, 7098, 7101, 7293-7296, 10375, 10382, 10384, 10394-10408, 10457-10502, and 10812, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267).
[0600]ln some embodiments, anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 10720-10727, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267).
[0601]ln some embodiments, anti-CD25 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, 4143-4442, 5641-5945, 7351-7354, 7359-7362, 2268-2830, 8819-9114, 9417-9422, 9437, 9439, 9442- 9659, 9992-10102, 10214-10276, and 10545-10585, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267).
[0602]ln some embodiments, anti-CD25 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, 4126-4130, 7351-7354, 7359-7362, 9417-9422, 9437, 9439, 10214-10276, and 10545-10585, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267). [0603]ln some embodiments, antl-CD25 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence seiected from any one of SEQ ID NOs: 4126-4130, 4443-4725, 7359- 7362, 7660-7930, 9417-9422, 9439, 10214-10245, and 10545-10585, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 7266) or VPAG (SEQ ID NO: 7267).
[0604]ln some embodiments, anti-CD25 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4126-4130, 7359-7362, and 9417-9422, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ, ID NO: 7266) or VPAG (SEQ ID NG: 7267).
[0605]ln some embodiments, anti-TNFR2 and anti-CD25 single-domain antibodies (e.g,, VHH) of the present disclosure are modified to enhance binding to staphylococcal protein A (SpA) or streptococcal protein G (SpG). Binding of SpA and SpG to antibodies or antibody fragments can be useful in the manufacturing process of the antibodies or antibody fragments. The high-affinity interaction of the IgG Fc region with SpA and SpG has been extensively exploited and became the gold standard for monoclonal antibody purification (Bjbrck and Kronvall, 1984). Other non-Fc containing antibody fragments, such as VHHs and Fabs do not have the capacity to bind to SpA or SpG via their Fc regions. However, sequence-dependent interaction with SpA has been demonstrated for these non-Fc containing antibody fragments (Graille et al., 2000; Henry et al., 2016). This characteristic circumvents potential use of affinity tags fused to the drug candidate for affinity chromatography that have the disadvantage as being regarded as a sequence liability, as it may impact protein immunogenicity as well as protein structure and stability and could compromise functionality. The interaction of the singledomain antibodies (e.g., VHH) to SpA relies on an alternative binding mode, with a 1-5 pM affinity, which is comparable to the 0.2 -3 pM measured for VH-SpA interactions (To et al., JBC, 2005; Henry et al., Pios One, 2016).
[0606]ln some embodiments, anti-TNFR2 and anti-CD25 single-domain antibodies (e.g., VHH) of the present disclosure have, or are modified to have a SpA-binding motif. For example, The VHH-SpA interface has been mapped to thirteen residues, which duster within the framework at the back side of the V-body, distant to the CDRs (Graille et al, 2000, Henry et al., 2016). In the absence of a VHH-SpA costructure, superposition of a SpA-Fab crystal structure and a VHH allows for visualizing the binding mode. Based on a structural and functional analysis, the thirteen residues of the VHH-SpA interface have been characterized to be intolerant to substitutions (residues Glyl5, Argl9, Tyr59, Gly65, and Arg66), tolerant to specific substitutions (residues Thr/Lys/Arg57, Thr68, Gln81, Asn82a, and Ser82b) or generally tolerant to a variety of substitutions (residues Serl7, Lys64, and Ser70) (all residue positions refer to Kabat numbering) (Henry et al., Pios One, 2016). Thus, a SpA-binding motif included in an anti- TNFR2 and anti-CD25 single-domain antibody (e.g., VHH) of the present disclosure may include one or more, or all of the thirteen residues.
[0607]ln some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure comprise one or more modifications at N-terminus to prevent formation of a pyroglutamate and product heterogeneity. In one embodiment, the amino acid residue Glu at the first position of the single-domain antibodies (e.g., VHH) is replaced with Asp (EID).
[060S]ln some embodiments, an anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 93-640, 2805-3363, 6651-6697, 6931-6992, 7174-7197, 10380, 10381, 10383, 10385, and 10386, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID). [0609]ln some embodiments, an anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NQs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 6636, 6640, 6644, 7090, 7090, 10380, 10381, 10383, 10385, and 10386, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID).
[0610]ln some embodiments, an anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 6643-6650, 7092, 7095, 7098, 7101, 7293-7296, 641-1127, 6698-6744, 10375, 10382, 10384, 10394-10408, 10457- 10502, 10812, 10720-10727, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID).
[0611]ln some embodiments, an anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID Nos: 81-92, 6648-6650, 7092, 7095, 7098, 7101, 7293-7296, 7101, 10375, 10382, 10384, 10394-10408, 10457-10502, and 10812 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the singledomain antibody (e.g., VHH) is replaced with Asp (EID).
[0612]!n some embodiments, an anti-TNFR2 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ, ID Nos: 10720-10727, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID).
[0613]ln some embodiments, an anti-CD25 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino add sequence selected from any one of SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, 4143-4442, 5641-5945, 7351-7354, 7368-7659, and 8819-9114, 9438, 9442-9551, 9992-10102, and 10246-10276, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID).
[0614]ln some embodiments, an anti-CD25 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, and 7351-7354, 9438, and 10246-10276, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID). [0615]ln some embodiments, an anti-CD25 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4126-4130, 4443-4725, 7359- 7362, 9439, 7660-7930, 9417-9422, 9552-9659, 10214-10245, and 10545-10585, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID).
[0616]ln some embodiments, an anti-CD25 single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 4126-4130, 7359-7362, 9439, 9417-9422, 0214-10245, and 10545-10585, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (EID).
Alternative protein scaffolds
[0617]ln some embodiments, anti-TNFR2 antigen-binding domains and/or anti-CD25 antigen-binding domains and/or multispecific antigen-binding proteins of the present disclosure can adopt an alternative protein scaffold. Such alternative protein scaffold may be a single chain polypeptidic framework, optionally with a reduced size (e.g., less than about 200 amino acids), that contains a highly structured core associated with variable domains of high conformational tolerance allowing insertions, deletions, or other substitutions. Such antigen-binding proteins may be generated by grafting CDRs or variable regions described herein onto a suitable protein scaffold. The structure of alternative scaffolds may vary, but preferably are of human origin for those developed as therapeutics.
[0618]Alternative protein scaffolds of the present disclosure can be based either on a conventionai immunoglobulin (ig) backbone, or are derived from a completely unrelated protein. These variable domains can be modified to create novel binding interfaces toward any targeted antigen, in some embodiments, an alternative protein scaffold of the present disclosure can be derived from Protein A, e.g., the Z-domain thereof (affibodies), lmmE7 (immunity proteins), BPTI/APPI (Kunitz domains), Ras- binding protein AF-6 (PDZ-domains), charybdotoxin (Scorpion toxin), CTLA-4, Min-23 (knottins), lipocalins (anticalins), neokarzi nostatin, a fibronectin domain (used in "adnectin"), an ankyrin repeat (AR) domain (used in "DARPins"), avidity multimers (also known as "avimers"), or thioredoxin (Skerra, A., Curr. Opin, Biotechnol. 18:295-304 (2005); Hosse et al., Protein Sci. 15:14-27 (2006); Nicaise et al., Protein Sci. 13:1882-1891 (2004); Nygren and Uhlen, Curr. Opin. Struc. Biol. 7:463-469 (1997), all of which are hereby incorporated by reference in their entirety). [0619] Antica H ns are a suitable type of non-lg based alternative scaffolds for use in the antigen-binding molecules of the present disclosure, Anticalins are a class of engineered ligand-binding proteins that are based on the lipocalin scaffold. Lipocalins are a family of proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. Lipocalins have limited sequence homology, but share a common tertiary structure architecture based on eight antiparallel ^-barrels. Lipocalins contain four exposed loops built on the rigid ^-barrel structure. Exemplary anticalin proteins that are commonly used are about a size of about 180 amino acids and a mass of about 20 kDa.
[0620]DARPins are another suitable non-lg based alternative scaffold that can be used in the antigenbinding molecules of the present disclosure. DARPins are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. They are derived from natural ankyrin repeat (AR) proteins, which usually contain a 33 amino acid protein motif consisting of two a-helices separated by loops, which repeats mediate protein-protein interactions. DARPins can be generated using combinatorial AR libraries constructed based on the 33 amino acid AR motif with seven randomized positions. DARPin libraries can be screened using ribosome display, and library members typically are well produced in Escherichia coii, do not aggregate, and display high thermodynamic stability. Preferably, DARPins contain two to four of these motifs flanked by N- and C- terminal capping motifs to shield hydrophobic regions and allow increased solubility.
[0621]The avimer structure can also be used as a protein backbone to generate a suitable non-lg based alternative scaffold. Avimers typically consist of two or more peptide sequences of 30 to 35 amino acids each, connected by peptide linker. The individual sequences are derived from A-domains of various membrane receptors and have a rigid structure, stabilized by disulfide bridges and calcium. Each A- domain can bind to a certain epitope of the target protein. The combination of domains binding to different epitopes of the same protein increases affinity to this protein, an effect known as avidity. [0622]Proteins derived from fibronectin III (FN3) domains can also be used to generate a suitable non-lg based alternative scaffold (also known as "monobody"). For example, the tenth fibronectin type III domain (FN10) of human fibronectin corresponds to a p-sandwich with seven ^-strands and three connecting loops showing structural homologies to Ig domains without disulfide bridges. In some cases, the connecting loops of FN10, each about 15 to 21 amino acids in length, can be randomized and the domains displayed on both phage and yeast to select for a scaffold with the desirable properties.
Adnectins™ Is an exemplary scaffold generated using 10th FN3 domains randomized and displayed in this way. Another exemplary scaffold comprising FN3 domains is a Centyrin™. Centryrins™ contain the consensus sequence of FN3 domains of human Tenascin C (TNC), which is found in the extracellular matrix of various tissues. Centyrin™ scaffolds have loops that have structural homology to antibody variable domains (i.e., CDR1, CDR2 and CDR3), and are small (about 10 kDa), simple, and highly stable single domain proteins that do not contain cysteine, disulfides or glycosylated residues. Centyrin™ possess excellent biophysical properties such as stability to heat, pH, denaturant and organic solvents, reversible unfolding and monodispersity. Another recent exemplary FN3-based scaffold that can be used in the present disclosure is fluctuation-regulated affinity proteins (FLAPs), as described in See et al., 2020. Biotechnology journal 15(12):e2000078, which is incorporated herein by reference in its entirety.
Fusions and Conjugates
[0623]ln one aspect, provided herein are multispecific antigen-binding proteins and conjugates comprising one or more anti~TNFR2 antigen-binding domains (e.g., antibody such as a single-domain antibody) and one or more anti-CD25 antigen-binding domains (e.g., antibody such as a single-domain antibody) that are linked, directly or indirectly, to one or more additional domains or moieties. The one or more anti-TNFR2 antigen-binding domains can specifically bind TNFR2. The one or more anti-CD25 antigen-binding domains can specifically bind CD25.
Fusion or Conjugation to Fc regions
[0624]Sn some embodiments, a multispecific antigen-binding protein or conjugate of the present disclosure comprises one or more anti-TNFR2 antigen-binding domains (e.g., antibody such as a singledomain antibody) and one or more anti-CD25 antigen-binding domains (e.g., antibody such as a singledomain antibody) provided herein operably linked to a dimerization domain such as an immunoglobulin Fc region. An immunoglobulin Fc region may be linked indirectly or directly to the one or more anti- TNFR2 antigen-binding domains (e.g., antibody such as a single-domain antibody) and one or more anti- CD25 antigen-binding domains (e.g., antibody such as a single-domain antibody), in some embodiments, a multispecific antigen-binding protein or conjugate of the present disclosure comprises one, two, three, four, five, six or more anti-TNFR2 antigen-binding domains and one, two, three, four, five, six or more anti-CD25 antigen-binding domains provided herein operably linked to an Fc region.
[0625]ln some embodiments, the present disclosure provides a multispecific antigen-binding protein or conjugate that comprises (1) one or more anti-TNFR2 antigen-binding domains, such as one or more anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13; (2) one or more anti-CD25 antigen-binding domains, such as one or more anti-CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 10, or Table 12-1; and (3) an Fc region. [0626JI n some embodiments, the present disclosure provides a muitispecific antigen-binding protein or conjugate that comprises (1) one or more anti-TNFR2 antigen-binding domains, such as one or more anti-TNFR2 VHH antibodies selected from T-001, T-002, T-003, T-004, T-004.VTV, T-005hul, T-006, T- 007, T-008hul, T-008hul.A3, T-007hul with CDR2 G/A, CDR3 T/S, T-009Hul, T-009Hul.NAR, T-OlOHul, T-OlOHul.NAR, T-009Hul_QGR, T-009Hul_SGR, T-009Hul_QGR_.N/C, T-009Hul__NAR_N/C, T- 009Hul_SGR_N/C, T-007Hul.S3.QGR, T-007Hul.S3.EGR, T-007Hul.S3.SGR, T-007Hul.S3.NSR, T- 007Hul.S3.NTR, T-007Hul.S3.SAR, T-007Hul.EQ.QGR, T-007Hul.EQ.EGR, T-007Hul.EQ.SGR, T- 007Hul.EQ.S3.NSR, T-011, T-012, T-013, T-014, T-015, T-016, T-017, T-018, T-019, T-020, T-021, T- OlShul.Al, T-016hul.G2, T-016hul.A3, T-016hul.A4, T-016hul.A5, T-O16hul.A6, T-016hul.A7, T- 016hul.A9, T-016hul.A10, T-016hul.All, T-016Hul.A5.A7, T-016Hul.A5.Al, T-016Hul.A5.A3, T- 016Hul.A5.A4, T-016Hul.S5.A4, T-016Hul.A5.A6, T-016Hul.A7.Al, T-016Hul.A7.A3, T-016Hul.A7.A4, T-016Hul.A7.A6, T-016Hul.Al.A3, T-016Hul.Al.A4, T-016Hul.Al,A6, T-016Hul.A3.A4, T-016Hul.A3.A6, T-016Hul.A4.A6, T-008Hul.Gl, T-008Hul.G2, T-008Hul.A3, T-008Hul.A4, T-008Hul.A5, T-008Hul.A6, T-008HU1.A7, T-008HU1.G8, T-008Hul.A9, T-008Hul.A10, T-008Hul.All, T-009Hul_SGR,Gl, T- Q09Hul. SGR.G2, T-009Hul_SGR.A3, T-009Hul_ SGR,A4, T-009Hul...SGR.A5, T-009Hul_.SGR.A6, T- 009Hul_SGR.A7, T-009Hul_.SGR.G8, T-009Hul_SGR.A9, T-009Hul_.SGR.A10, and T-009Hul„SGR.All; (2) one or more anti-CD25 antigen-binding domains, such as one or more anti-CD25 VHH antibodies selected from C-001, C-002, C-003, C-004, C-005, C-006, C-007, C-008, C-009, C-010, C-009Hul.l8, C- OlOHul.18, C-009HU1.A8, C-010Hul.A8, C-009Hul.L8, C-010Hul.L8, C-OUHul, C-012Hul, C-013Hul, C- 014HU1, C-OlSHul, C-OOSHul, C-016Hul, C-017Hul, C-018HU1, C-019Hul, C-020HU1, C-021HU1, C- 022Hul, C-023HU1, C-024Hul, C-025Hul, C-026Hul, C-027Hul, C-028Hul, C-029Hul, C-030Hul, C- 031HU1, C-032HU1, C-033Hul, C-034Hul, C-035Hul, C-036Hul, C-037Hul, C-038Hul, C-039Hul, C- 040HU1, C-041HU1, C-005HU1.A1, C-005Hul.A2, C-005HU1.A3, C-005HU1.A4, C-005HU1.A5, C- 005HU1.A6, C-005HU1.A7, C-005Hul.A8, C-005Hul.A9, C-005Hul.A10, C-005Hul.All, C-005Hul.A12, C- 005HU1.A13, C-005HU1.A14, C-005Hul.A15, C-010Hul.L8.Gl, C-010Hul.L8.G2, C-010Hul.L8.A3, C- 010Hul.L8.A4, C-010Hul.L8.A5, C-010Hul.L8.A6, C-010Hul.L8.A7, C-010Hul.A8, C-010Hul.L8.A9, C- 010Hul.L8.A10, C-010Hul.L8.All, C-010Hul.L8.A12, C-010Hul.L8.A13, C-010Hul.L8.A14, C- 010Hul.L8.A15, C-010Hul.L8.A16, C-010Hul.L8.A17, C-010Hul.L8.A18, C-010Hul.L8.A19, C- 010Hul.L8.A20, C-010Hul.L8.A21, C-010Hul.L8.A22, C-010Hul.L8.A23, C-010Hul.L8.A24, C- 010Hul.L8.A25, C-010Hul.L8.A26, and C-010Hul.L8.A27; and (3) an Fc region.
[0627]ln some embodiments, the present disclosure provides a multispecific antigen-binding protein or conjugate that comprises (1) a means for binding an epitope within human TNFR2 bound by any one of the exemplary anti-TNFR2 VHH antibodies listed in Table 1-1, Table 1-2, Table 5, Table 11, or Table 13; (2) a means for binding an epitope within human CD25 bound by any one of the exemplary anti-CD25 VHH antibodies listed in Table 1-3, Table 1-4, Table 9, Table 19, or Table 12-1; and (3) an Fc region. [O62SJ8 n some embodiments, the present disclosure provides a multispecific antigen-binding protein or conjugate that comprises (1) a means for binding an epitope within human TNFR2 bound by an antibody selected from T-001, T-002, T-003, T-004, T-004.VTV, T-005hul, T-006, T-007, T-008hul, T-008hul.A3, T- 007hul with CDR2 G/A, CDR3 T/S, T-009Hul, T-009HU1.NAR, T-OlOHul, T-OlOHul.NAR, T-009Hul_QGR, T-009HU 1_ SGR, T-009Hul_.QGR_N/C, T-009Hul„NAR„N/C, T-009Hul_.SGR__N/C, T-007Hul .S3.QGR, T- 007Hul.S3.EGR, T-007Hul.S3.SGR, T-007Hul.S3.NSR, T-007Hul.S3.NTR, T-007Hul.S3.SAR, T- 007Hul.EQ.QGR, T-007 Hui. EQ. EG R, T-007Hul.EQ.SGR, T-007Hul.EQ.S3.NSR, T-011, T-012, T-013, T- 014, T-015, T-016, T-017, T-018, T-019, T-020, T-021, T-016hul.Al, T-016hul.G2, T-016hul.A3, T- 016hul.A4, T-016hul.A5, T-016hul.A6, T-016hul.A7, T-016hul.A9, T-016hul.A10, T-016hul.All, T- 016Hul.A5.A7, T-016Hul.A5.Al, T-016Hul.A5.A3, T-016Hul.A5.A4, T-016Hul.S5.A4, T-016Hul.A5.A6, T-016Hul.A7.Al, T-016Hul.A7.A3, T-016Hul.A7.A4, T-016Hul.A7,A6, T-016Hul.Al.A3, T-016Hul.Al.A4, T-016Hul.Al.A6, T-016Hul.A3.A4, T-016Hul.A3.A6, T-016Hul.A4.A6, T-008Hul.Gl, T-008Hul.G2, T- 008HU1.A3, T-008HU1.A4, T-008Hul.A5, T-008Hul.A6, T-008Hul.A7, T-008Hul.G8, T-008Hul.A9, T- 008HU1.A10, T-008Hul.AH, T-009Hul„SGR.Gl, T-009Hul_SGR.G2, T-009Hul__SGR.A3, T- 009Hul .SGR.A4, T-009Hul_.SGR.A5, T-009Hul_SGR.A6, T-009Hul_.SGR.A7, T-009Hul_.SGR.G8, T- 009Hul_SGR.A9, T-009Hul__SGR.A10, and T-009Hul_SGR.All; (2) a means for binding an epitope within human TNFR2 bound by an antibody selected from C-001, C-002, C-003, C-004, C-005, C-006, C-007, C- 008, C-009, C-010, C-009HU1.I8, C-010Hul.l8, C-009Hul.A8, C-O10Hul.A8, C-009Hul.L8, C-010Hul.L8, C- OHHul, C-012HU1, C-013HU1, C-014Hul, C-015Hul, C-005Hul, C-016Hul, C-017Hul, C-018Hul, C- 019HU1, C-020HU1, C-021Hul, C-022Hul, C-023Hul, C-024HU1, C-025Hul, C-026HU1, C-027HU1, C- 028HU1, C-029HU1, C-030Hul, C-031Hul, C-032Hul, C-033Hul, C-034Hul, C-035Hul, C-036Hul, C- 037HU1, C-038HU1, C-039Hul, C-040Hul, C-041Hul, C-005Hul.Al, C-005Hul.A2, C-005Hul.A3, C- 005HU1.A4, C-005HU1.A5, C-005Hul.A6, C-005Hul.A7, C-005Hul.A8, C-005Hul.A9, C-005Hul.A10, C- 005Hul.AH, C-005HU1.A12, C-005Hul.A13, C-005Hul.A14, C-005Hul.A15, C-010Hul.L8.Gl, C- 010Hul.L8.G2, C-010Hul.L8.A3, C-010Hul.L8.A4, C-010Hul.L8.A5, C-010Hul.L8.A6, C-010Hul.L8.A7, C- 010HU1.A8, C-010Hul.L8.A9, C-010Hul.L8.A10, C-010Hul.L8.All, C-010Hul.L8.A12, C-010Hul.L8.A13, C-010Hul.L8.A14, C-010Hul.L8.A15, C-010Hul.L8.A16, C-010Hul.L8.A17, C-010Hul.L8.A18, C- 010Hul.L8.A19, C-010Hul.L8.A20, C-010Hul.L8.A21, C-010Hul.L8.A22, C-010Hul.L8.A23, C- Fc region. [0629]A "Fc region" as used herein refers to a portion of a heavy chain constant region comprising CH2 and CHS. in some embodiments, an Fc region comprises a hinge, CH2, and CHS. in various embodiments, when an Fc region comprises a hinge, the hinge can mediate dimerization between two Fc-containing polypeptides. An Fc region included in a multispecific antigen-binding protein or conjugate may be an Fc region from any species, or derived from any species, including, but not limited to, human, mouse, rat, monkey (e.g., cyno), camel, llama, shark, goat, rabbit, and/or bovine. In various embodiments, an Fc region included in a multispecific antigen-binding protein or conjugate of the present disclosure is a human immunoglobulin Fc region, or is derived from a human immunoglobulin Fc region, in some embodiments, the immunoglobulin Fc region is of IgG, IgE, IgM, IgD, IgA or lg¥ isotype. In some embodiments, the immunoglobulin Fc region is an IgG isotype, such as IgGl, lgG2, lgG3, or lgG4 subclass. The immunoglobulin Fc region may comprise a variant or fragment of a native IgG Fc region. [0630]ln some embodiments, an Fc region included in a multispecific antigen-binding protein or conjugate described herein may be a murine (e.g., a mouse or a rat) immunoglobulin Fc region, or derived from a murine immunoglobulin Fc region. In some embodiments, an Fc region included in a multispecific antigen-binding protein or conjugate described herein may be a cyno immunoglobulin Fc region, or derived from a cyno immunoglobulin Fc region.
[0631]A native Fc region typically possesses an effector function, including but not limited to, Fc receptor binding; Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (for example B-cell receptor); and B-cell activation, etc. Such effector functions generally require the Fc region to be combined with a binding domain (for example, an antibody variable domain) and can be assessed using various assays.
[0632]ln some embodiments, a multispecific antigen-binding protein or conjugate of the present disclosure can comprise a dimer of Fc regions. In some embodiments, an Fc region mediates dimerization of the anti-TNFR2-binding domains and/or anti-CD25-binding domains at physiological conditions, such as when expressed from a cell, such that a dimer is formed that doubles the number of anti-TNFR2 binding domains and/or anti-CD25-binding domains. For example, a multispecific antigenbinding protein comprising one VHH domain and an Fc region is monovalent as a monomer, but the Fc region can mediate dimerization; as a result, the multispecific antigen-binding protein is bivalent (i.e., having two VHH domains per molecule). Similarly, in some embodiments, two VHH domains (2x) are fused to an IgG Fc region and as a result of dimerization, the multispecific antigen-binding protein is tetravalent (i.e., having four VHH domains per molecule). In some embodiments, three VHH domains (3x) are fused to an IgG Fc region and as a result of dimerization, the multispecific antigen-binding protein is hexavalent (i.e., having six VHH domains per molecule), In some embodiments, four VHH domains (4x) are fused to an IgG Fc region and as a result of dimerization, the multispecific antigenbinding protein is octavalent (i.e., having eight VHH domains per molecule).
[0633]ln some embodiments, the CH3 domain of the Fc region can be used as homodimerization domain, such that the resulting multispecific antigen-binding protein may be formed from two identical polypeptides. In other cases, the CH3 dimer interface region of the Fc region can be mutated to enable heterodimerization. For example, a heterodimerization domain can be incorporated into multispecific antigen-binding protein such that the construct is a heterodimeric multispecific antigen-binding protein. [0634]When a dimer of Fc regions is used in a multispecific antigen-binding protein or conjugate of the present disclosure, the first and second Fc regions may be of the same IgG isotype such as, e.g., IgGl/IgGl, lgG2/lgG2, lgG4/lgG4. Alternatively, the first and second Fc regions may be of different IgG isotypes such as, e.g., lgGl/lgG2, lgGl/lgG4, lgG2/lgG4, etc.
[O635jln some embodiments, the Fc region included in a multispecific antigen-binding protein or conjugate of the present disclosure can be mutated or modified. In some embodiments, the mutations include one or more amino acid substitutions to reduce an effector function of the Fc region. Various examples of mutations to Fc regions to alter, such as reduce, effector function are known, including any as described below, in general, the numbering of the residues in an immunoglobulin heavy chain or portion thereof, such as an Fc region, is according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991),
[O636J8 n some embodiments, the human IgG Fc region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). Non-limiting examples of amino add modifications that can alter ADCC and/or CDC are described in Alegre et al, 1992 J Immunol, 148: 3461-3468; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Shields et al., 2001 JBC, 276(9): 6591-6604; Lazar et al., 2006 PNAS, 103(11): 4005-4010; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164; Moore et al., 2010 mAbs, 2(2): 181-189; and Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11, each of which is incorporated herein by reference in its entirety.
[0637]ln some embodiments, an Fc region included in a multispecific antigen-binding protein or conjugate of the present disclosure exhibits reduced effector functions (such as CDC and ADCC). Various in vitro and/or in viva cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A), Pro239Gly (P329G), Asn325Glu (N325E), and/or Ala327Ser (A327S). in some embodiments, modifications within the Fc region reduce binding to Fc-receptor-gamma receptors (FcyRs) while have minimal impact on binding to the neonatal Fc receptor (FcRn).
[0640]ln some embodiments, the human IgGl Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent glycosylation of the multispecific antigen-binding protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fc region of the multispecific antigenbinding protein is modified at amino acid Leu235 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the multispecific antigen-binding protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu234Ala (L.234A). In some embodiments, the Fc region of the multispecific antigenbinding protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the Fc region of the multispecific antigen-binding protein is altered at both amino acids 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A/L235A) or Leu234Val and Leu235Ala (L234V/L235A). In some embodiments, the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 297, e.g., Leu234Ala, Leu235Ala, Asn297Ala (L234A/L235A/N297A). In some embodiments, the Fc region of the multispecific antigenbinding protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro239Ala (L234A/L235A/P329A). In some embodiments, the Fc region of the multispecific antigen-binding protein is modified at amino acid Asp265 (Kabat Numbering) to alter Fc receptor interactions, e.g., Asp265Ala (D265A). In some embodiments, the Fc region of the multispecific antigen-binding protein is modified at amino acid Pro329 (Kabat Numbering) to alter Fc receptor interactions, e.g., Pro329Ala (P329A) or Pro329Gly (P329G). In some embodiments, the Fc region of the multispecific antigen-binding protein is altered at both amino acids 265 and 329, e.g., Asp265Ala and Pro329Ala (D265A/P329A) or Asp265Ala and Pro329Gly (D265A/P329G). In some embodiments, the Fc region of the multispecific antigenbinding protein is altered at amino acids at 234, 235, and 265, e.g., Leu234Ala, Leu235Ala, Asp265Ala (L234A/L235A/D265A). In some embodiments, the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro329Gly
(L234A/L235A/P329G). In some embodiments, the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, 265 and 329, e.g., Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A/L235A/D265A/P329G). In some embodiments, the Fc region of the multispecific antigen-binding protein is altered at Gly235 to reduce Fc receptor binding. For example, wherein Gly235 is deleted from the multispecific antigen-binding protein. In some embodiments, the human IgGl Fc region is modified at amino acid Gly236 to enhance the interaction with CD32A, e.g., Gly236Ala (G236A). In some embodiments, the human IgGl Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).
[0641]ln some embodiments, the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Gly, Leu235Ser, Gly236Arg (L234G/L235S/G236R). in some embodiments, the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Thr, Giy236Arg (L234S/L235T/G236R). In some embodiments, the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Val, Gly236Arg (L234S/L235V/G236R). in some embodiments, the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Gln, Gly236Arg (L234T/L235Q/G236R). In some embodiments, the Fc region of the multispecific antigen-binding protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Thr, Gly236Arg (L234T/L235T/G236R), In some embodiments, the Fc region of the multispecific antigenbinding protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Thr, Leu235Thr, Pro329Gly (L234A/L235A/P329G). In some embodiments, the Fc region of the multispecific antigen-binding protein is altered at amino acids at 252, 254, and 256, e.g,, Met252Tyr, Ser254Thr, Thr256Glu (M252Y/S254T/T256E).
[0642]ln some embodiments, the Fc region of the multispecific antigen-binding protein is lacking an amino acid at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (1235). In some embodiments, the Fc region of the multispecific antigenbinding protein is lacking an amino acid at one or more of the following positions Glu233 (E233), Leu234 (L234), or Leu235 (L235) and is modified at one or more of the Asp265 (D265), Asn297 (N297), or Pro329 (P329) to reduce Fc receptor binding. For example, an Fc region included in a TNFR2 binding polypeptide is derived from a human Fc domain, and comprises a three amino acid deietion in the lower hinge corresponding to IgGl E233, L234, and L235. In some embodiments, such Fc polypeptides do not engage FcyRs and thus are referred to as "effector silent" or "effector null." For example, Fc deletion of these three amino acids reduces the complement protein Clq binding. In some embodiments, a polypeptide with an Fc region with Fc deletion of these three amino acids retains binding to FcRn and therefore has extended half-life and transcytosis associated with FcRn mediated recycling.
[0643]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino add sequence: IgGl L234A, L235A (also known as "LALA" variant) (mutations bolded in the sequence below)
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDiA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:
4045).
[0644]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence: igGl L234A, L235A, and P329A (aiso known as "LALAPA" variant) (mutations bolded in the sequence below)
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:
4046),
[0645]ln one embodiment, the variant of human IgGl Fc region having the amino acid sequence of SEQ
ID NO: 4045 or 4046 further comprises a lysine residue at the C-terminus,
[0646]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence:
IgGl D265A, N297A and P329A (also known as "DANAPA" variant) (mutations bolded in the sequence below)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST YRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:
4047).
[0647]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence:
IgGl L234A, L235A, and G237A (also known as "LALAGA" variant) (mutations bolded in the sequence below)
DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:
4048),
[0648]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence:
IgGl L234G/L235S/G236R (mutations bolded in the sequence below) DKTHTCPPCPAPEGSRGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:
4054).
[0649]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence:
IgGl L234S/L235T/G236R (mutations bolded in the sequence below)
DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:
4055).
[0650]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence:
IgGl L234S/L235V/G236R (mutations bolded in the sequence below)
DKTHTCPPCPAPESVRGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:
4056).
[0651]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence: igGl L234T/L235Q/G236R (mutations bolded in the sequence below)
DKTHTCPPCPAPETQRGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:
4057).
[0552]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence: igGl L234T/L235T/G236R (mutations bolded in the sequence below)
DKTHTCPPCPAPETTRGPSVFLFPPKPKDTLMISRTPEVICWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYICTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:
4058). [0653]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgGl Fc region, having an amino acid sequence:
IgGl L234A/L235A/P329G (mutations bolded in the sequence below)
DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:
4059),
[0654jln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human igGl Fc region, having an amino acid sequence:
IgGl M252Y/S254T/T256E (mutations bolded in the sequence below)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYIOTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:
4060),
[0655]ln some embodiments, the human IgG Fc region is modified to enhance FcRn binding. Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively) (Kabat numbering, Dall’Acqua et al 2006, J. Biol Chem Vol, 281(33) 23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157-159), or Met252lle, Thr256Asp, Met428Leu (M252I, T256D, M428L, respectively) (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest), Asn434Ala (N434A), Asn434Trp (N434W), Thr256Asp, Thr307Gln (T256D/T307Q), Thr256Asp, Thr307Trp (T256D/T307W), Met252Tyr, Thr256Asp (M252Y/T256D), Thr307Gln, Gln311Val, Ala378Val (T307Q/Q311V/A378V), Thr256Asp, His286Asp, Thr307Arg, Gln311Val, Ala378Val (T256D/H286D/T307R/Q311V/A378V), or Leu309Asp, Gln311His, Asn434Ser (L309D/Q311H/N434S) (see, Ko et al., BioDrugs (2021) 35:147-157).
[0656]ln some embodiments, the Fc region lacks or has reduced fucose atached to the N-linked glycan- chain at N297, There are numerous ways to prevent fucosylation, including but not limited to production in a FUT8 deficient cell line; addition inhibitors to the mammalian cell culture media, for example Castanospermine; and metabolic engineering of the production cell line.
[0657]ln some embodiments, the Fc domain included in a multispecific antigen-binding protein or conjugate of the present disclosure Is derived from a human Fc domain and comprises mutations M252Y and M428V. In some embodiments, the mutated or modified Fc polypeptide includes the following mutations: M252Y and M428L using the Kabat numbering system. In some embodiments, such mutations enhance binding to FcRn at the acidic pH of the endosome (near 6.5), while iosing detectable binding at neutral pH (about 7.2), allowing for enhanced FcRn mediated recycling and extended half-life. [0658]ln some embodiments, the Fc domain included in a multispecific antigen-binding protein or conjugate is derived from a human Fc domain and comprises mutations to induce heterodimerization. In some embodiments, such mutations include those referred to as "knob" and "hole" mutations. For example, having an amino acid modification within the CH3 domain at Thr366, which when replaced with a bulkier amino acid, e.g., Try (T366W), is able to preferentially pair with a second CH3 domain having amino acid modifications to less bulky amino acids at positions Thr366, Leu368, and Tyr407, e.g., Ser, Ala, and Vai, respectively (T366S/L368A/Y407V). In some embodiments, the "knob" Fc domain comprises the mutation T366W. In some embodiments, the "hole" Fc domain comprises mutations T366S, L368A, and Y407V. Heterodimerization via CH3 modifications can be further stabilized by the introduction of a disulfide bond, for example by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on opposite CH3 domains (Reviewed in Carter, 2001 Journal of Immunological Methods, 248: 7-15). In some embodiments, Fc domains used for heterodimerization comprise additional mutations, such as the mutation S354C on a first member of a heterodimeric Fc pair that forms an asymmetric disulfide with a corresponding mutation Y349C on the second member of a heterodimeric Fc pair. In some embodiments, one member of a heterodimeric Fc pair comprises the modification H435R or H435K to prevent protein A binding while maintaining FcRn binding, In some embodiments, one member of a heterodimeric Fc pair comprises the modification H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the hole Fc domain comprises the modification H435R or H435K (referred to as "hole-R" in some instances when the modification is H435R), while the knob Fc domain does not. In some instances, the hole-R mutation improves purification of the heterodimer over homodimeric hole Fc domains that may be present.
[0659]ln some embodiments, the human IgG Fc region is modified to prevent dimerization. In these embodiments, the multispecific antigen-binding proteins of the present disclosure are monomeric. For example, modification at residue Thr366 to a charged residue, e.g., Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Giu (T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3 dimerization.
[0660]ln some embodiments, the immunoglobulin Fc region of the multispecific antigen-binding protein is of human lgG3 isotype, or a variant thereof, in one embodiment, the lgG3 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent to glycosylation of the antibody, e.g., Asn297Aia (N297A) or Asn297Asp (N297D). in some embodiments, the human lgG3 Fc region is modified at amino acid 435 to extend the half-life, e.g., Arg435His (R435H). In some embodiments, the human lgG3 Fc region lacks Lys447 (EU index of Kabat et al 1991).
[0661]ln some embodiments, the immunoglobulin Fc region of the multispecific antigen-binding protein is of human lgG4 isotype, or a variant thereof.
[0662]As a non-limiting example, an immunoglobulin Fc region of human lgG4 isotype may have an amino acid sequence:
ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 10728)
[0663]! n some embodiments, the immunoglobulin Fc region of human lgG4 isotype may comprise the amino acid sequence set forth in SEQ ID NO: 10728, or a similar sequence thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0664]ln some embodiments, the human lgG4 Fc region comprises one or more mutations selected from, e.g., Ser228Pro (S228P), Leu235Glu (L235E), Leu235Ala (L235A), Phe234Ala (F234A), and/or Pro329Gly (P329G) according to EU numbering.
[0665]ln one embodiment, the human lgG4 Fc region is modified at amino acid 235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the human lgG4 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent to glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human lgG4 Fc region is lacks Lys447 (EU index of Kabat et al 1991).
[0666]ln some embodiments, the lgG4 Fc region of the fusion protein is altered at amino acids at 228 and 235, e.g., Ser228Pro, Leu235Glu or Leu235Ala (S228P/L235E or S228P/L235A). In some embodiments, the lgG4 Fc region of the fusion protein is altered at amino acids at 228, 234 and 235, e.g., Ser228Pro, Phe234Ala, Leu235Glu or Leu235Ala (S228P/F234A/L235E or S228P/F234A/L235A). In some embodiments, the lgG4 Fc region of the fusion protein is altered at amino acids at 228, 235, and 329, e.g., Ser228Pro, Leu235Glu and P329G (S228P/L235E/P329G).
[0667)1 n various embodiments, the Fc domain comprises an amino acid sequence according to SEQ ID NO: 4049, 4053, and/or 6365, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In various embodiments, the Fc domain is encoded by a nucleic acid sequence that encodes an amino acid sequence according to SEQ ID NOs: 4049, 4053, and/or 6365, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0668]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 S228P, L235E (mutations bolded in the sequence below)
ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4049).
[0669]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 S228P, L235A (mutations bolded in the sequence below)
ESKYGPPCPPCPAPEFAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4050).
[0670]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 S228P, F234A, L235E (mutations bolded in the sequence below)
ESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD iAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4051).
[0671]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human lgG4 Fc region, having an amino acid sequence: igG4 S228P, F234A, L235A (mutations bolded in the sequence below)
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4052).
[0672]ln one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human lgG4 Fc region, having an amino acid sequence: igG4 P329G, S228P, L235E (mutations bolded in the sequence below) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DI AVE WESNGQP EN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG N VFSCSVM HEALH N HYTQKSLSLSLG K (SEQ I D NO: 4053).
[0673]ln one embodiment, the Immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human igG4 Fc region, having an amino acid sequence: ESKYGPPCPSCPESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK (SEQ ID NO: 6365).
[0674]Additional lgG4 heavy chain modifications suitable for use in the multispecific antigen-binding proteins or conjugates of the present disclosure include those described in Tables 1 and 2 of Dumet et al., mAbs, 11:8, 1341-1350, which is incorporated herein by reference in its entirety.
[O675J8 n some embodiments, the multispecific antigen-binding protein or conjugate contains an immunoglobulin hinge region, in some embodiments, the hinge region serves as a linker to connect one or more anti-TNFR2 binding domains and/or one or more anti-CD25 antigen-binding domains (e.g., VHHs) to the Fc region. In other embodiments, the multispecific antigen-binding protein can comprise a linker in addition to the hinge region to connect the one or more anti-TNFR2 binding domains and/or one or more anti-CD25 antigen-binding domains (e.g., VHHs) to the Fc region. The hinge region can be selected from any of the human igG subclasses. For example, the multispecific antigen-binding protein may contain a modified IgGl hinge having the sequence of EPKSSDKTHTCPPC (SEQ ID NO: 3923), wherein the Cys220 that typically forms a disulfide bond with the C-terminal cysteine of the light chain is mutated to serine, e.g., Cys220Ser (C220S). In other embodiments, the multispecific antigen-binding protein contains a truncated hinge having a sequence DKTHTCPPC (SEQ ID NO: 3924). In other embodiments, the fusion protein contains a truncated hinge having a sequence DKTHTCPPCP (SEQ ID NO: 10729).
[0676]ln some embodiments, the multispecific antigen-binding protein or conjugate has a modified hinge from igG4, which is modified to prevent or reduce strand exchange, e.g., Ser228Pro (S228P), having the sequence ESKYGPPCPPC (SEQ ID NO: 3925).
[0677]in alternative embodiments, a multispecific antigen-binding protein or conjugate of the present disclosure may comprise sequences other than an Fc region to achieve muitimerization (e.g., dimerization). For example, an amino acid sequence containing at least one cysteine residue may be included to facilitate dimerization of two polypeptides by formation of a disulfide bond between the two polypeptides. In some embodiments, such multimerizing domain may comprise one or more cysteine residues, or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif. [0678jFc mutations suitable for use in the multispecific antigen-binding proteins disclosed herein are also discussed in, e.g., Wilkinson et al., Fc-engineered antibodies with immune effector functions completely abolished. PLoS One. 2021; WO2021234402A2; US 8,969,526; EP3632065B1; and US 7,083,784, each of which is incorporated herein by reference.
Fusion or Conjugation to Half-Life Extension Moieties
[0679]in some embodiments, a multispecific antigen-binding protein or conjugate of the present disclosure may comprise one or more other moieties which provide the multispecific antigen-binding protein or conjugate with increased (in vivo) half-life, in vivo half-life extension means, that the multispecific antigen-binding protein or conjugate has an increased half-life in a mammal, such as a human subject, after administration.
[0680]Non-limiting examples of half-life extension moieties suitable for use in the present disclosure include polyethylene glycol (PEG) molecules, serum proteins or fragments thereof, binding domains that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.
[0681]ln some embodiments, a multispecific antigen-binding protein or conjugate of the present disclosure may comprise a binding moiety that can bind to serum albumin, such as human serum albumin, or a serum immunoglobulin, such as IgG. In one embodiment, a multispecific antigen-binding protein or conjugate of the present disclosure may comprise a binding moiety that can bind to human serum albumin. In one embodiment, the binding moiety is a single-domain antibody (e.g., VHH).
[0682]For example and without limitation, albumin binders that are described in, e.g., WO 04/041865, WO 06/122787, W02012/175400, WO 2012/175741, WO2015/173325, W02017/080850, WO2017/085172, WO2018/104444, W02018/134235, WO2018/134234, each of which Is Incorporated herein by reference is its entirety, can be used in the multispecific antigen-binding protein or conjugate of the present disclosure.
Fusion or Conjugation to Cytokines
[0683]!n some embodiments, a multispecific antigen-binding protein or conjugate of the present disclosure may comprise one or more cytokine molecules. Non-limiting exemplary cytokine molecules that may be conjugated with include interleukin-2 (I L-2), transforming growth factor beta (TGF-[S), thymic stromal lymphopoietin (TSLP), or a variant or combination thereof,
[0684]The cytokine IL-2 plays a major role in the activation and function of Tregs. Incorporating IL-2 into the multispecific antigen-binding protein or conjugate of the present disclosure may enhance the ability of the anti-TNFR2 antigen-binding protein to promote Tregs expansion and stabilization.
[0685]IL-2-containing multispecific antigen-binding proteins may be prepared as described in e.g., US 10,174f091; WQ2014/023752, W02019/246404, each of which is incorporated by reference in its entirety.
[0686]i n one embodiment, the IL-2 molecule used in the multispecific antigen-binding proteins or conjugates of the present disclosure is a wild-type IL-2 having the amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR PRDLISNINVIVLELKGSETTFMCE¥ADETATIVEFLNPWITFCQSIISTLT (SEQ ID NO: 3926)
[0687]ln some embodiments, the IL-2 molecule used in the multispecific antigen-binding proteins or conjugates of the present disclosure is a variant of IL-2 having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3926.
[0688]ln one embodiment, the IL-2 molecule used in the multispecific antigen-binding proteins or conjugates of the present disclosure is a variant of IL-2 with a N88D mutation (bolded in the sequence below), having the amino acid sequence:
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR PRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWiTFCQSIISTLT (SEQ ID NO: 3927)
[0689]Qther suitable IL-2 variants that can be used in the multispecific antigen-binding proteins or conjugates of the present disclosure include those described in US 10,174,091, US 10,174,092, US 11,091,526, US 11,091,527, WO2016/164937, US 9,580,486, US 7,105,653, US 9,616,105, US 9,428,567, US2017/0051029, US2014/0286898A1, WO2014/153111, W02010/085495, WO2016/014428, WO2016/025385, and US2006/0269515, each of which are incorporated by reference in its entirety.
Fusion or Conjugation to Other Moieties
[0690] Multispecific antigen-binding proteins provided herein may be operably linked, directly or indirectly, to a second moiety, such as but not limited to, a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytokine, a cytotoxic agent, a small molecule drug, a chemotherapeutic agent, a therapeutic agent, a diagnostic agent, or a combination thereof. [0691]ln some embodiments, a conjugate of the present disclosure comprises a label, which can generate a detectable signal. Such conjugates can be used for research or diagnostic purposes, such as for the in vivo detection of cancer. Preferably, the label is capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope (such as 3H, 14C, 32P, 35S, 1231, 1251, 1311); a fluorescent (fluorophore) or chemiluminescent (chromophore) compound (such as fluorescein isothiocyanate, rhodamine or luciferin); an enzyme (such as (5- galactosidase, alkaline phosphatase, or horseradish peroxidase); an imaging agent; or a metal ion. In some embodiments, the label is a radioactive atom for scintigraphic studies, for example 99Tc or 1231, or a spin label for nuclear magnetic resonance (NMR) imaging, such as zirconium-89, iodine-123, iodine- 131, indium-ill, fluorine-19, carbon-13, nltrogen-15, oxygen-17, gadolinium, manganese or iron. Zirconium-89 may also be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011/056983).
[0692]IMu Itispecific antigen-binding proteins of the present disclosure may be conjugated to another moiety, such as an epitope tag, e.g., for the purpose of purification or detection. Examples of such molecules that are useful in protein purification include those that present structural epitopes capable of being recognized by a second molecule. This is commonly employed in protein purification by affinity chromatography, in which a molecule is immobilized on a solid support and exposed to a heterogeneous mixture containing a target protein conjugated to a molecule capable of binding the immobilized compound. Non-limiting examples of epitope tag molecules that can be conjugated to multispecific antigen-binding proteins of the present disclosure, e.g., for the purposes of molecular recognition include a poly-histidine tag (His-tag), a myc-tag, human influenza hemagglutinin (HA) tag, a FLAG-tag, maltose-binding protein, glutathione-S-transferase, biotin, and streptavidin. Conjugates containing the epitopes presented by these molecules are capable of being recognized by complementary molecules such as maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, streptavidin, or biotin, respectively. For example, one can purify a muitispecific antigen-binding protein of the present disclosure that has been conjugated to an epitope tag from a complex mixture of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc.) by treating the mixture with a solid phase resin containing a complementary molecule that can selectively recognize and bind the epitope tag of the TNFR2 antibody or fragment thereof. Examples of solid phase resins include agarose beads, which are compatible with purifications in aqueous solution. [0693]ln some embodiments, a conjugate of the present disclosure may comprise a multispecific antigen-binding protein described herein conjugated to a therapeutic agent, which can be cytotoxic, cytostatic or otherwise provides some therapeutic benefit, in some embodiments, the cytotoxic agent is a drug, a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate). Such conjugates may be applicable to, e.g., the treatment or prevention of a disease associated with autoreactive cytotoxic T-cell activity. In some embodiments, antibody drug conjugates described herein may allow targeted delivery of a drug moiety to a target tissue (e.g., tumors). [0694]ln some embodiments, a conjugate of the present disclosure comprises a toxin, in some embodiments, the toxin includes, for example, bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al., J. Nat. Cancer Inst. 92(19):1573-1581 (2000); Mandler et al„ Bioorganic & Med. Chem. Letters 10:1025-1028 (2000); Mandler et al,, Bioconjugate Chem. 13:786-791 (2002)), maytansinolds (EP 1391213; Liu et al.. Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode et al., Cancer Res. 58:2928 (1998); Hinman et al., Cancer Res, 53:3336-3342 (1993)). The toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DMA binding, or topoisomerase inhibition. Examples of other therapeutic agents that can be conjugated to a multispecific antigen-binding protein of the present disclosure are described herein (see "Treatment Methods and Other Uses" section), [0695]ln some embodiments, multispecific antigen-binding proteins of the present disclosure may be fused or conjugated to one or more moieties that facilitate delivery to the central nervous system (CNS)/brain. The moiety that can facilitate delivery of a multispecific antigen-binding protein described herein to the central nervous system (CNS)/brain can be for example, a peptide, a polypeptide, small molecule, a lipid, or a synthetic polymer. Various approaches to deliver single-domain antibodies into the brain are described in Pothin et al., Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.
[0696jAs a non-limiting example, a multispecific antigen-binding protein of the present disclosure may be fused or conjugated to a moiety (e.g., an antibody) that binds to the transferrin receptor (Tf R) or insulin receptor. The transferrin receptor (Tf R) is highly expressed by brain capillary endothelial cells (BCECs) forming the blood-brain barrier (BBS) and has been utilized as a target for brain drug delivery. Monoclonal antibodies binding to the TfR, such as clone RI7, have been shown to internalize into BCECs in vivo. As another example, a multispecific antigen-binding protein of the present disclosure may be conjugated to hydrophobic fatty acid moieties, such as C18 fatty acid (stearic acid), C16 fatty acid (palmitic acid) or C8 fatty acid (octanoic acid) moieties; or amphiphilic block copolymer moieties, such as polyethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (pluronics or poloxamers) or poly(2- oxasolines). Various faty add moieties and block copolymer moieties that can be utilized for brain delivery of proteins are described in, e.g., Yi and Kabanov, J Drug Target. 2013; 21(10): 940-955, which is incorporated herein by reference in its entirety.
[0697] Exam pie methods for attaching a moiety, such as a label, to a binding protein include those described in Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); Nygren, J. Histochem. and Cytochem. 30:407 (1982); Wensel and Meares, Elsevier, N.Y. (1983); and Colcher et al., Meth. Enzymol., 121 :802-16 (1986). Additional suitable methods for preparing the conjugates of the present disclosure include those described in, e.g., WO 2009/067800, WO 2011/133886, and US2014322129, incorporated by reference herein in their entirety. [0698]ln some embodiments, the attachment between a multispecific antigen-binding protein and a second moiety can be covalent or non-covalent, e.g., via a biotin-streptavidin non-covalent interaction. In some embodiments, a second moiety can be attached to a multispecific antigen-binding protein described herein using any of various molecular biological or chemical conjugation and linkage methods known in the art and described below. In some embodiments, linkers such as peptide linkers, cleavable linkers, non-cleavable linkers or linkers that aid in the conjugation reaction, can be used to link or conjugate a second moiety to a multispecific antigen-binding protein described herein.
[0699]ln some embodiments, a multispecific antigen-binding protein is conjugated to one or more second moieties, e.g., about 1 to about 20 moieties per molecule, optionally via a linker, in some embodiments, the one or more second moieties can be the same or different. The linker may be composed of one or more linker components. For covalent attachment of an antibody and the second moiety, the linker typically has two reactive functional groups, i.e., bivalency in a reactive sense. Bivalent linker reagents which are useful to attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups have been described in, e.g., Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, p 234-242.
[0700]ln some embodiments, a linker used in a conjugate of the present disclosure may include 6- maleimidocaproyl ("MC"), maleimidopropanoyl ("MP")/ valine-citrulline ("val-cit"), a alaninephenylalanine ("aia-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-Succinimidyl 4-(2- pyridylthiojpentanoate ("SPP"), N-Succinimidyl 4-(N~maleimidomethyl)cyclohexane-i carboxylate ("SMCC"), or N-Succinimidyl(4-iodo-acetyl)aminobenzoate ("STAB"), or a combination thereof.
[0701]ln some embodiments, a linker used in a conjugate of the present disclosure may comprise amino acid residues. Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues used in an amino acid linker component may include naturally occurring amino acids, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
(0702]Conjugates of a multispecific antigen-binding protein and second moiety (e.g., cytotoxic agent) can be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2- pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCi), active esters {such as disuccinimidyl substrate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,S- diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
[0703]Conjugates of the present disclosure can be prepared by a variety of methods, For example, the conjugation method may include: (1) reaction of a nucleophilic group of a VHH domain with a bivalent linker reagent, to form VHH-Linker, via a covalent bond, followed by reaction with a drug moiety; or (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form drug-linker, via a covalent bond, followed by reaction with the nucleophilic group of a VHH domain.
[0704]Nucleophilic groups on proteins including antibodies (e.g., VHH domains), include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups (e.g., lysine), (iii) side chain thiol groups (e.g., cysteine), and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (I) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Additional nucleophilic groups can be introduced into proteins (e.g., antibodies such as VHH domains) through the reaction of lysines with 2- iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into a protein (e.g., antibody such as a VHH domain) by introducing one, two, three, four, or more cysteine residues.
[0705]Conjugates, such as antibody drug conjugates, may also be produced by modification of an antibody, such as a VHH domain, to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. The sugars of glycosylated antibodies may be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups which may lead with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g., by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium metaperiodate, resulting in production of an aldehyde in place of the first amino acid. Such aldehyde can be reacted with a drug moiety or linker nucleophile.
[0706] Likewise, nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (I) active esters such as NHS esters, HOBi esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (ill) aldehydes, ketones, carboxyl, and maleimide groups.
[0707]Alternatively, a multispecific antigen-binding protein containing a VHH domain and cytotoxic agent may be made, e.g., by recombinant DNA techniques or peptide synthesis. A DNA sequence may be engineered to comprise respective regions encoding the two portions of the multispecific antigenbinding protein either adjacent to one another or separated by a region encoding a linker peptide which does not impair the desired properties of the multispecific antigen-binding protein. The DNA sequence can be then transfected into a host cell that expresses the multispecific antigen-binding protein. The multispecific antigen-binding protein can be recovered from the cell culture and purified using techniques known in the art.
Linkers
[0708]ln some embodiments, the one or more polypeptides of the multispecific antigen-binding proteins of the present disclosure are operably linked via peptide linkers. A peptide linker can range from 2 amino acids to 60 or more amino acids, and in certain aspects a peptide linker ranges from 3 amino adds to 50 amino acids, from 4 to 30 amino adds, from 5 to 25 amino acids, from 10 to 25 amino acids, 10 amino adds to 60 amino acids, from 12 amino acids to 20 amino acids, from 20 amino adds to 50 amino adds, or from 25 amino acids to 35 amino acids in length. [0709]ln some embodiments, a peptide linker, e.g., a peptide linker separating two VHH domains or an VHH domain and a heavy chain constant region, is at least 5 amino acids, at least 6 amino acids or at least 7 amino acids in length and optionally is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids or up to 60 amino acids in length.
[0710]ln some embodiments, the linker ranges from 5 amino acids to 50 amino acids in length, e.g., ranges from 5 to 50, from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, or from 5 to 20 amino adds in length. In other embodiments of the foregoing, the linker ranges from 6 amino adds to 50 amino acids in length, e.g., ranges from 6 to 50, from 6 to 45, from 6 to 40, from 6 to 35, from 6 to 30, from 6 to 25, or from 6 to 20 amino acids in length. In yet other embodiments of the foregoing, the linker ranges from 7 amino acids to 50 amino adds in length, e.g., ranges from 7 to 50, from 7 to 45, from 7 to 40, from 7 to 35, from 7 to 30, from 7 to 25, or from 7 to 20 amino adds in length.
[0711]ln some embodiments, charged (e.g., charged hydrophilic linkers) and/or flexible linkers are used. Examples of flexible linkers that can be used in the multispecific antigen-binding proteins of the disclosure include those disclosed by Chen et al, 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et a/., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible linkers are or comprise repeats of glycines and serines (termed "GS-linker" herein), e.g., a monomer or multimer of GnS (SEQ ID NO: 4013) or SG„ (SEQ ID NO: 4014), where n is an integer from 1 to 10, e.g., 1 2, 3, 4, 5, 6, or 7, 8, 9 or 10. In one embodiment, the linker is or comprises a monomer or multimer of repeat of G<S (SEQ ID NO: 3969), e.g., (GGGGS)n (SEQ ID NO: 4015), In one embodiment, the linker is or comprises a monomer or multimer of repeat of G3S (SEQ ID NO: 10730), e.g., (GGGS)n (SEQ, ID NO: 10731).
[0712]Polyglycine linkers can suitably be used in the multispecific antigen-binding proteins of the disclosure. In some embodiments, a peptide linker used herein comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Giy) (SEQ ID NO: 4016), five consecutive glycines (5Gly) (SEQ ID NO: 4017), six consecutive glycines (6Gly) (SEQ ID NO: 4018), seven consecutive glycines (7Gly) (SEQ ID NO: 4019), eight consecutive glycines (8Gly) (SEQ ID NO: 4020), or nine consecutive glycines (9Gly) (SEQ. ID NO: 4021).
[0713]ln some embodiments, a GS-linker used herein comprises an amino acid sequence selected from GGSGGS (SEQ ID NO: 4022), i.e., (GGS)2 (SEQ ID NO: 4022); GGSGGSGGS (SEQ ID NO: 4023), i.e., (GGS)3 (SEQ ID NG: 4023); GGSGGSGGSGGS (SEQ ID NO: 4024), i.e., (GGS)« (SEQ ID NO: 4024); GGSGGSGGSGGSGGS (SEQ ID NO: 4025), i.e., (GGS)s(SEQ ID NO: 4025); and GGGS (SEQ ID NO: 10732). In some embodiments, the multispecific antigen-binding proteins can include a combination of a GS-linker and a glycine linker.
[0714]ln one embodiment, two or more VHHs are linked via a GGGGS (SEQ ID NO: 3969) linker. In one embodiment, two or more VHHs are linked via a GGGGSGGGGS (SEQ ID NO: 4026) linker. In one embodiment, two or more VHHs are linked via a GGGGSGGGGSGGGGS (SEQ iD NO: 3970) linker, in one embodiment, two or more VHHs are linked via a GGGGSGGGGSGGGGSGGGGS (SEQ, ID NO: 3976) linker. In one embodiment, two or more VHHs are linked via a GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3977) linker. In one embodiment, two or more VHHs are linked via a GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 4001) linker. In one embodiment, two or more VHHs are linked via a GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 4002) linker. In one embodiment, two or more VHHs are linked via a GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 4012) linker, In one embodiment, two or more VHHs are linked via a GGGGSPAPAPAPAPGGGGS (SEQ ID NO: 6361) linker. In one embodiment, two or more VHHs are linked via a GGGGSPAPAPAPAPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 6362) linker. In one embodiment, two or more VHHs are linked via a GGGGSESKVGPPCPSCP (SEQ ID NO: 4008) linker.
[0715]! n one embodiment, a VHH and an Fc region are linked via a GGGGSESKYGPPCPSCP (SEQ ID NO: 4008) linker. In one embodiment, a VHH and an Fc region are linked via a GGGGS (SEQ ID NO: 3969) linker. In one embodiment, a VHH and an Fc region are linked via a GGGGSGGGGS (SEQ ID NO: 4026) linker.
[0716]!n some embodiments, the one or more polypeptides of the multispecific antigen-binding proteins of the present disclosure are operably linked via a "rigid" peptide linker. Such peptidic linker may comprise a proline-rich peptide. In one embodiment, a rigid peptide linker comprises PAPAPAPAPAPAPAPAP (SEQ ID NO: 4009). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAP (SEQ ID NO: 4010). In one embodiment, a rigid peptide linker comprises PAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 4011). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 4012). in one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGS (SEQ ID NO: 10733). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPGGGGS (SEQ ID NO: 6361). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAP APAPAPAPGGGGS (SEQ ID NO: 6362). In one embodiment, a rigid peptide linker comprises A(EAAAK)nA (SEQ ID NO: 4027), where n is any integer, e.g„ 1 2, 3, 4, 5, 6, or 7, 8, 9 or 10. [0717]Other exemplary peptide linkers that can be used in the multispecific antigen-binding proteins described herein are shown in Table 2.
Table 2. Exemplary Peptide Linker Sequences
Exemplary muhisssecific antigen-binding proteins of the present disclosure
[0718] N on-l Smiting examples of multispecific antigen-binding proteins (e.g., bivalent or tetravaient constructs with/without Fc regions, i L-2 fusion constructs) are disclosed in the "Examples" and "List of Sequences" sections below.
[0719]ln various embodiments, a multispecific antigen-binding protein of the present disclosure comprises one or more anti-TNFR2 binding domains as described herein and one or more anti-CD25 binding domains as described herein, joined by one or more linkers as described herein in Table 2, and optionally further comprising a Fc domain as described herein.
[0720]in various embodiments, a multispecific antigen-binding protein of the present disclosure comprises one or more anti-TNFR2 binding domains and one or more anti-CD25 binding domains, joined by one or more linkers as described herein in Table 2, wherein the one or more anti-TNFR2 binding domains and the one or more anti-CD25 binding domains comprise amino acid sequences selected from SEQ ID NOs: 4129, 4130, 81, 85, 83, 88, 92, 4128, 7, 90, 4120, 19, 35, 11, 4116, 15, and/or 4, or similar sequences thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto
[0721]! n a specific embodiment, the anti-TNFR2 binding domains comprise amino acid sequences selected from SEQ ID NOs: 4, 7, 11, 15, 19, 35, 81, 83, 85, 88, 90, and 92.
[0722]ln a specific embodiment, the anti-CD25 binding domains comprise amino acid sequences selected from SEQ ID NOs: 4116, 4120, 4128, 4129, and 4130,
[0723]ln various embodiments, a multispecific antigen-binding protein of the present disclosure comprises one or more anti-TNFR2 binding domains and one or more anti-CD2.5 binding domains, joined by one or more linkers as described herein in Table 2, wherein the one or more anti-TNFR2 binding domains and/or the one or more anti-CD25 binding domains comprise amino acid sequences selected from the following, from N-terminus to C-terminus, or similar sequences thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto:
1) SEQ ID NOs: 7, 7, 7, and 7;
2) SEQ ID NOs: 4120, 7, 7, 7, and 7;
3) SEQ ID NOs: 4120, 19, 19, 19, and 19;
4) SEQ ID NOs: 19, 19, 19, and 19;
5) SEQ ID NOs: 35, 35, 35, 35, and 35;
6) SEQ ID NOs: 4120, 11, 11, 11, and 11;
7) SEQ ID NOs: 11, 11, 11, and 11;
8) SEQ ID NOs: 4129, 81, and 81;
9) SEQ ID NOs: 4129, 83, and 83;
10) SEQ ID NOs: 4129, 81, and 83;
11) SEQ ID NOs: 4129, 88, and 88;
12) SEQ ID NOs: 4129, 92, and 92;
13) SEQ ID NOs: 4129, 88, and 90;
14) SEQ ID NOs: 4130, 81, and 81;
15) SEQ ID NOs: 4130, 83, and 83;
16) SEQ ID NOs: 4130, 88, and 88;
17) SEQ ID NOs: 4130, 92, and 92;
18) SEQ ID NOs: 4128, 81, and 81;
19) SEQ ID NOs: 4128, 83, and 83;
20) SEQ ID NOs: 4128, 83, and 88;
21) SEQ ID NOs: 4128, 92, and 92;
22) SEQ ID NQs: 4129, 4130, and 88;
23) SEQ ID NOs: 4129, 4130, and 92;
24) SEQ ID NOs: 7 and 7;
25) SEQ ID NOs: 15 and 15;
26) SEQ ID NOs: 15, 15, 15 and 15;
27) SEQ ID NOs: 4 and 4; 28) SEQ ID NOs: 4, 4, 4, and 4;
29) SEQ ID NOs: 19 and 19;
30) SEQ ID NOs: 19, 19, 19, and 19;
31) SEQ ID NOs: 35, 35, 35, and 35;
32) SEQ ID NOs: 11 and 11; and
33) SEQ ID NOs: 11, 11, 11, and 11.
[0724]ln various embodiments, a multispecific antigen-binding protein of the present disclosure comprises one or more anti-TNFR2 binding domains and one or more anti-CD25 binding domains, joined by one or more linkers as described herein in Table 2, and the multispecific antigen-binding protein further comprises an Fc domain as described herein, wherein the one or more anti-TNFR2 binding domains and the one or more anti-CD25 binding domains comprise amino acid sequences selected from SEQ ID NOs: 4129, 4130, 81, 85, 83, 88, 92, 4128, 7, 90, 4120, 19, 35, 11, 4116, 15, and/or 4, or similar sequences thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0725]! n a specific embodiment, the anti-TNFR2 binding domains comprise amino acid sequences selected from SEQ ID NOs: 4, 7, 11, 15, 19, 35, 81, 83, 85, 88, 90, and 92.
[0726]!n a specific embodiment, the anti-CD25 binding domains comprise amino acid sequences selected from SEQ ID NOs: 4116, 4120, 4128, 4129, and 4130.
[0727]! n various embodiments, the Fc domain comprises an amino acid sequence according to SEQ ID NO: 4049, 4053, and/or 6365, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In various embodiments, the Fc domain is encoded by a nucleic acid sequence that encodes an amino acid sequence according to SEQ ID NOs: 4049, 4053, and/or 6365, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0728]!n various embodiments, a multispecific antigen-binding protein of the present disclosure comprises one or more anti-TNFR2 binding domains and one or more anti-CD25 binding domains, joined by one or more linkers as described herein in Table 2, and the multispecific antigen-binding protein further comprises an Fc domain as described herein, wherein the one or more anti-TNFR2 binding domains and the one or more anti~CD25 binding domains comprise amino acid sequences selected from SEQ ID NOs: 4129, 4130, 81, 85, 83, 88, 92, 4128, 7, 90, 4120, 19, 35, 11, 4116, 15, and/or 4, or similar sequences thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and wherein the Fc domain comprises an amino add sequence according to SEQ ID NO: 4049, 4053, and/or 6365, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0729]ln various embodiments, a multispecific antigen-binding protein of the present disclosure comprises one or more anti-TNFR2 binding domains and one or more anti-CD25 binding domains, joined by one or more linkers as described herein in Table 2, and the multispecific antigen-binding protein further comprises an Fc domain as described herein, wherein the one or more anti-TNFR2 binding domains and the one or more anti-CD25 binding domains comprise amino acid sequences selected from the following, from N-terminus to C-terminus, or similar sequences thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto:
1) SEQ ID NOs: 83, 83, 83, and 4049;
2) SEQ ID NOs: 83, 83, and 4049;
3) SEQ ID NOs: 81, 83, and 4049;
4) SEQ ID NOs: 88, 90, and 4049;
5) SEQ ID NOs: 83, 83, 4049, and 4129;
6) SEQ ID NOs: 81, 83, 4049, and 4129;
7) SEQ ID NOs: 88, 90, 4049, and 4129;
8) SEQ ID NOs: 83, 83, 4049, and 4130;
9) SEQ ID NOs: 81, 83, 4049, and 4130;
10) SEQ ID NOs: 4129, 4049, 83, and 83;
11) SEQ ID NOs: 4129, 4049, 81, and 83;
12) SEQ ID NQs: 83, 4049, and 83;
13) SEQ ID NOs: 4129, 83, 4049, and 83;
14) SEQ ID NOs: 4130, 83, 4049, and 83;
15) SEQ ID NOs: 4129, 4049, and 88;
16) SEQ ID NOs: 4129, 4049, and 92;
17) SEQ ID NOs: 88, 4049, and 4129; ) SEQ ID NOs: 92, 4049, and 4129;) SEQ ID NOs: 88 and 4049; ) SEQ ID NOs: 92 and 4049; ) SEQ ID NOs: 4130, 4049, and 88;) SEQ ID NOs: 4130, 4049, and 92;) SEQ ID NOs: 4129, 88, and 4049;) SEQ ID NOs: 4129, 92, and 4049;) SEQ ID NOs: 4130, 88, and 4049;) SEQ ID NOs: 4130, 92, and 4049;) SEQ ID NOs: 4128, 88, and 4049;) SEQ ID NOs: 4128, 92, and 4049;) SEQ ID NOs: 81, 81, 81, and 4049;) SEQ ID NOs: 88, 88, and 4049; ) SEQ ID NOs: 92, 92, and 4049; ) SEQ ID NOs: 88, 85, and 4049; ) SEQ ID NOs: 81, 85, and 4049; ) SEQ ID NOs: 7, 7, and 6365; ) SEQ ID NOs: 7, 6365, and 7; ) SEQ ID NOs: 4116, 4053, and 7;) SEQ ID NOs: 15, 15, and 6365; ) SEQ ID NOs: 15, 6365, and 15; ) SEQ ID NOs: 4116, 4053, and 15;) SEQ ID NOs: 4, 4, and 6365; ) SEQ ID NOs: 4, 6365, and 4; ) SEQ ID NOs: 4116, 4053, and 4;) SEQ ID NOs: 19, 19, and 6365; ) SEQ ID NOs: 19, 6365, and 19; ) SEQ ID NOs: 4116, 4053, and 19;) SEQ ID NOs: 35, 6365, and 35; ) SEQ ID NOs: 11, 11, and 6365; ) SEQ ID NOs: 11, 6365, and 11; and) SEQ ID NOs: 4116, 4053, and 11; [0730]ln various embodiments, a multispecific antigen-binding protein of the present disclosure comprises one or more anti-TNFR2 binding domains and one or more anti-CD25 binding domains, joined by one or more linkers as described herein in Table 2, and the multispecific antigen-binding protein further comprises an Fc domain (e.g., IgGl LALAPA) as described herein, wherein the one or more anti- TNFR2 binding domains comprise an amino acid sequences selected from SEQ ID NOs: 10461, 10468, 10469, 10470, 10477, and 10812, or similar sequences thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and wherein the one or more anti-CD25 binding domains comprise an amino acid sequence selected from SEQ ID NOs: 9439, 9422, 10572, and 10575, or similar sequences thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0731]ln various embodiments, a multispecific antigen-binding protein of the present disclosure comprises one or more anti-TNFR2 binding domains and one or more anti-CD25 binding domains, joined by one or more linkers as described herein in Table 2, and the multispecific antigen-binding protein further comprises an Fc domain (e.g,, IgGl LALAPA) as described herein, wherein the one or more anti- TNFR2 binding domains comprise a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 10414, 10421, 10422, 10423, 10429, and 10811; and wherein the one or more anti-CD25 binding domains comprise a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9436; a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NQ: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9416; a CORI comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10531; or a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10534.
[0732]ln various embodiments, a multispecific antigen-binding protein of the present disclosure comprises one or more anti-TNFR2 binding domains and one or more anti-CD25 binding domains, joined by one or more linkers as described herein in Table 2, and the multispecific antigen-binding protein further comprises an Fc domain (e.g., IgGl LALAPA) as described herein, wherein the one or more anti- TNFR2 binding domains and the one or more anti-CD25 binding domains comprise amino acid sequences selected from the following, from N-terminus to C-terminus, or similar sequences thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto:
1) SEQ ID NOs: 10461, 10461, 4046, and 9439;
2) SEQ ID NOs: 10468, 10468, 4046, and 9439;
3) SEQ ID NOs: 10469, 10469, 4046, and 9439;
4) SEQ ID NOs: 10470, 10470, 4046, and 9439;
5) SEQ ID NOs: 10477, 10477, 4046, and 9439;
6) SEQ ID NOs: 10812, 10812, 4046, and 9439;
7) SEQ ID NOs: 10461, 10461, 4046, and 9422;
8) SEQ ID NOs: 10468, 10468, 4046, and 9422;
9) SEQ ID NOs: 10469, 10469, 4046, and 9422;
10) SEQ ID NOs: 10470, 10470, 4046, and 9422;
11) SEQ ID NOs: 10477, 10477, 4046, and 9422;
12) SEQ ID NOs: 10812, 10812, 4046, and 9422;
13) SEQ ID NOs: 10461, 10461, 4046, and 10572;
14) SEQ ID NOs: 10468, 10468, 4046, and 10572;
15) SEQ ID NOs: 10469, 10469, 4046, and 10572;
16) SEQ ID NOs: 10470, 10470, 4046, and 10572;
17) SEQ ID NOs: 10477, 10477, 4046, and 10572;
18) SEQ ID NOs: 10812, 10812, 4046, and 10572;
19) SEQ ID NOs: 10461, 10461, 4046, and 10575;
20) SEQ ID NOs: 10468, 10468, 4046, and 10575;
21) SEQ ID NOs: 10469, 10469, 4046, and 10575;
22) SEQ ID NOs: 10470, 10470, 4046, and 10575;
23) SEQ ID NOs: 10477, 10477, 4046, and 10575; or
24) SEQ ID NOs: 10812, 10812, 4046, and 10575.
[0733]ln various embodiments, a multispecific antigen-binding protein of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 6401-6520 and 10792-10310, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0734]ln various embodiments, a multispecific antigen-binding protein of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 10792-10810, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0735]ln various embodiments, a multispecific antigen-binding protein of the present disclosure comprises a fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 3933-3964, and 7055-7085, 7255-7265, 3930-3968, 7278, 7297- 7332, 10372, 10373, 10376-10379, 10503, 10705, 10707, 10709, 10711, 10713, 10715, 10717, and 10719, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0736]lt is to be understood that although the exemplary multispecific antigen-binding proteins described herein contain non-humanized VHH amino acid sequences, such non-humanized VHH amino acid sequences can be replaced with any of the humanized VHH amino acid sequences described herein (e.g., in Tables 1-1, 1-2, 1-3, 1-4, 11, 12-1, and 13).
[0737]ln some embodiments, the multispecific antigen-binding protein described herein may further comprise a signal sequence at its N-terminus. Signal sequences may be present in the precursor molecule of the multispecific antigen-binding protein and may be removed after the protein is secreted from the host cell during production. In some embodiments, the signal sequence is MAVMAPRTLVLLLSGALALTQTWA (SEQ ID NO: 3928) or a fragment or variant thereof. In some embodiments, the signal sequence is MYRMQLLSCIALSLALVTNS (SEQ ID NO: 3929), or a fragment or variant thereof.
Polynucleotide Molecules
[073S]ln another aspect, provided herein are polynucleotide molecules encoding the multispecific antigen-binding proteins described herein. Polynucleotide molecules encoding polypeptide portion(s) of a conjugate of the present disclosure are also encompassed within the present disclosure.
[0739]ln some embodiments, a polynucleotide molecule of the present disclosure comprises a nucleotide sequence that encodes an anti-TNFR2 VHH amino acid sequence selected from SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 93-640, 2805-3363, 6636, 6640, 6644, 7090, 6651-6697, 6931- 6992, 7222-7254, 7174-7197, 10380, 10381, 10383, 10385, and 10386, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0740]ln some embodiments, a polynucleotide molecule of the present disclosure encoding an anti- TNFR2 VHH comprises the nucleotide sequence of any one of SEQ ID NOs: 48-59, 6645-6647, 7091, 7094, 7097, 7100, 3364-3922, 6993-7054, 7198-7221, and 10588-10661, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0741]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure comprises a nucleotide sequence that encodes a humanized anti~TNFR2 VHH amino acid sequence selected from SEQ ID NOs: 81-92, 6648-6650, 7092, 7095, 7098, 7101,7293-7296, 641-1127, 6698-6744, 10375, 10382, 10384, 10394-10408, 10457-10502, and 10812, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0742]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized anti~TNFR2 VHH amino acid sequence of SEQ ID NO: 7095, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In an embodiment provided herein, the polynucleotide molecule encoding a humanized anti- TNFR2 VHH amino acid sequence of SEQ ID NO: 7095 comprises the nucleotide sequence of SEQ ID NO: 7094, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0743]!n an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized anti-TNFR2 VHH amino acid sequence of SEQ ID NO: 7098, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In an embodiment provided herein, the polynucleotide molecule encoding a humanized anti- TNFR2 VHH amino acid sequence of SEQ ID NO: 7098 comprises the nucleotide sequence of SEQ ID NO: 7097, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity,
[0744]in an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized anti-TNFR2 VHH amino acid sequence of SEQ ID NO: 7101, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In an embodiment provided herein, the polynucleotide molecule encoding a humanized anti- TNFR2 VHH amino acid sequence of SEQ ID NO: 7101 comprises the nucleotide sequence of SEQ ID NO: 7100, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity,
[0745]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NQ: 10394, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0746]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NG: 7294, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0747]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10395, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[074S]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10396, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0749]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10397, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0750]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10398, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0751]in an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10399, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0752]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10400, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0753]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10401, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0754]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10402, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0755]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10403, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0756]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10404, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0757]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10405, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0758]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10406, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0759]in an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10407, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0760]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10408, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0761]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10457, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0762]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10458, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0763]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10459, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0764]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10460, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0765]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10461, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0766]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10462, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0767]in an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10463, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0768]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10464, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0769]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10465, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0770]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10466, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0771]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10467, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0772]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10468, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0773]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10469, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0774]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10470, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0775]in an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10471, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0776]in an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10472, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0777]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10473, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0778]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NG: 10474, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0779]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NG: 10475, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0780]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NG: 10476, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0781]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NG: 10477, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0782]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10478, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0783]in an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10479, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0784]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10480, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0785]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10481, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0786]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10482, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0787]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10483, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [078S]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 7101, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0789]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10484, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0790]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10485, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0791]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10486, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0792]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10487, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0793]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10488, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0794]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10489, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0795]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10490, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0796]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10491, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0797]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10492, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0798]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10493, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0799]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10494, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0800]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10495, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0801]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10496, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0802]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10497, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0803]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10498, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0804]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10499, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0805]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10500, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0806]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10501, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0807]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10502, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0808]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10720, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0809]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10721, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0810]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10722, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0811]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10723, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0812]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10724, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0813]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10725, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0814]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10726, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0815]in an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10727, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0816]ln some embodiments, a polynucleotide molecule of the present disclosure encodes an anti-CD25 VHH amino acid sequence selected from SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, 4126-4130, 4143- 4725, 5641-5945, 7351-7354, 7359-7362, 7368-7930, 8819-9014, 9417-9422, 9437, 9439, 9442-9659, 9992-10102, 10214-10276, and 10545-10585, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0817]ln some embodiments, a polynucleotide molecule of the present disclosure comprises a nucleotide sequence that encodes an anti-CD25 VHH amino acid sequence selected from SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, 4143-4442, 5641-5945, 7351-7354, 7368-7659, 8819-9114, 9437, 9442- 9551, 9992-10102, 10246-10276, and 10545-10585, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0818]ln some embodiments, a polynucleotide molecule of the present disclosure encoding an anti- CD25 VHH comprises the nucleotide sequence of any one of SEQ ID NOs: 4121-4125, 5946-6250, 7355- 7358, 9115-9410, 9438, 10103-10213, and 10662-10703, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0819]ln an embodiment provided herein, s polynucleotide molecule of the present disclosure comprises a nucleotide sequence that encodes a humanized VHH amino acid sequence selected from SEQ ID NOs: 4126-4130, 4443-4725, 7359-7362, 7660-7930, 9417-9422, 9439, 10214-10245, 9652-9659, and 10545-10585, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0820]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10375, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0821]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10382, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0822]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10384, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0823]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10380, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0824]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10381, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0825]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10383, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0826]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10385, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0827]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10386, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0828]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10545, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0829]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10546, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0830]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10547, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0831]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10548, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0832]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10549, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0833]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10550, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0834]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10551, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0835]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10552, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0836]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10553, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0837]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10554, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0838]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10555, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0839]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10556, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0840]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10557, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0841]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10558, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0842]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10559, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0843]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10560, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0844]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10561, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0845]in an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10562, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0846]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10563, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0847]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10564, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0848]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10565, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0849]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10566, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0850]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 9420, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0851]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10567, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0852]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10568, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0853]in an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10569, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0854]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10570, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0855]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10571, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0856]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10572, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0857]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10573, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [085S]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10574, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0859]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10575, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0860]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10576, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0861]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10577, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0862]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10578, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0863]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10579, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0864]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10580, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0865]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10581, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0866]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10582, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0867]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10583, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0868] In an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10584, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0869]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence of SEQ ID NO: 10585, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0870]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 3933-3964, and 7055- 7085, 7255-7265, 3930-3968, 7278, 7297- 7332, 10372, 10373, 10376-10379, 10503, 10705, 10707, 10709, 10711, 10713, 10715, 10717, and 10719, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0871]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encoding a fusion protein comprises the nucleotide sequence of any one of SEQ ID NOs: 10704, 10706, 10708, 10710, 10712, 10714, 10716, and 10718, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0872]ln various embodiments, provided herein are nucleic acid sequences encoding a multispecific antigen-binding protein of the present disclosure which comprises one or more anti-TNFR2 binding domains and one or more anti-CD25 binding domains, joined by one or more linkers as described herein in Table 2, wherein the one or more anti-TNFR2 binding domains and the one or more anti-CD25 binding domains comprise amino acid sequences selected from SEQ ID NOs: 4129, 4130, 81, 85, 83, 88, 92, 4128, 7, 90, 4120, 19, 35, 11, 4116, 15, and/or 4, or similar sequences thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0873]ln a specific embodiment, the nucleic acid sequence encoding the one or more anti-TNFR2 binding domain encodes amino acid sequences selected from SEQ ID NOs: 4, 7, 11, 15, 19, 35, 81, 83, 85, 88, 90, and 92.
[0874] in a specific embodiment, the nucleic acid sequence encoding the one or more anti-CD25 binding domains encodes amino acid sequences selected from SEQ ID NOs: 4116, 4120, 4128, 4129, and 4130, [0875)1 n various embodiments, provided herein are nucleic add sequences encoding a multispecific antigen-binding protein of the present disclosure which comprises one or more anti-TNFR2 binding domains and one or more anti-CD25 binding domains, joined by one or more linkers as described herein in Table 2, wherein the one or more anti-TNFR2 binding domains and the one or more anti-CD25 binding domains comprise amino acid sequences selected from the following, from N-terminus to C-terminus, or similar sequences thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at ieast 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto:
1) SEQ ID NOs: 7, 7, 7, and 7;
2) SEQ ID NOs: 4120, 7, 7, 7, and 7;
3) SEQ ID NOs: 4120, 19, 19, 19, and 19;
4) SEQ ID NOs: 19, 19, 19, and 19;
5) SEQ ID NOs: 35, 35, 35, 35, and 35;
6) SEQ ID NOs: 4120, 11, 11, 11, and 11;
7) SEQ ID NOs: 11, 11, 11, and 11;
8) SEQ ID NOs: 4129, 81, and 81;
9) SEQ ID NQs: 4129, 83, and 83;
10) SEQ ID NOs: 4129, 81, and 83;
11) SEQ ID NOs: 4129, 88, and 88;
12) SEQ ID NOs: 4129, 92, and 92;
13) SEQ ID NOs: 4129, 88, and 90;
14) SEQ ID NOs: 4130, 81, and 81;
15) SEQ ID NOs: 4130, 83, and 83;
16) SEQ ID NOs: 4130, 88, and 88;
17) SEQ ID NOs: 4130, 92, and 92;
18) SEQ ID NOs: 4128, 81, and 81;
19) SEQ ID NOs: 4128, 83, and 83;
20) SEQ ID NOs: 4128, 88, and 83;
21) SEQ ID NOs: 4128, 92, and 92;
22) SEQ ID NOs: 4129, 4130, and 88;
23) SEQ ID NOs: 4129, 4130, and 92;
24) SEQ ID NOs: 7 and 7; 25) SEQ ID NOs: 15 and 15;
26) SEQ ID NOs: 15, 15, 15 and 15;
27) SEQ ID NOs: 4 and 4;
28) SEQ ID NOs: 4, 4, 4, and 4;
29) SEQ ID NOs: 19 and 19;
30) SEQ ID NOs: 19, 19, 19, and 19;
31) SEQ ID NOs: 35, 35, 35, and 35;
32) SEQ ID NOs: 11 and 11; and
33) SEQ ID NOs: 11, 11, 11, and 11; .
[0876]! n various embodiments, provided herein are nucleic acid sequences encoding a multispecific antigen-binding protein of the present disclosure that comprises one or more anti-TNFR2 binding domains and one or more anti-CD25 binding domains, joined by one or more linkers as described herein in Table 2, and the multispecific antigen-binding protein further comprises an Fc domain as described herein, wherein the one or more anti-TNFR2 binding domains and the one or more anti-CD25 binding domains comprise amino acid sequences selected from SEQ ID NOs: 4129, 4130, 81, 85, 83, 88, 92, 4128, 7, 90, 4120, 19, 35, 11, 4116, 15, and/or 4, or similar sequences thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0877]ln a specific embodiment, the nucleic acid sequence encoding the anti-TNFR2 binding domains encodes amino acid sequences selected from SEQ ID NOs: 4, 7, 11, 15, 19, 35, 81, 83, 85, 88, 90, and 92. [0878]ln a specific embodiment, the nucleic acid sequence encoding the anti-CD25 binding domains encodes amino acid sequences selected from SEQ ID NOs: 4116, 4120, 4128, 4129, and 4130.
[0879]! n various embodiments, the nucleic acid sequence encoding the Fc domain encodes an amino acid sequence according to SEQ ID NO: 4049, 4053, and/or 6365, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, in various embodiments, the Fc domain is encoded by a nucleic acid sequence that encodes an amino acid sequence according to SEQ ID NOs: 4049, 4053, and/or 6365, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. [0880]ln various embodiments, provided herein are nucleic acid sequences encoding a multispecific antigen-binding protein of the present disclosure that comprises one or more anti-TNFR2 binding domains and one or more anti-CD25 binding domains, joined by one or more linkers as described herein in Table 2, and the multispecific antigen-binding protein further comprises an Fc domain as described herein, wherein the one or more anti-TNFR2 binding domains and the one or more anti-CD25 binding domains comprise amino acid sequences selected from SEQ ID NOs: 4129, 4130, 81, 85, 83, 88, 92, 4128, 7, 90, 4120, 19, 35, 11, 4116, 15, and/or 4, or similar sequences thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and wherein the Fc domain comprises an amino acid sequence according to SEQ ID NO: 4049, 4053, and/or 6365, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0881]ln various embodiments, provided herein are nucleic acid sequences encoding a multispecific antigen-binding protein of the present disclosure that comprises one or more anti-TNFR2 binding domains and one or more anti-CD25 binding domains, joined by one or more linkers as described herein in Table 2, and the multispecific antigen-binding protein further comprises an Fc domain as described herein, wherein the one or more anti-TNFR2 binding domains and the one or more anti-CD25 binding domains comprise amino acid sequences selected from the following, from N-terminus to C-terminus, or similar sequences thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto:
50) SEQ ID NOs: 83, 83, 83, and 4049;
51) SEQ ID NOs: 83, 83, and 4049;
52) SEQ ID NOs: 81, 83, and 4049;
53) SEQ ID NOs: 88, 90, and 4049;
54) SEQ ID NOs: 83, 83, 4049, and 4129;
55) SEQ ID NOs: 81, 83, 4049, and 4129;
56) SEQ ID NQs: 88, 90, 4049, and 4129;
57) SEQ ID NOs: 83, 83, 4049, and 4130;
58) SEQ ID NOs: 81, 83, 4049, and 4130;
59) SEQ ID NOs: 4129, 4049, 83, and 83;
60) SEQ ID NOs: 4129, 4049, 81, and 83;
61) SEQ ID NOs: 83, 4049, and 83; ) SEQ ID NOs: 4129, 83, 4049, and 83;) SEQ ID NOs: 4130, 83, 4049, and 83;) SEQ ID NOs: 4129, 4049, and 88; ) SEQ ID NOs: 4129, 4049, and 92; ) SEQ ID NOs: 88, 4049, and 4129; ) SEQ ID NOs: 92, 4049, and 4129; ) SEQ ID NOs: 88 and 4049; ) SEQ ID NOs: 92 and 4049; ) SEQ ID NOs: 4130, 4049, and 88; ) SEQ ID NOs: 4130, 4049, and 92; ) SEQ ID NOs: 4129, 88, and 4049; ) SEQ ID NOs: 4129, 92, and 4049; ) SEQ ID NOs: 4130, 88, and 4049; ) SEQ ID NOs: 4130, 92, and 4049; ) SEQ ID NOs: 4128, 88, and 4049; ) SEQ ID NOs: 4128, 92, and 4049; ) SEQ ID NOs: 81, 81, 81, and 4049;) SEQ ID NOs: 88, 88, and 4049; ) SEQ ID NOs: 92, 92, and 4049; ) SEQ ID NOs: 88, 85, and 4049; ) SEQ ID NOs: 81, 85, and 4049; ) SEQ ID NOs: 7, 7, and 6365; ) SEQ ID NOs: 7, 6365, and 7; ) SEQ. ID NOs: 4116, 4053, and 7; ) SEQ ID NOs: 15, 15, and 6365; ) SEQ ID NOs: 15, 6365, and 15; ) SEQ ID NOs: 4116, 4053, and 15; ) SEQ ID NOs: 4, 4, and 6365; ) SEQ ID NOs: 4, 6365, and 4; ) SEQ ID NOs: 4116, 4053, and 4; ) SEQ ID NOs: 19, 19, and 6365; ) SEQ ID NOs: 19, 6365, and 19; 94) SEQ ID NOs: 4116, 4053, and 19;
95) SEQ ID NOs: 35, 6365, and 35;
96) SEQ ID NOs: 11, 11, and 6365;
97) SEQ ID NOs: 11, 6365, and 11; and
98) SEQ ID NOs: 4116, 4053, and 11;
[0882]ln an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a multispecific antigen-binding protein comprising an amino add sequence selected from SEQ ID NOs: 6401-6520 and 10792-10810, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0883]ln an embodiment provided herein, a polynucleotide molecule comprises a nucleic acid sequence selected from SEQ ID NOs: 6521-6632, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
[0884]A polynucleotide molecule may be used to transform/transfect a host cell or host organism, e.g., for expression and/or production of a polypeptide. Suitable hosts or host cells for production of a multispecific antigen-binding polypeptide described herein include any suitable fungal, prokaryotic, or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. A host or host cell comprising a polynucleotide molecule encoding a multispecific antigen-binding protein described herein is also encompassed by the present disclosure.
[0885JA polynucleotide molecule may be for example DNA, RNA, or a hybrid thereof, and may also comprise (e.g., chemically) modified nucleotides, like locked nucleic acids (LNA) or peptide nucleic acids (PNA). In some embodiments, the polynucleotide is single-stranded. In some embodiments, the polynucleotide is double-stranded. In one embodiment, the polynucleotide is in the form of doublestranded DNA (e.g., plasmid). In some embodiments, the polynucleotide is in the form of a singlestranded RNA (e.g., mRNA).
[0886]Techniques for generating polynucleotides may include, for example but not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more "mismatched" primers. Alternatively, polynucleotides of the present disclosure may be isolated from a suitable natural source. Polynucleotide sequences encoding naturally occurring (poly)peptides can for example be subjected to site-directed mutagenesis, to generate a polynucleotide molecule encoding polypeptide with sequence variation.
Vectors
[0887]Also provided herein are vectors comprising the polynucleotide molecules encoding the multispecific antigen-binding proteins, or other relevant polypeptides of the present disclosure. A "vector" as used herein is a vehicle suitable for carrying genetic material into a host cell. A vector can include a nucleic acid vector, such as a plasmid or mRNA, or nucleic acids embedded into a bigger structure, such as a liposome or viral vector.
[0888]A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters, enhancers, terminators) that regulate the expression of a polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, ^-galactosidase). For DNA-based vectors, this usually includes the presence of elements for transcription (e.g., a promoter and a polyA signal) and translation (e.g., Kozak sequence). In some embodiments, the vector is an expression vector, i.e., a vector suitable for expressing an encoded polypeptide or construct under suitable conditions in a host ceil.
[0889]To express a multispecific antigen-binding protein (or fragments thereof) of the present disclosure, polynucleotides encoding partial or full-length polypeptide chains, e.g., obtained as described above (e.g., VHH, VHH-Fc), can be inserted into expression vectors such that the genes are operatively linked to one or more transcriptional and translational control sequences. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. Polynucleotides encoding the two or more polypeptide chains (when present and differ from one another) of a multispecific antigen-binding protein of the present disclosure can be inserted Into separate vectors, or, optionally, incorporated into the same expression vector.
[0890]ln addition to polynucleotides encoding the polypeptide chain(s) of a multispecific antigenbinding protein, the recombinant expression vectors of the invention may include regulatory sequences that control the expression of genes encoding the polypeptide chain(s) in a host cell. The design of the expression vector, including the selection of regulatory sequences, may depend on the choice of the host cell to be transformed and/or the desired level of protein expression. For example, suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. Additional examples of viral regulatory elements, and sequences thereof, include those described in, e.g., U.S. Pat. Nos. 5, 168,062; 4,510,245; and 4,968,615; the disclosures of each of which are incorporated herein by reference.
[0891]Recombinant expression vectors of the present disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. A selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., US4,399,216; US 4,634,665; and US 5,179,017; the disclosure of each of which is incorporated herein by reference in its entirety). For example, typically the selectable marker gene confers resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin, or cytotoxic drugs, such as G418, puromycin, blasticidin, hygromycin or methotrexate, to a host cell into which the vector has been introduced. Suitable selectable marker genes can include the dihydrofolate reductase (DHFR) gene (for use in DHFR deficient host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
[0892]Vectors of the present disclosure may further include sequence elements that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g,, 5' and 3' untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
[0893]Viral vectors can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell). Viral vectors are particularly useful for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents to induce gene integration. Examples of suitable viral vectors include a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses (AAV) such as AAV2, AAVS, AAV9), negative strand RIMA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpes virus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), baculovirus, coronavirus, and poxvirus (e.g,, vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding polypeptides of the present disclosure include, for example Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include, but are not limited to, avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M.1996. Fundamental Virology, DMKDN Fields, PM Howley, ed. (Philadelphia, Lippincott-Raven Publishers): 763-843., the disclosure of which is incorporated herein by reference). Other examples of viral genomes useful in the compositions and methods of the present disclosure include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline sarcoma virus, feline leukemia virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus. Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.
Host Cells
[0894]ln one aspect, the present disclosure also provides host cells or host organisms that comprise the polynucleotides or vectors encoding the muitispecific antigen-binding proteins, or other relevant polypeptides described herein. Suitable host cells or host organisms can be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host ceils can also include cells transfected in vivo with a polynucleotide(s) or vector provided herein.
[0895]Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris}; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), COS cells, SP2/0 cells, and 293 and CHO cells, and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Exemplary prokaryotic cells include bacterial cells such as Escherichia coii. Preparation Methods
[0896]The present disclosure also provides methods of producing the multispecific antigen-binding proteins or conjugates described herein.
[0897]ln some embodiments, a method may comprise transforming/transfecting a host cell or host organism with a polynucleotide encoding a multispecific antigen-binding protein or other relevant polypeptide(s) described herein, expressing the multispecific antigen-binding protein or other relevant polypeptide(s) in the host, optionally followed by one or more isolation and/or purification steps. [0898]When recombinant expression vectors encoding one or more polypeptide(s) of a multispecific antigen-binding protein or conjugate of the present disclosure are introduced into mammalian host cells, the host cells are cultured for a period of time sufficient to allow for expression of the protein(s) or polypeptide(s) in the host cells or secretion of the protein(s) or polypeptide(s) into the culture medium in which the host cells are grown. Protein(s) or polypeptide(s) can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as VHH domains.
[0899]Once a protein or polypeptide of the present disclosure has been produced by recombinant expression, it can be purified by any method known in the art for purification of a protein or polypeptide, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for TNFR2 and CD25 after Protein A or Protein G selection, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the proteins or polypeptides of the present disclosure can be fused to heterologous polypeptide sequences described herein (e.g., His-tag) or otherwise known in the art to facilitate purification or to produce therapeutic conjugates below). Once isolated, a protein or polypeptide of the present disclosure can, if desired, be further purified, e.g., by high performance liquid chromatography, or by gel filtration chromatography, such as on a Superdex™ column.
Pharmaceutical Compositions and formulations
[0900]The present disclosure also provides a composition comprising multispecific antigen-binding proteins, or conjugates of the present technology, at least one polynucleotide molecule encoding the same, at least one vector comprising such a polynucleotide molecule, or at least one host cell comprising the polynucleotide molecule or vector. The composition may be a pharmaceutical composition. The composition may further comprise at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
[0901]As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions.
[0902] Exam pies of suitable formulations include, but are not limited to, solutions, suspensions, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Phdomain Sci Technol 52:238- 311.
[0903]A pharmaceutical composition of the present disclosure may be formulated according to its intended route of administration. Examples of suitable routes of administration include, e.g., intravenous, subcutaneous, intratumoral, oral (e.g., buccal, sublingual), intranasal, inhalation, intraocular, intramuscular, intradermal, transdermal (i.e., topical), intraperitoneal, transmucosal, vaginal, and rectal administration, or injection to the CNS/brain (e.g., intraspinal, intracerebral, or intrathecal administration). Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; fixed oils; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as phosphates, acetates, or citrates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of plastic or glass. [0904]Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include, for example, physiological saline, bacteriostatic water, Cremophor EL®, or phosphate buffered saline (PBS). The composition is preferably sterile and has a proper fluidity. In most embodiments, the composition is stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the contamination by microorganisms can be achieved by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. [0905]Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and/or freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously steri le-f iltered solution thereof.
[OSOSjOral compositions may include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, capsules, or liquid forms. Formulation in tablet and liquid forms may be used for protease insensitive VHHs. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pilis, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystailine cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
[0907]For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
[0908jSystemic administration can also be by transmucosal or transdermal means. Fortransmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. [0909]The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. [0910]For brain delivery, compounds of the present disclosure may be formulated to facilitate crossing of the blood-brain barrier. For example, multispecific antigen-binding proteins or conjugates of the present disclosure may be encapsulated into brain targeted liposomes, lipid nanoparticles, lipid microparticles, or lipid microcapsules for brain delivery. Example liposomes delivery systems are described in Pothin et al,, Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.
[O911J8 n some embodiments, the active compounds are prepared with carriers that can protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US 4,522,311, which is incorporated herein by reference in its entirety.
[0912]8t is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure is dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
[0913]The pharmaceutical compositions (or components thereof) can be included in a kit, container, pack, or dispenser together with instructions for administration. These pharmaceutical compositions can be included in diagnostic kits with instructions for use.
[0914]Pharmaceutical compositions are administered in an amount effective for treatment or prophylaxis of the specific indication. The therapeutically effective amount is typically dependent on the weight of the subject being treated, the physical or health condition of the subject, the extensiveness of the condition to be treated, or the age of the subject being treated. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 50 pg/kg body weight to about 50 mg/kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 100 pg/kg body weight to about 50 mg/kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 100 pg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering a pharmaceutical composition of the present disclosure may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well- known methods in the art (e.g., Mordent! et «/., 1991, Phdomainaceut. Res. 8:1351).
[0915]ln some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 10 mg to about 1,000 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 500 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 300 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 200 mg per dose.
[0916]ln some embodiments wherein the antigen-binding proteins of the present disclosure are administered as a viral vector (e.g., an AAV), dose ranges and frequency of administration of the viral vector described herein can vary depending on the nature of the viral vector, and the medical condition, as well as parameters of a specific patient and the route of administration used. In some embodiments, viral vector compositions can be administered to a subject at a dose ranging from about 1x10s plaque forming units (pf u) to about IxlO15 pfu, depending on mode of administration, the route of administration, the nature of the disease and condition of the subject. In some cases, the viral vector compositions can be administered at a dose ranging from about 1x10s pfu to about lxio15 pfu, or from about Ixio10 pfu to about lxio15 pfu, or from about lxio8 pfu to about lxio12 pfu. A more accurate dose can also depend on the subject in which it is being administered. For example, a lower dose may be required if the subject is juvenile, and a higher dose may be required if the subject is an adult human subject. In certain embodiments, a more accurate dose can depend on the weight of the subject. In certain embodiments, for example, a juvenile human subject can receive from about lxio8 pfu to about lxio19 pfu, while an adult human subject can receive a dose from about lxio10 pfu to about lxlOK pfu. [0917]Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al, 1987, J, Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, intraspinal, intracerebral, intrathecal and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
[0918]A pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded. [0919)1 n certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds,), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.
[0920]The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous, intramuscular, intratumoral, intraperitoneal, intraspinal, intracerebral, and intrathecal injections, drip infusions, etc. in one embodiment, the injectable preparations may be prepared, e.g., by dissolving, suspending, or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc,, which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e,g,, sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.
[0001]Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the multispecific antigen-binding proteins described herein may be about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, the multispecific antigenbinding proteins described herein may be contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms,
[0921]The pharmaceutical composition may be administered as needed to a subject. In some embodiments, an effective dose of the pharmaceutical composition is administered to a subject one or more times. In various embodiments, an effective dose of the pharmaceutical composition is administered to the subject once a month, less than once a month, such as, for example, every two months, every three months, or every six months. In other embodiments, an effective dose of the pharmaceutical composition is administered more than once a month, such as, for example, every two weeks, every week, twice per week, three times per week, daily, or multiple times per day. An effective dose of the pharmaceutical composition is administered to the subject at least once. In some embodiments, the effective dose of the pharmaceutical composition may be administered multiple times, including for periods of at least a month, at least six months, or at least a year. In some embodiments, the pharmaceutical composition is administered to a subject as needed to alleviate one or more symptoms of a condition.
[0922]ln some embodiments, a pharmaceutical composition of the present disclosure may be administered to a subject at levels lower than that required to achieve the desired therapeutic effect and the dosage may be gradually increased until the desired effect is achieved. Alternatively, a pharmaceutical composition of the present disclosure may be administered at a high dose and subsequently administered progressively lower doses until a therapeutic effect is achieved. In general, a suitable daily dose of an antigen-binding protein of the invention is an amount of the antibody which is the lowest dose effective to produce a therapeutic effect.
[0923]Pharmaceutical compositions of the present disclosure may optionally include more than one active agent. For example, compositions of the present disclosure may contain a multispecific antigenbinding protein conjugated to, admixed with, or administered separately from another pharmaceutically active molecule, e.g., Treg cell, or an additional agent that is useful for induction of Treg cell expansion. For instance, a multispecific antigen-binding protein may be admixed with one or more additional active agents, such as IL-2 or TNFa, to treat an immunological disease, e.g., a disorder described herein. Alternatively, pharmaceutical compositions of the present disclosure may be formulated for coadministration or sequential administration with one or more additional active agents that can be used to attenuate CD8+ T-cell growth. Examples of additional active agents that can be used to attenuate cytotoxic T-cell proliferation and that can be conjugated to, admixed with, or administered separately from a multispecific antigen-binding protein of the present disclosure include cytotoxic agents, e.g., those described herein.
Treatment Methods and Other Uses
[0924)1 n one aspect, provided herein is a method of using multispecific antigen-binding proteins or conjugates of the present disclosure to stimulate the proliferation of a population of regulatory T (Treg) cells (e.g., CD4+, CD25+, F0XP3+ Treg cells). This response may also have the effect of reducing populations of cytotoxic T-lymphocytes (e.g., CD8+ T-cells) that are often associated with mounting an inappropriate immune response that can cause an immunological disorder. In addition, multispecific antigen-binding proteins or conjugates of the present disclosure may synergize with existing Treg proliferating agents, such as IL-2 and TNFa.
[0925]Also provided herein is a method of using multispecific antigen-binding proteins or conjugates of the present disclosure to activate and/or enhance suppressive function (e.g., inhibition of effector T/B cell function or proliferation or antigen presenting cell function) of a population of Treg ceils.
[0926]Further provided herein is a method of using multispecific antigen-binding proteins or conjugates of the present disclosure to stabilize immunosuppressive phenotype (including stable expression of e.g., FOXP3, HELIOS, CTLA-4) of a population of Treg cells.
[0927]! n various embodiments of the above methods, the methods may comprise contacting the population of regulatory T cells with a multispecific antigen-binding protein or conjugate described herein. The methods may be carried out in vitro or in vivo. When such methods are carried out in vivo, the methods further comprise administering the multispecific antigen-binding protein or conjugate described herein into a subject.
[0928]Tregs are a subset of T cells that play a crucial role in peripheral self-tolerance and the prevention of autoimmunity. Historically, Tregs have been identified as a CD4 subset that specifically express CD25, the high affinity IL-2 receptor alpha chain (Sakaguchi et al., 1995). Subsequently, FOXP3 transcription factor was identified as CD4 Treg's master regulator (Hori et al., 2003). In fact, FOXP3 deficiency leads to systemic autoimmunity in both mouse and human in which it causes the Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome due to Tregs deficiency and unregulated effector T cell function (Bennett et al., 2001). CD4 Tregs can differentiate during T cell development (thymic "tTregs") or in the periphery (peripheral "pTregs") under non-inflammatory T cell receptor stimulation (Wing et al., 2019). Numerous subsets have been described including naive and memory Tregs (Sakaguchi et al., 2020), Th-like Tregs (Halim et al., 2017) as well as CD8 Tregs (Mishra et al., 2021; Niederlova et al., 2021). CD4 Tregs regulate immune response through various mechanisms including the secretion of regulatory cytokines (e.g., IL-10, IL-35, TGF-P), IL-2 scavenging, adenosine production, direct cytotoxicity and dendritic cell regulation (Vignali et al., 2008). The terms "regulatory T cells" or "Treg" as used herein are meant to encompass all the above-described subsets of regulatory T cells.
[0929]Tregs have enhanced affinity for MHC Il-presented self-antigen peptide and have a TCR repertoire that is non-overlapping with effector CD4 T cells (Fazilleau et al., 2007; Hsieh et al., 2006; Pacholczyk et al., 2006). Therefore, self-antigen recognition in the periphery can induce tTregs activation (Moran et al., 2011), Importantly however, once activated, Tregs can suppress effector cells that have different antigen specificity through bystander suppression (Thornton and Shevach, 2000; Yeh et al., 2017; Yu et al., 2005) by regulating antigen presenting cells or soluble factors.
[0930]8t has been shown that over time, Tregs retain some plasticity and can lose F0XP3 expression, These so called "ex-Tregs" have increased level of FOXP3 promoter methylation and lower FOXP3 expression compared to Tregs and can acquire effector function (Zhou et al., 2009). In Tregs, the demethylation of FOXP3 promoter, particularly in the "Treg-specific demethylated region" (TSDR) (Huehn et al., 2009), stabilizes gene expression. Likewise, human Tregs exposed to IL-2 4- inflammatory cytokines have been shown to lose F0XP3 expression while upregulating RORg and IL-17, a feature associated with TH17 cells. Instability of the Treg phenotype in the presence of inflammatory cytokines can be referred to as "Treg fragility" and is of crucial relevance for therapeutic purpose in autoimmune diseases. Indeed, to induce a long-lasting therapeutic benefit, It is important to stabilize the phenotype and function of Tregs and prevent their conversion to pathogenic cells that further contribute to disease.
[0931]ln some embodiments, multispecific antigen-binding proteins or conjugates of the present disclosure may be capable of stimulating the proliferation of a population of Treg cells by between 1% and 100% relative to untreated cells (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), as measured, e.g., by fluorescence activated cell sorting (FACS) analysis. In certain embodiments, multispecific antigen-binding proteins or conjugates of the present disclosure may be capable of reducing the growth of a population of CD8* T-cells, e.g., by about 10% to about 200% relative to untreated cells (e.g,, 10%, 20%, 30%, 40%, 50%, 75%, 100%, 125%, 150%, 175%, or 200%).
[0932]ln some embodiments, multispecific antigen-binding proteins of the present disclosure can be used to promote the proliferation of a population of Treg cells and thus enhance the immunomodulatory activity of these cells. Multispecific antigen-binding proteins of the present disclosure can therefore be used to attenuate an aberrant cell-mediated or humoral immune response associated with a variety of human diseases, such as autoimmune disorders, asthma, allergic reactions, and diseases associated with allograft tolerance. For example, multispecific antigen-binding proteins of the present disclosure may be administered to suppress cytotoxic T-cell and B-cell activity, thereby attenuating the response of a subject to a self or benign antigen. Multispecific antigen-binding proteins of the present disclosure can be administered to a mammalian subject, such as a human, to attenuate an aberrant immune response, such as a response against a self or non-threatening antigen. Alternatively, multispecific antigen-binding proteins of the present disclosure can be used to expand a population of Treg cells ex vivo that have been extracted, e.g., from a patient or an MHC-matched donor. After inducing proliferation of these Treg cells in culture by contacting with a multispecific antigen-binding protein of the present disclosure, these cells can subsequently be administered to a subject, e.g., using adoptive cell transfer techniques known in the art or described herein. In this way, multispecific antigen-binding proteins of the present disclosure may synergize with existing techniques to suppress humoral and cell-mediated immune responses as a treatment modality for patients suffering from a variety of immunological disorders.
[0933]in some embodiments, multispecific antigen-binding proteins of the present disclosure are capable of interacting with and promoting signal transduction events mediated by TNFR2 and CD25. Multispecific antigen-binding proteins of the present disclosure may be able to induce conformational changes within TNFR2 that lead to receptor trimerization. This spatial configuration has been shown to render TNFR2 active for MAPK/TRAF 2/3 signal transduction, which subsequently leads to activation of NF-xB-mediated transcription of genes involved in Treg cell growth and escape from apoptosis (Faustman, et al., Nat Rev Drug Discov. 9:482-493 (2010), the disclosure of which is incorporated herein by reference).
[0934]ln some embodiments, multispecific antigen-binding proteins of the present disclosure may be capable of increasing the transcription and/or expression of various genes. For example, multispecific antigen-binding proteins of the present disclosure may induce the expression of one or more of Akt, clAP2, Etk, TRAF2, VEGFR2, P13K, genes encoding proteins involved in the angiogenic pathway, IKK complexes, RIP, NIK, MAP3K, genes encoding proteins involved in the NF-xB pathway, NIK, JNK, AP-1, a MEK (e.g., MEK1, MEK7), MKK3, NEMO, IL2R, Foxp3, 112, TNF, and lymphotoxin (e.g., lymphotoxin a and lymphotoxin (3). The increase in expression of these genes can be detected using established molecular biology techniques known in the art, e.g., by detecting an increase in mRNA levels by Northern biot analysis or reverse-transcription PCT (RT-PCR) methods, or by detecting an increase in protein levels by immunoblot analysis or ELISA-based techniques. In some embodiments, multispecific antigen-binding proteins of the present disclosure may be capable of promoting the activity of one or more proteins associated with the TNFR2 signaling pathway (or related signaling pathways that are activated as a result of TNFR2 signaling). For instance, multispecific antigen-binding proteins of the present disclosure may be capable of promoting an increase in the phosphorylation of one or more proteins, such as Akt, clAP2, Etk, TRAF2, VEGFR2, P13K, proteins involved in the angiogenic pathway, IKK complexes, RIP, NIK, MAP3K, proteins involved in the NF-KB pathway, NIK, JNK, AP-1, a MEK (e.g., MEK1, MEK7), MKK3, NEMO, IL2R, Foxp3, 112, TNF, and lymphotoxin (e.g., lymphotoxin a and lymphotoxin P). An increase in the phosphorylation of one or more proteins that occurs, e.g., as a result of treatment of a subject or of a sample of cells isolated from a subject can be detected using standard molecular biology techniques known in the art, such as by immunobiot analysis or ELISA-based techniques.
[0935]in some embodiments, multispecific antigen-binding proteins of the present disclosure increase expression of one or more proteins selected from a protein in the NF-kB pathway, FOXP3, HELIOS, EZH2, HLA-DR, ICAM-1, OX-40, ICOS, and CCR8.
[0936]! n another aspect, provided herein is a method of inhibiting an immune response mediated by a B cell or a CD8+ T cell in a subject, the methods including the step of administering to the subject an effective amount of a multispecific antigen-binding protein, a conjugate, a polynucleotide molecule, a vector, or a host cell described herein.
[0937]ln another aspect, multispecific antigen-binding proteins, conjugates, polynucleotide molecules, vectors, and/or host cells described herein, or pharmaceutical compositions thereof, are useful for the (prophylactic or therapeutic) treatment of a wide array of diseases or disorders. Accordingly, the present technology provides a multispecific antigen-binding protein, a conjugate, a polynucleotide molecule, a vector, or a host cell for use as a medicament. Also provided is a (prophylactic and/or therapeutic) method of treating a disease or disorder, wherein said method comprises administering, to a subject in need thereof, a pharmaceutically active amount of a multispecific antigen-binding, a conjugate, a polynucleotide molecule, a vector, or a host cell described herein.
[0938jThe diseases or disorder that can be treated with the compositions and methods described herein include, but are not limited to, immunological diseases (e.g., autoimmune diseases), inflammatory diseases, cancers, cardiovascular diseases (e.g., atherosclerosis, heart failure, left heart failure with reduced ejection fraction, left heart failure with preserved ejection fraction, right ventricular failure, congestive heart failure, restrictive cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, idiopathic cardiomyopathy, hypertension), and infertility and pregnancy-associated diseases (e.g., recurrent pregnancy loss, pre-eclampsia, preterm labor, fetal growth restriction, intrauterine growth restriction).
[0939]Examples of immunological diseases that can be treated with the compositions and methods described herein include, but are not limited to, autoimmune diseases, allergies, asthma, neurological diseases, metabolic diseases (e.g., diabetes), macular diseases (e.g., macular degeneration), muscular atrophy, diseases related to miscarriage, vascular diseases (e.g., atherosclerosis), diseases related to bone loss (e.g., bone loss as a result of menopause or osteoporosis), blood disorders (e.g., hemophilia), musculoskeletal disorders, diseases related to growth receptor expression or activity, obesity, graft- versus-host disease (GVHD), or allograft rejections.
[0940]ln some embodiments, the compositions and methods described herein are used to treat an autoimmune disease. In some embodiments, the autoimmune disease is selected from lupus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpastures disease, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lichen sclerosis, !gG4-related disease, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disease, pemphigus vulgaris or related blistering skin disease, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, premature ovarian failure, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, primary ovarian insufficiency, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthritis, stiff-man syndrome, type I diabetes, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (Granulomatosis with polyangiitis) or other immune vasculitis.
[0941]ln some embodiments, the compositions and methods described herein are used to treat lupus, in some embodiments, the lupus is systemic lupus erythematosus (SLE), cutaneous lupus (including acute cutaneous lupus, chronic cutaneous lupus erythematosus, or discoid lupus erythematosus (DLE) and subacute cutaneous lupus erythematosus), lupus nephritis, neonatal lupus, or drug-induced lupus. [0942jln some embodiments, the autoimmune disease is atopic dermatitis, psoriasis, systemic lupus erythematosus, or arthritis.
[0943]! n some embodiments, the compositions and methods described herein are used to treat allergy. In some embodiments, the allergy is an allergic conjunctivitis, chemical allergy, cosmetic allergy, drug allergy, dust allergy, food allergy, hay fever, hives, mold allergy, pet allergy, poison ivy allergy oak allergy, or seasonal allergy.
[0944]ln some embodiments, the compositions and methods described herein are used to treat a neurological condition. In some embodiments, the neurological condition is a brain tumor, a brain metastasis, a spinal cord injury, schizophrenia, epilepsy, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Huntington's disease, Parkinson's disease, or stroke,
[0945]! n some embodiments, the compositions and methods described herein are used to treat a graft rejection. Without wishing to be bound by theory, multispecific antigen-binding proteins of the present disclosure may treat graft rejections, e.g., by binding TNFR2 receptors on the surface of autoreactive CD8+ T-cells that bind antigens presented on the surface of the graft and inducing apoptosis in these CD8+ T-cells, or by inducing the expansion of Treg cells that may subsequently eliminate autoreactive CD8+- T-cells. Examples of graft rejections that can be treated with the compositions and methods described herein include, without limitation, skin graft rejection, bone graft rejection, vascular tissue graft rejection, ligament graft rejection (e.g., anterior cruciate ligament graft rejection, anterior sacroiliac ligament graft rejection, caudal cruciate ligament graft rejection, cranial cruciate ligament graft rejection, cricothyroid ligament graft rejection, dorsal radiocarpal ligament graft rejection, inferior pubic ligament graft rejection, lateral collateral ligament graft rejection, medial collateral ligament graft rejection, palmar radiocarpal ligament graft rejection, patellar ligament graft rejection, periodontal ligament graft rejection, posterior cruciate ligament graft rejection, posterior sacroiliac ligament graft rejection, radial collateral ligament graft rejection, sacrospinous ligament graft rejection, sacrotuberous ligament graft rejection, superior pubic ligament graft rejection, suspensory ligament of the breast graft rejection, suspensory ligament of the lens graft rejection, ulnar collateral ligament graft rejection) and organ graft rejection (e.g,, heart, lung, kidney, liver, pancreas, intestine, and thymus graft rejection).
[0946]! n some embodiments, the compositions and methods described herein are used to treat a graft- versus-host disease. In some embodiments, the graft-versus-host disease arises from a bone marrow transplant or one or more blood cells such as B-cells, T-cells, basophils, common myeloid progenitor cells, common lymphoid progenitor cells, dendritic cells, eosinophils, hematopoietic stem cells, neutrophils, natural killer cells, megakaryocytes, monocytes, or macrophages.
[0947]ln some embodiments, the compositions and methods described herein are used to treat an inflammatory disease. The inflammatory disease may be acute or chronic inflammation. In some embodiments, the inflammatory disease is selected from osteoarthritis, atopic dermatitis, endometriosis, polycystic ovarian syndrome, inflammatory bowel disease, fibrotic lung disease, and cardiac inflammation.
[0948jln some embodiments, the compositions and methods described herein are used to treat a cancer. In some embodiments, the cancer is an adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dube syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumor (including childhood germ cell tumor), gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal carcinoma, HIV/AIDS-related cancerjuvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B-cell prolymphocytic leukemia and hairy cell leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic T-cell lymphocytic leukemia, eosinophilic leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer (e.g., nonsmall cell lung cancer, small cell lung cancer), hodgkin lymphoma, non-hodgkin lymphoma, lynch syndrome, mastocytosis, medulloblastoma (including childhood medulloblastoma), melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or MYH)-associated polyposis, myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma (including childhood neuroblastoma), neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian, fallopian tube, and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, soft tissue sarcomas, skin cancer (non-melanoma), small bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Waldenstrom macroglobulinemia (lymphoplasmacytic lymphoma), Werner syndrome, Wilms tumor, or xeroderma pigmentosum.
[0949]ln some embodiments, multispecific antigen-binding proteins of the present disclosure can also be used to treat a patient in need of organ repair or regeneration, e.g., by inducing the proliferation of cells within a damaged tissue or organ. While not wishing to be bound by any theory, it is contemplated that agonistic multispecific TNFR2 and/or CD25 antibodies may stimulate organ repair or regeneration, e.g., by binding TNFR2 on the surface of cells within damaged tissue to induce TRAF2/3- and/or NF-KB- mediated ceil proliferation. Examples of tissues and organs that may be induced to regenerate by the use of multispecific antigen-binding proteins of the present disclosure include the blood vessels including the aorta, bone, cranial nerves, ear, eye, embryonic structures, heart, heart, hematopoietic system, kidney, small intestine, large intestine, liver, lung, nerves, olfactory gland, pancreas, pituitary gland, peripheral nervous system, central nervous system, spinal cord, salivary gland, structures of the head, testes, thymus, and tongue.
[0950jAdditionai diseases that can be treated with the compositions and methods of the present disclosure include genetic diseases with an immunological phenotype. Exemplary genetic diseases with an immunological phenotype are described in, e.g., Table 52 of Tangye et al., Journal of Clinical Immunology volume 42, pagesl473-1507 (2022), which is incorporated herein by reference in its entirety.
[0951]ln some embodiments, patients receiving multispecific antigen-binding protein treatment of the present disclosure can be monitored for their responsiveness to the treatment. For example, a physician may monitor the response of a mammalian subject (e.g., a human) to treatment with multispecific antigen-binding proteins of the present disclosure by analyzing the quantity of IFNy secreted by CD8-J- T- cells within a particular patient. For example, a composition of the present disclosure may be capable of reducing IFNy secretion by between 1% and 100% (e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%). Alternatively, a physician may monitor the responsiveness of a subject (e.g., a human) to treatment with a composition of the present disclosure by analyzing the Treg cell population in the lymph of a particular subject. For example, a physician may withdraw a sample of blood from a mammalian subject (e.g., a human) and determine the quantity or density of a population of Treg cells (e.g., CD4+ CD25+ FOXP3+ Treg cells or CD17+ Treg cells) using established procedures, such as FACS analysis. In such embodiments, high counts of Treg cells can be indicative of efficacious therapy, while lower Treg cell counts may indicate that the patient is to be prescribed or administered higher dosages of the multispecific antigen-binding protein of the present disclosure until, e.g., an ideal Treg cell count is achieved. In addition, a physician of skill in the art may monitor the effect of treatment by administration of a composition of the present disclosure to a subject suffering from an immunological disorder, such as an autoimmune disease described herein, by analyzing the quantity of autoreactive CD8+ T-cells within a lymph sample isolated from the patient. Multispecific antigen-binding proteins of the invention may attenuate the proliferation of autoreactive T-cells, e.g., by binding TNFR2 and CD25 at the surface of an autoreactive T-cell and inducing apoptosis, and/or by stimulating the expansion of Treg cells that subsequently eliminate autoreactive T lymphocytes. Treatment with multispecific antigenbinding proteins may lead to reduced quantities of autoreactive T-cells within the lymph isolated from a patient receiving treatment, and a rapid decline in the population of autoreactive T-cells in a lymph sample isolated from such a patient may indicate effective treatment. In cases where a lymph sample isolated from a patient exhibits an autoreactive T-cell count that has not declined in response to agonistic TNFR2 and/or anti~CD25 antibody therapy, a physician may prescribe the patient higher doses of the antibody or an antigen-binding fragment thereof or may administer the multispecific antigenbinding proteins with higher frequency, e.g., multiple times per day, week, or month. [0952]Multispecific antigen-binding proteins described herein may be administered as a monotherapy or in combination with one or more additional therapeutic agents.
[0953]ln some embodiments, multispecific antigen-binding proteins of the present disclosure may also be admixed, conjugated, or administered with, or administered separately from, another agent that promotes Treg cell proliferation. Additional agents that can be used to promote Treg cell expansion include, e.g., IL-2 and TN Fa, the cognate ligand for TNFR2 and CD25.
[0954]ln some embodiments, pharmaceutical compositions of the invention may be formulated for coadministration or sequential administration with one or more additional active agents that can be used to inhibit CD8+ T-cell growth. Examples of additional active agents that can be used to inhibit cytotoxic T-cell proliferation and that can be conjugated to, admixed with, or administered separately from a multispecific antigen-binding protein of the present disclosure include cytotoxic agents, e.g., those described herein.
[0955]Exemplary cytotoxic agents that can be conjugated to, admixed with, or administered separately from multispecific antigen-binding proteins of the present disclosure include, but not limited to, 13-cis retinoic acid, 14-hydroxy-retro-retinol, 2-chloro-2'-deoxyadenosine, 2-Chloro-2'-arabino-fluoro-2'- deoxyadenosine, 2-chlorodeoxyadenosine, 2-chlorodeoxyadenosine (2-Cda), 2 -deoxycoformycin, 3- methyl TTNEB, 6-mercaptopurine, 6-thioguanine, 9-aminocamptothecin, 9-cis retinoic acid, aclarubicin, acodazole hydrochloride, acronine, adozelesin, adozelesin, adriamycin, aldesleukin, all-trans retinoic acid, all-trans retinol, altretamine, ambomycin, ametantrone acetate, aminoglutethimide, amsacrine, amsacrine, anastrozole, anisomycin, anthramycin, acivicin, asparaginase, asperlin, azacitidine, azacitidine, azetepa, azotomycin, AZQ, batimastat, benzodepa, bicalutamide, Bis (platinum), bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, busulfan, cactinomycin, calusterone, camptothecin, caracemide, carbetimer, carboplatin, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, CEP-751, chlorambucil, chlorambucil, cirolemycin, cisplatin, cisplatin, cladribine, combretestatin a-4, Cl-973, CPT-11, crisnatol mesylate, cyclophosphamide, cyclophosphamide, cytarabine, cytarabine, daca (n-[2-(dimethyl-amino) ethyl] acridine-4-carboxamide), dacarbazine, dactinomycin, Dactinomycin (Actinomycin D), darubicin, daunomycin, Daunomycin, daunorubicin hydrochloride, decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate, diacarbazine (DTIC), diaziquone, docetaxel, dolasatins, doxorubicin, Doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, DWA 2114R, edatrexate, eflornithine hydrochloride, ellipticine, elsamitrucin, enloplatin, enpromate, epipropidine, Epi rubicin, epirubicin hydrochloride, erbulozole, esorubicin hydrochloride, estramustine, estramustine phosphate sodium, etanidazole, ethiodized oil i 131, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine (2-F-ara-AMP), fludarabine phosphate, fluorodeoxyuridylate, fluorouracil, flurocitabine, fosquidone, fostriecin sodium, gemcitabine, gemcitabine, gemcitabine hydrochloride, gold 1S8AU , homocamptothecin, hPRL-G129R, hydroxyurea, hypoxanthine, idarubicin hydrochloride, ifosfamide, ifosfamide, ilmofosine, interferon y- 1b, interferon a-2b, interferon o-nl, interferon a-n3, interferon a-2a, interferon P-la, iproplatin, irinotecan hydrochloride, JM216, JM335, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, linomide, lometrexol sodium, lomustine, losoxantrone, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin, mitomycin, mitomycin C, mitosper, mitotane, mitoxantrone, mitoxantrone hydrochloride, mitozolomide, mycophenolic acid, N-{2- chloroethyi)-N' cyclohexyl-N-nitrosourea (CCNU), N-(2-chloroethyl)-N'-(diethyl) ethyiphosphonate-N- nitrosourea (fotemustine), N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU), N- (4-hydroxyphenyl) retinamide, N,N'-Bis (2-chloroethyl)-N-nitrosourea (BCNU), nitrogen mustard (mechlor ethamine), N-methyl-Nnitrosourea (MNU), nocodazole, nogalamycin;ormaplatin, N-propargyl- 5,8-dideazafolic acid, ormaplatin, oxaliplatin, oxisuran, paclitaxel, pegaspargase, peliomycin, pentamustine, peploycinsulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, pyrazoloacridine, raltitrexed, rhizoxin, rhizoxin d, riboprine, rogletimide, safingol, safingol hydrochloride, semustine, simtrazene, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, streptozotocin, strontium chloride Sr 89, sulfur mustard, sulofenur, talisomycin, taxane, taxoid, tecogalan sodium, tegafur, teloxantrone hydrochloride, temoporfin, temozolomide, teniposide, teniposide 9-amiro camptothecin, teroxirone, testolactone, thiamiprine, thioguanine, thiotepa, thiotepa, thymitaq, tiazofurin, tirapazamine, tomudex, tomudex, TOP-53, topotecan, topotecan hydrochloride, toremifene citrate, trestolone acetate, trichostatin A, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tubulozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine, vinblastine sulfate, vincristine, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, or zorubicin hydrochloride, , [0956]Other therapeutic agents that can be conjugated to, admixed with, or administered separately from multispecific antigen-binding proteins of the present disclosure include, but are not limited to, 2f deoxycoformycin (DCF), 1,25 di hydroxyvitamin D3, 5-ethynyluracil, 9-dioxamycin, abiraterone, acylfulvene, adecypenol, ALL-TK antagonists, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, anagrelide, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, antiandrogen, prostatic carcinoma, anti-dorsalizing morphogenetic protein-1, antiestrogen, antineoplaston, antisense oligonucleotides, a phidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, ara-CDP-DL-PTBA, argininedeaminase, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivatives, balanol, BCR/ABL antagonists, benzochlorins, benzoylstaurosporine, beta lactam derivatives, beta- alethine, betaclamycin B, betulinic acid, bFGF inhibitor, bisantrene, bisaziridinylspermine, bisnafide, bistratene A, bleomycin A2, bleomycin B2, breflate, budotitane, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives (e.g., 10-hydroxy-camptothecin), canarypox IL-2, capecitabine, carboxamide-amino-triazole, carboxyamidotriazole, CaRest M3, CARN 700, cartilage derived inhibitor, casein kinase inhibitors (ICOS), castanospermine, cecropin B, cetrorelix, chlorins, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, clomifene analogues, clotrimazole, collismycin A , collismycin B, combretastatin A4, combretastatin analogue, conagenin, crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabine ocfosfate, cytolytic factor, cytostatin, dacliximab, dehydrodidemnin B, deslorelin, dexifosfamide, dexrazoxane, dexverapamil, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, , diphenyl spiromustine, discodermolide, docosanol, dolasetron, d oxifl uridine, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur, epithilones, epothilones (A, R~H; B, R=Me), epristeride, erythrocyte gene therapy, estramustine analogue, estrogen agonists, estrogen antagonists, etoposide 4'-phosphate (etopofos), exemestane, fadrozole, filgrastim, finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, glutathione inhibitors, hepsulfam, heregulin, hexamethylene bisacetamide, homoharringtonine (HHT), hypericin, ibandronic acid, idarubicin, idoxifene, idramantone, ifepristone, ilomastat, imidazoacridones, imiquimod, immunostimulant peptides, insulin-like growth factor-1 receptor inhibitor, interferon agonists, interferons, interleukins, iobenguane, iododoxorubicin, ipomeanol, irinotecan, iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, leukemia inhibiting factor, leukocyte alpha interferon, leuprolide+estrogen+progesterone, leuprorelin, levamisole, liarozole, linear polyamine analogue, lipophilic disaccharide peptide, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lombricine, lometrexol, lonidamine, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides, mannostatin A, marimastat, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, meterelin, methioninase, metoclopramide, MIF inhibitor, miltefosine, mirimostim, mismatched double stranded RNA, mithracin, mitoguazone, mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast growth factor-saporin, mofarotene, molgramostim, monoclonal antibody, human chorionic gonadotrophin, monophosphoryl lipid A+myobacterium cell wall sk, mopidamol, multiple drug resistance gene inhibitor, multiple tumor suppressor 1-based therapy, mustard anticancer agent, mycaperoxide B, mycobacterial cell wall extract, myriaporone, N- acetyldinaline, nafarelin, nagrestip, naloxone+pentazocine, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidant, nitrullyn, N-substituted benzamides, O6-benzylguanine, octreotide, okicenone, oligonucleotides, onapristone, ondansetron, oracin, oral cytokine inducer, osaterone, oxaliplatin, oxaunomycin, paclitaxel analogues, paclitaxel derivatives, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine, peldesine, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perillyl alcohol, phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piritrexim, placetin A, placetin B, plasminogen activator inhibitor, platinum complex, platinum compounds, platinum-triamine complex, podophyllotoxin, propyl bis-acridone, prostaglandin J2, proteasome inhibitors, protein A-based immune modulator, protein kinase C inhibitor, protein kinase C inhibitors, microalgal, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurins, pyridoxylated hemoglobin polyoxyethylene conjugate, raf antagonists, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-GAP inhibitor, retelliptine demethylated, rhenium Re 18S etidronate, ribozymes, Ril retinamide, rnerbarone, rohitukine, romurtide, roquinimex, rubiginone B 1, ruboxyl, saintopin, SarCNU, sarcophytol A, sargramostim, Sdi 1 mimetics, senescence derived inhibitor 1, sense oligonucleotides, signal transduction inhibitors, signal transduction modulators, single chain antigenbinding protein, sizofiran, sobuzoxane, sodium borocaptate, sodium phenylacetate, solverol, somatomedin binding protein, sonermin, sparfosic acid, spicamycin D, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-cell division inhibitors, stipiamide, stromelysin inhibitors, sulfinosine, superactive vasoactive intestinal peptide antagonist, suradista, suramin, swainsonine, synthetic glycosaminoglycans, tallimustine, tamoxifen methiodide, tauromustine, tazarotene, tellurapyrylium, telomerase inhibitors, tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide, thiocoraline, thrombopoietin, thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, tin ethyl etiopurpurin, titanocene dichloride, topsentin, toremifene, totipotent stem cell factor, translation inhibitors, tretinoin, triacetyluridine, triciribine, tropisetron, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists, variolin B, velaresol, veramine, verdins, vinorelbine, vinxaltine, vitaxin, zanoterone, zilascorb, or zinostatin stimalamer,
[0957]ln some embodiments, multispecific antigen-binding proteins of the present disclosure may be admixed, conjugated, or administered with, or administered separately from, an anti-inflammatory agent. Exemplary anti-inflammatory agents useful in conjunction with the compositions and methods of the invention include steroids, colchicine, hydroxychloroquine, sulfasalazine, dapsone, methotrexate, mycophenolate mofetil, azathioprine, cyclosporine, sirolimus, everolimus, azathioprine, leflunomide, mycophenolate, inhibitors of IL-1/IL-2/IL-4/IL5/IL-6/IL-13/IL-17/IL- 23/TNF/compiement/BAFF/interferon/JAK/CD28/lgE/lntegrins/T cell costimulation pathway or B-cell depleting agents.
[0958]ln some embodiments, multispecific antigen-binding proteins of the present disclosure may be admixed, conjugated, or administered with, or administered separately from, an immunotherapy agent. Exemplary immunotherapy agents useful in conjunction with the compositions and methods of the invention include an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-Ll agent, an anti-PD-L2 agent, a TNFa cross-linking agent, a TRAIL cross-linking agent, an anti-CD27 agent, an anti-CD30 agent, an anti- CD40 agent, an anti-4-lBB agent, an anti-GITR agent, an anti-OX40 agent, an anti-TRAI LR1 agent, an anti-TRAI LR2 agent, an anti-TWEAKR agent, an anti-TLIA agent, an anti-LIGHT agent, an anti-BTLA agent, an anti-LAG3 agent, an anti-Siglecs agent, an anti-ICOS ligand agent, an anti-B7-H3 antibody; an anti-B7- H4 agent; an anti-VISTA agent; an anti-TMIGD2 agent; an anti-BTNL2 agent; an anti-CD48 agent; an anti- KIR agent; an anti-LIR agent; an anti-ILT agent; an anti-NKG2D agent; an anti-NKG2A agent; an anti-MICA agent; an anti-MICB agent; an anti~CD244 agent; an anti-CSFl R agent; an anti-IDO agent; an anti-TGFp agent; an anti-CD39 agent; an anti-CD73 agent; an anti-CXCR4 agent; an anti-CXCL12 agent; an anti- SIRPA agent; an anti-CD47 agent; an anti-VEGF agent; and an anti-neuropilin agent and, e.g,, agents directed toward the immunological targets described in Table 1 of Mahoney et al., Cancer immunotherapy, 14:561-584 (2015), the disclosure of which is incorporated herein by reference. The immunotherapy agent described herein may be, for example, an antibody, a small molecule, or a chimeric antigen receptor.
[0959]ln some embodiments, multispecific antigen-binding proteins of the present disclosure can also be admixed with, co-administered with, or administered separately from Bacillus Calmette-Guerin (BCG), a bacterial strain that has been used to treat a variety of immunological disorders, such as type I diabetes, multiple sclerosis, scleroderma, Sjogren's disease, systemic lupus erythematosus. Grave's disease, hypothyroidism, Crohn's disease, colititis, an autoimmune skin disease, and rheumatoid arthritis, among others. For instance, multispecific antigen-binding proteins of the present disclosure may be included in a therapeutic regimen in combination with BCG for the treatment of an immunological disorder (e.g., one of those described above, such as type I diabetes or rheumatoid arthritis). The multispecific antigen-binding protein may be co-administered with BCG, e.g., by an injection route described herein. Alternatively, the multispecific antigen-binding protein may be administered separately from a BCG-containing composition. The use of BCG to treat immunological disorders has been described, e.g., in US 6,660,487; and US 6,599,710; the disclosures of each of which are incorporated herein by reference in its entirety.
EXAMPLES
[0960]The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.
Example 1, Camelid immunization with TNFR2
[0961]Three alpacas were immunized by four subcutaneous injections with recombinant human TNFR2 (10417-H08H, Sino Biologica Is) and complete/incomplete Freund's or Gerbu FAMA adjuvant using standard protocols to elicit a humoral immune response that included the generation of antigen-specific conventional and heavy-chain only (VHH) antibodies.
[0962]Before the first and after the third injection, serum was prepared from blood samples. Antibody induction was monitored by comparing antigen-specific antibody titers in the sera before and after immunization by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well Maxisorp plates were coated with human TNFR2 (10417-H08H, Sino Biologicals), blocked and incubated with diluted serum samples. TNFR2-specific antibodies were bound by alkaline phosphatase-conjugated goat anti-alpaca IgG (H+L) (Jackson ImmunoResearch, Cat. No. 128-055-160) and detected using p-Nitrophenyl Phosphate.
Example 2. Phage library constractton for generating anti-TNFR2 VHH libraries
[0963]Four to ten days after the fourth injection in accordance with procedures described in Example 1, blood samples were collected, and four to six days after the fourth injection a bone marrow sample was aspirated. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood or bone marrow following density gradient purification with Ficoll-PaqueTM Plus. Total RNA was extracted from freshly isolated PBMCs.
[0964]To generate anti-TNFR2 VHH immune libraries, total RNA was reverse transcribed to cDNA using random hexamer primers. Conventional and heavy chain IgH cDNA fragments were amplified by polymerase chain reaction (PCR) using primers annealing to the IgH leader sequence region and the CH2 region. The resulting amplicons represented the VHH and VH cDNAs, respectively. The VHH fragment was isolated and used as template for a nested PCR to introduce appropriate endonuclease recognition sites for cloning into the pQ81 phagemid in frame with gene III. Libraries were transformed into electrocompetent E. coli TGI cells, in total, six libraries were built, with 95.5% to 100% VHH insert frequency and maximum library sizes between 4.2x10s and 2.4xl03. Phage for phage display was prepared following standard protocols.
[0965]Binders to human and mouse TNFR2 were enriched from VHH immune libraries by two rounds of phage display. The general panning strategy is illustrated in Figure 2A using the panning substrates listed Table 3. For the Table below: Hu, Human; Ms, mouse; PBS, phosphate buffered saline; Cat., catalog; MW, molecular weight; Calc., Calculated; Seq., Sequence; N-term., N-terminal; aa, amino acid.
Table 3. Panning substrates for anti-TNFRZ VHH
[0966]For the first panning round, libraries originating from the first harvested blood sample and the first harvested bone marrow sample of the same animal were pooled in equal parts (at the phage level), resulting in three pooled input libraries per antigen. Each library was panned under four conditions (two antigen concentrations and two ways of antigen immobilization) with human TNFR2, resulting in 12 panning reactions. For the second rounds of panning, six output samples (enriched libraries) from the first round were chosen and served as input libraries for the second round. Preferentially, the enriched libraries from the higher panning substrate concentration were chosen to preserve maximum diversity. Pannings of the second round were performed with three antigen concentrations of human and mouse antigen to result 24 conditions. Three antigen concentrations for TN FR2 were further chosen to recover binders with lower affinity. This panning regimen was implemented to identify binders that crossreacted with human and mouse TNFR2.
[0967] I n some cases, binders with medium affinity were preferred since higher valency may drive avidity. Thus, the antigen concentrations in the second panning round were the same, one tenth and one hundredth of the first round.
[0968]Phages were produced according to QVQ Holding B.V. (QVQ) standard operating procedures (SOPs) and phage titers were determined to ensure at least 10-fold excess over the maximum diversity of the libraries. Panning substrates were commercially purchased (see Table 1). The panning substrates were immobilized either by direct coating on enzyme-linked immunoassay (ELISA) plates or by binding of biotinylated antigen on neutravidin-coated ELISA plate. Glycerol stocks were prepared from all outputs and are stored at -80°C.
[0969]Panning outputs were analyzed by random clone picking/periplasmic extract (PE)-ELISA/Sanger sequencing (QVQ) and next-generation sequencing (NGS; Genewiz/PipeBio).
[0970]For random colony picking, rescued outputs of the first and second panning rounds were plated out and 920 random single clones (equal numbers of colonies from each condition) were selected to create masterplates (96-well format). From the masterplates, expression cultures in deep-well plates were inoculated to produce periplasmic extracts containing monoclonal VHH. Periplasmic extracts were used to determine binding of individual VHHs to human, mouse and cynomolgus antigen by ELiSA. For conditions where the panning substrate was biotinylated and captured by neutravidin, background binders were identified by ELISA with neutravidin. Ail masterplates were sequenced by the Sanger method.
[0971]For NGS analysis, minipreps from input libraries and outputs after the first and second rounds of panning were prepared, amplified by PCR and sequenced by NGS.
Example 3, Next-generation sequencing of candidates [0972]Following two rounds of panning, phages were eluted and corresponding phagemid DNA was extracted. Identification of initial V-body candidates was performed in a parallelized fashion, employing a random colony picking, as well as a next-generation sequencing (NGS) approach, as orthogonal techniques to yield a particularly diverse set of initial candidates. Prior to NGS techniques, random colony picking was the prevalent method for initial hit identification, which involved transformation of a phagemid pool (from a panning elution) and selection of individual bacterial colonies to isolate single clones. Following this approach, 920 single colonies were randomly picked from the 36 samples of the second panning round (Figure 28). Then, individual clones were expressed and subject to ELISA screening against the target antigen TNFR2 to select for antigen binding V-bodies, which were further functionally characterized,
[0973] All panning eluates were sequenced using NGS. In brief, the entire anti-TNFR2 VHH region was PCR-amplified from isolated phagemid pools by primers annealing to universal phagemid sequences 5' and 3s of the VHH-encoding region. In a second step, the generated amplicons were fused to sequencing-compatible and sample-specific barcodes. By fusing unique barcodes, it was possible to multiplex hundreds of different samples. Following the preparation of 51 samples, an Illumina NovaSeq 6000 with an SP flowcell was employed for sequencing, yielding 250 base pair (bp) reads from each direction and a total of ™600 million reads. To account for differences in the number of expected unique sequences in the library, and both panning rounds, each library was sequenced with a total of 20 million reads, compared to the first and second round of panning with 2 million reads each. This strategy allowed for covering sufficient sequence space in the libraries, as well as in the panning eluates. A spikein of 30% of a standard PhiX reference genome control into the sequencing reaction helped to provide a technical quality control for assessing sequencing accuracy. The NGS raw data contained multiplexed sequencing reads, which were de-multiplexed based on the sample-specific barcodes. The demultiplexed data containing unmerged sequencing reads were then processed by employing an NGS analysis platform. In brief, forward and reverse sequence pairs were merged by their overlapping sequence, thereby generating a full anti-TNFR2 VHH sequence from two half sequences (Figure 3). The framework regions, CDRs, and sequence-specific liabilities were then annotated for the merged V-body sequences.
[0974]Based on CDR3 identity, anti-TNFR2 V-body sequences were clustered, allowing for a detailed analysis of V-body enrichment during phage display, sequence diversity, CDRS length distribution and cluster abundance. Identified V-bodies can be classified into eleven distinct clusters, as follows: Group A, Group B, Group C, Group D, Group G, Group J, Group K, Group L, Group M , Group N, and Group O. The following Table 4-1 to Table 4-33 display the amino acid frequency distribution at each amino acid (AA) position (IMGT) for CDR1, CDR2 and CDR3 for the eleven clusters. Table 5 provides the sequence identifiers of amino acid sequences of the complementarity determining regions (CDR1, CDR2 and CDR3), amino acid and DNA sequences of the full-length anti-TNFR2 VHH domain for the identified anti- TNFR2 V-bodies.
Table 4~1 anti TNFR2 CDR1 ammo add frequency distribution for Group A
Table 4-2 ants TOFR2 CDR2 amino acid frequency distribution for Group A Table 4~3 anti TNFR2 CDR3 amino add frequency distribution for Group A
Table 4-4 anti TNFR2 CDR1 amine add frequency distribution for Group B
Position i Table 4-5 anti TMFR2 CDR2 amino add frequency distribution for Group B
Table 4-6 anti TIMFR2 CDR3 amino acid frequency distribution for Group B Table 4-7 anti TMFR2 CDR1 ammo add frequency distribution for Group C
Table 4-8 anti TNFR2 CDR2 amino add frequency distribution for Group C Table 4-9 anti TMFR2 CD$3 ammo add frequency distribution for Group C
UJ w 4^
3l63571.84v9
Table 4-10 anti TNFR2 CDR1 amino acid frequency distribution for Group D
Table 4-11 anti TNFR2 CDR2 amino add frequency distribution for Group D Table 4-12 anti TNFR2 CQR3 amino acid frequency distribution for Group D
Position
Table 4-13 anti TNFR2 CDR1 ammo add frequency distribution for Group G
Position i Table 4-14 anti TIMFR2 CDR2 ammo add frequency distribution for Group G
5.000075
3163S71849v Table 4-15 anti TN FR2 CDR1 amino add frequency distribution for Group J
Table 4-17 anti TNFR2 CDR2 amino add frequency distribution for Group J Table 4-18 anti TNFR2 CDR3 amino acid frequency distributism for Group J
Table 4-19 a nti TiMFRZ CDR1 amino add frequency distribution for Group K Position Table 4-20 a nti ThlFRl CDR2 amino add frequency distribution for Group K
Table 4-21 anti TIMFR2 CDR3 amino add frequency distribution for Group K Table 4-22 anti TNFR2 CDR1 amino add frequency distribution for Group L
Position
Table 4-23 anti TNFR2 CDR2 amino add frequency distribution for Group L Table 4-24 anti TNFR2 CDR3 amino add frequency distribution for Group L
Table 4-25 anti TIMFR2 CDR1 amino acid frequency distribution for Group M Tabie 4-26 anti TIMFR2 CM2 amino acid frequency distribution for Grouq iVI
Table 4-27 anti TNFR2 CDR3 amino acid frequency distribution for Groups M Table 4-28 anti TMFR2 €DR1 ammo acid frequency distribution for Group
Table 4-29 anti TMFR2 CDR2 ammo acid frequency distribution for Group M Table 4-30 anti TMFR2 €DR3 ammo acid frequency distribution for Group
Table 4-31 anti TMFR2 CDR1 amino add frequency distribution for Group O Table 4-32 a nt! TMFR2 CD$2 ammo add frequency distribution for Group 0
Tabie 4-33 anti "HMFR2 CDR3 ammo acid frequency distribution for Group O
3163S7184v9
SZOOOO! Tsble 5. Sequence Identifiers of aoti-lWR2 V-bodies identified from panning
Example 4. Flow cytometry binding of anti-T^FR2 V-bodies to TMFR2
[0975]To measure the binding of V-bodies to cell-displayed TNFR2 from human, cynomolgus or mouse, HEK293 cells were transfected with plasmids encoding for respective antigens. After 48 to 72 hours, binding was measured by incubation of His-tagged anti-TNFR2 V-bodies with cells at various fixed concentrations, followed by washing and detection with Alexa488 fluorophore-labelled anti-His antibodies. To detect the binding of anti-TNFR2 V-bodies to specific cysteine- rich domains (CRDs) of TNFR2, binding to HEK293 transfected with constructs where a single CRD was exchanged for camel CRD was measured. Lack of binding to a particular construct indicated binding to the domain which was exchanged.
[0976]For generation of data depicted in Figures 4A-4B, HEK293T cells were transiently transfected with a plasmid encoding human TNFR2 (hTNFR2; hTNFR2__pcDNA3.4.dna). After 48 hours, HEK293T cells were harvested and incubated with 1 pM purified His-tagged (myc-his tag) VHHs, including a control VHH against an irrelevant antigen. VHH binding was then detected using an Alexa488-labeled anti-His tag antibody and measured by flow cytometry (iQue).
[0977]For generation of data depicted in Figures 5A-SB HEK293T ceils were transiently transfected with a plasmid encoding human TNFR2 (hTNFR2; hTNFR2__pcDNA3.4.dna). After 48 hours, HEK293T cells were harvested and incubated with 100 nM purified His-tagged (myc-his tag) VHHs, including a control VHH against an irrelevant antigen. Anti-TNFR2 VHH binding was then detected using an Alexa488-labeled anti-His tag antibody and measured by flow cytometry (IQue).
[0978]For generation of data depicted in Figures 6A-6B, HEK293T cells were transiently transfected with a plasmid encoding cynomolgus TNFR2 (cTNFR2; cTNFR2_pcDNA3.4.dna) or mouse TNFR2 (mTNFR2; mTNFR2_pcDNA3.4.dna). After 48 hours, HEK293T cells were harvested and incubated with 100 nM purified His-tagged (myc-his tag) VHHs, including a control VHH against an irrelevant antigen. Anti- TNFR2 VHHs binding was then detected using a Alexa488-labeled anti-His tag antibody and measured by flow cytometry (iQue).
(0979)Figure 7 shows testing of human TNFR2 V-body binding across a range of concentrations for T- 002, T-014, T-001, T-006, T-007, and T-003. V-bodies were tested at the following molar concentrations: 100 nM, 50 nM, 12.5 nM, 6.25 nM, 3.12 nM and 1.55 nM. For generation of data depicted in Figure 7, HEK293T ceils were transiently transfected with a plasmid encoding human TNFR2 (hTNFR2; hTNFR2_pcDNA3,4.dna). After 48 hours, HEK293T cells were harvested and incubated with increasing molar concentrations of purified His-tagged (myc-his tag) VHHs, including a control VHH against an irrelevant antigen. Anti-TNFR2 VHH binding was then detected using an Alexa488-labeled anti-His tag antibody and measured by flow cytometry (IQue). The bar histogram (Figure 7) shows the percentage of Alexa488 positive cells for T-002, T-014, T-001, T-006, T-007, and T-003.
Example 5. Surface plasmon resonance binding affinities of the [0980]Binding affinities of the anti-TNFR2 V-bodies to their respective target was determined by surface plasmon resonance (SPR) using a Caterra LSA instrument. A schematic diagram depicting the experimental setup of the present Example is shown in Figure 8. Affinity purified anti-TNFR2 V-bodies were covalently crosslinked onto an LSA HC200M chip using EDC/Sulfo NHS. The interaction with human, cynomolgus, and mouse TNFR2 (extracellular domain) (V-body coupling concentration: human, 1 pM; cynomolgus/mouse, 4 pM) was measured under physiological conditions (Running Buffer: HBST- 50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 % (w/v) BSA, 0.05% (v/v) Tween20, 25°C) using eight different antigen concentrations (3-fold serial dilutions, start at 200 nM). Resulting sensorgrams (Figures 9A-9F) were analyzed and equilibrium-binding affinities (KDS) were calculated using Carterra's data analysis software. Figure 10 shows a summary of binding affinities of 16 anti-TNFR2 V-bodies including T-002, T- 006, T-001, T-007, T-014, and T-003. Seven of the 16 V-body candidates tested (including T-002, T-006, T-001, T-007, T-014, and T-003) were shown to bind human TNFR2 with 1-2-digit nM affinity. Nine and zero V-bodies demonstrated cross-reactivity to cynomolgus TNFR2 (96% identical to hTNFR2 sequence) and mouse TNFR2 (63% identical to hTNFR2 sequence), respectively.
Example 6. MR2-1 competition of anti-TNFR2 V-bodies
[0981]To investigate whether humanized TNFR2 V-bodies targeted the epitope recognized by a MR2-1 bivalent agonist, TNFR2 expressing HEK cells (clone 25) were incubated with or without MR2-1 (5 or 10 pg/mL) prior to (pre-) and/or during (co-) incubation with His-tagged TNFR2-specific V-bodies. After washing, V-body binding was detected by labelled anti-His antibody. Binding inhibition in the presence of MR2-1 indicated binding of TNFR2 V-bodies and MR2-1 bivalent agonist to an overlapping epitope. Specifically, T-013hul and T-018hul may recognize the same epitope as MR2-1. MR2-1 binding enhanced binding of T-015hul, T-017hul and T-Ollhul to TNFR2 (Figure 11).
Example 7. TMFR2 reporter assay
[0982JHEK293 nuclear factor kappa B (NF-xB) reporter (Luc) cells stably expressing TN FR2 were established by transfection and antibiotic selection. Several clones were derived from the cell pool. In these cells, TNFR2 signaling was measured by NF-KB-induced expression of firefly luciferase (Luc). Data showing agonism of multivalent constructs characterized on NF-kB reporter HEK293 cells stably expressing TNFR2 are displayed in Figures 12A-12D. Typically, luciferase activity was measured 16- to 24-hours after incubation with the anti-TNFR2 V-bodies. For assay control experiments, commercially available human TNFR2 agonist MR2-1 monoclonal antibodies were tested on NF-xB reporter (Luc) HEK293 reporter cell-line stably expressing TNFR2 (clone 25) versus parental cell line (PCL) (Figure 13), For the sample analysis (n = 1), 2 pL of purified sample (43 mM Citrate 148 mM HEPES, pH 6) was diluted using 10-fold serial dilutions (1:10) in assay medium. A bar graph showing protein concentration (mg/mL) for the anti-TNFR2 V-body constructs and respective controls (see Figure 14A, for sample details) is shown in Figure 148. A bar graph showing relative luciferase units (RLUs) for anti-TNFR2 V- body constructs and respective controls (see Figure 14A, for sample details) tested on a PCL control is shown in Figure 14C. Concentration range curve data generated using MR2-1 (Hycuit) and TN Fa controls revealed increasing RLU measured with increasing concentrations of MR2-1 (mol/L) and TN Fa (ng/mL) (Figures 15A-15B, respectively). Figures ISA- ISC show dot plots of RLU measured across increasing concentrations (mol/L) of control (control 12) and tetravalent anti-TNFR2 V-body fusion constructs comprising four V-bodies mounted onto an Fc of a lgG4 variant comprising S228P and L235E mutations. Figures 17A-17F show dot plots of RLU measured across increasing concentrations (mol/L) of control (control 10) and tetravalent anti-TNFR2 V-body fusion constructs comprising four V-bodies mounted onto an Fc of a lgG4 variant comprising S228P and L235E mutations. Figures 1SA-18C show dot plots of RLU measured across increasing concentrations (mol/L) of control (control 10) and tetravalent anti- TNFR2 V-body fusion constructs comprising four anti-TNFR2 V-bodies mounted onto an Fc of a lgG4 variant comprising S228P and L235E mutations. Figures 19A-19C show exemplary dot plots of RLUs measured across increasing concentrations (mol/L) of control (control 13) and IL-2N88D anti-TNFR2 V- body fusion constructs. Limit of detection is denoted as "LOD". A comparison of RLU measured across increasing concentrations (mol/L) of monospecific constructs 10 and 12 tested on NF-xB reporter (Luc) HEK293 reporter cell-line stably expressing TNFR2 (clone 8) is shown in Figure 20.
Example 8, Testing of TNFR2 agcnism by multivalent constructs
[0983]The ability of multivalent constructs to agonize TNFR2 on human regulatory T cells (Tregs) was demonstrated by the upregulation of Human Leukocyte Antigen, DR isotype (HLA-DR) and chemokine motif (C-C motif) receptor 8 (CCR8) upon activation of TNFR2. HLA-DR and CCR8 are markers expressed on highly suppressive Tregs, which have been shown to be upregulated by TNFR2 agonist MR2-1. A description of established Treg markers (e.g., forkhead box P3 (FoxP3), HLA-DR, CCR8, and OX-40) useful in, e.g., screening of multivalent constructs in first wave binders on primary ceils, is described in Table 6.
Table 6. Treg Markers for Screening Multivalent Constructs
[0984] For testing of multivalent constructs in Tregs, human Tregs were isolated from huffy coats using magnetic beads, and subsequently stimulated with anti-CD3 and anti-CD28 coated stimulation beads with IL-2, in the presence or absence of fixed or increasing concentrations of a control VHH or TNFR2- specific V-bodies.
[0985]An exemplary experimental timeline of TNFR2 stimulation by multivalent constructs on primary human peripheral blood mononuclear cells (PBMCs) and CD4+ CD25+ CD127dim Tregs is shown in Figure 21. Briefly, on Day 0, PBMCs were isolated and selected from three donors (Donors 1-3). Tregs were positively selected for CD4+ CD25^ CD127dim (Mitlenyi Cat. No. 130-094-775]. Fluorescence-activated cell sorting (FACS) analysis was used in cell quality control (QC) experiments to confirm cell populations based on CD4, CD25, CD127, FoxP3, and TNFR2 markers. For the assay set-up, a 96-well plate was seeded with 2xl04 cells/well CD4+ C25+ CD127dim Tregs or PBMCs (200 pl total culture volume). Cells were activated with anti-CD3/anti-CD28 coated stimulation beads (using a ratio of 1:2 for beadsxells) and IL-2 (5 lU/mL). Multivalent constructs and controls were added to each well at a 1:100 dilution. Controls were multivalent control V-body fusion constructs, IL-2 N88D fusion constructs, and TNFR2 agonist MR2-1. On Day 6, FACS analysis was performed for live/dead quantification of cells and assessment of cell populations based on expression levels and density of CD4, FoxP3, HLA-DR, and/or CCR8 markers. A bar graph of an overview of in-assay concentrations (nM) of multivalent (e.g., tetravalent Fc, Vb-Fc-Vb, rigid bivalent no Fc) binders is shown in Figure 22. An exemplary gating strategy for Treg markers useful in the practice of the present Example is shown in Figure 23. Briefly, Treg Donor 1 is shown as an example and an identical strategy was used for Treg Donor 2 and Donor 3. Live cell and CD4 gating was based on Fluorescence Minus One (FMO) control determination of the cutoff point between background fluorescence and positive cell populations. F0XP3, HLA-DR, CCR8, and OX-40 gating was based on the CD4 subset of IgG control-stained sample from the same donor. For FoxP3, the gate was set at approximately 0,2%. For OX-40, HLA-DR and CCR8, the gate was set at approximately 2%.
[0986]Findings from the present Example showed that multivalent anti-TNFR2 constructs increased expression of the Treg suppression marker HLA-DR (Figures 24A-B) and CCR8 (Figure 248). In particular, each of the multivalent formats tested herein resulted in increased HLA-DR and CCR8 expression for specific T-003 binder compared to control with formats, with 4 binders showing the strongest increase. IL-2 N88D fusion constructs increased HLA-DR expression however a tetravalent anti-TNFR2 V-body formal was required for an additional increase in HLA-DR expression compared to control. Further, tetravalent anti-TNFR2 V-bodies strongly increased expression of Treg suppression marker HLA-DR on Fox P3‘ Tregs (Figures 25A-25B).
[0987]Results additionally revealed anti-TNFR2 V-body fusion construct dose-dependent induction of Treg-suppression marker HLA-DR in FoxP3+ CD4+ Tregs (see, e.g., Figures 2S-29). Dose-dependent induction of Treg suppression marker HLA-DR expression across various concentrations of tetravalent anti-TNFR2 V-body Fc fusion construct 10 for Donor 1 (top panel) and Donor 2 (bottom panel) are shown in Figure 27. Tetravalent Fc constructs, irrespective of binder, showed dose-dependent induction of Treg suppression marker HLA-DR expression. Maximal induction of HLA-DR expression was binder dependent. Construct T-003__10 reached a higher plateau compared to monoclonal TNFR2 agonist MR2- 1. Dose-dependent induction of Treg suppression marker HLA-DR expression across various concentrations of rigid bivalent anti-TNFR2 V-body fusion construct 2 for Donor 1 (top panel) and Donor 2 (bottom panel) is shown in Figure 28. Rigid bivalent constructs at higher concentrations showed modest induction of Treg suppression marker HLA-DR expression with donor dependent differences in maximal expression compared to MR2-1. Dose-dependent induction of Treg suppression marker HLA-DR expression across various concentrations of tetravalent anti-TNFR2 V-body (Vb-Fc-Vb) fusion construct 12 for Donor 1 (top panel) and Donor 2 (bottom panel) is shown in Figure 29. Vb-Fc-Vb constructs demonstrated dose-dependent induction of Treg suppression marker HLA-DR expression. Maximal induction and potency was dose dependent.
[0988]Amino acid sequences of exemplary multivalent anti-TNFR2 antigen-binding domain constructs described in herein are provided beiow. In the sequences, VHHjsequences are indicated with a straight underline (e.g., VHH), Fc regions are indicated with bold letters (e.g., hlgG4-P329G SPLE or hlgGl LALAGA), IL-2 N88D sequence is indicated with italic letters (e.g., IL2N88D}, linker sequences are indicated with a wavy underline (e.g., Linker), and hinge regions are indicated with both a wavy underline and bold letters (e.g., hinge). T-002_2xVHH-Fc
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT ¥YLQMNSLKAEDSG¥YFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS LRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNTVYLQMNSLKAEDSGVYFC YYQALSSPNYGQTF'vVGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMIS RTF£¥TCVWD¥SQEDPEyQFNW¥VDG¥£¥HNAK¥KPREEQFNSTYRV¥S¥LTVLHQDWLNGKE¥KCK¥SNKGL
GSSIEKTISKAKGQPR£PQ¥¥TLPPSQE£MTKNQVSLTCLVKGF¥PSDIAV£W£SNGQP£NN¥KTTPP¥LDSDGSFFL YSRLTVD^SRWQEGNVFSCSVMHEALMNHYTQKSLSLSLGK (SEQ ID NO: 3933)
T-002„VHH-Fc-VHH
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEFE GGPSyFLFPPKPKDTLMISRYP£VTC¥WDVSQ£DP£¥QFNWY¥DGV£¥NNAkTKPRE£QFNSTYR¥VSVLTVLHQ DWLNGKEYKCKySNKGLGSSIERTISKAKGQPR£PQ¥¥TLPPSQE£MTKNQVSLTaVKGF¥PSDIA¥£WESNGQP£ NN¥KTrPPVLDSDGSFFL¥SRLTVDKSRWQEGN¥FSCSyMR£ALHNHYYQKSLSLSLGKGGGGSGGGGS£¥QLyES
GGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGREL¥AGIRSDGFTNYADS¥KGRFTISSDNVKNTVYLQMNS LKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 3935)
T-002JL2N88D-FC-VHH
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR PRDi./5D/Wy/VEE/./CGSE7TFMCEYAD£7"A7"/¥EFEA/RW'/7"FCQS//STETGGG,GSGGGGSGGG,GSGGGG5DKTHTCPPC PAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV LWmQDWLNGKE¥KCK¥SNKA£PAPOTISKAKGQPR£PQ¥¥T£PPSREEMTKNQ¥S£TCL¥KGF¥PSDIA¥EW£S NGQPENNYKTTPP¥LDSDGSFFL¥SKLTVDKSRWQQGN¥FSCSVMHEALHNI3¥TQKSLS£SPGGGGGSGGGGSEy
QLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNTVYL QMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 3937)
T"002_VHH-ngidJlnker-VHH
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQLVE SGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNTVYLQMN SLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 3938)
T-006__2xVHH”Fc
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLA QPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPE DTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEFEGGPSVFL
FPPKPKDTLMISRTPEVTCVWD¥SQ£DPE¥QFNWWDGVEVHNAKTKPREEQFNST¥RV¥S¥LTVLHQDWLNGK EYKC§(ySNKGLGSSIEKTISKAKGQPREPQV¥TLPPSQEEMTKNQ¥SLTCLVKGF¥PSDIAVEWESNGQPENN¥KTTP P¥LDSDGSFFL¥SRLT¥D§CSRWQEGNVFS€S¥MHEALHNHYTQKSLSLSLGK (SEQ ID NO: 3939)
T-006JYHH-R-VHH
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPC PAPEFEGGPSyFEFPPKPKDTLMISRTPE¥TCVWD¥SQEDPEVQFNWY¥DGVE¥HNAKTKPR£EQFNSTYRWSV LTVLHQDWLNGKEYKCKVSNKGLGSSIEKT§SKAKGQPREPQVYTLPPSQEEMTKNQVSETCL¥KGFYPSDIAyEWE SNGQPENNYKTTPPVLDSDGSFFFYSRLTVDICSRWQEGNVFSCSVMHFAEHNHYTQKSESLSLGICGGGGSGGGGSE yQL^SGGGLAQPGGSjM^AASGn^RYAMSWA^APGJ<GI^WySGISDDGSPIY¥ADSy|<GRFIlSRDNAK!iI VYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSS (SEQ ID NO: 3941)
T-006JL2N88D-Fc~VHH
APTSSSTKKTQLQLEHLLLDLQMILNG1NNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLR PRDLlSDINVIVLELKGSETTFMCEYADETATlVEFLNRWSTFCQSHSTLTGGGGSGGGGSGGGGSGGGGSWmTGPPG PAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVNNAKTKPREEQYNSTYRVVSV LWLNQDWLNGKEYKCKVSNKALPAPIERTISKAKGQPREPQ¥¥T1PPSREEMTKNQ¥SLTCLVKGF¥PSDIA¥EWES NGQPENNYKTTPP¥LDSDGSFFL¥SKLTVDKSRWQQGNVFSCSVMHEALHNI3YTQKSLSESPGGGGGSGGGGS£¥
QLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKNTV YLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSS (SEQ ID NO: 3943)
T-006_ VHH-rigidJinker-VHH
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTiSRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGS EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSS (SEQ ID NO: 3944)
T-001..2xVHH-Fc
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS LRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNTVYLQMNSLKPEDTAVYT CYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEFEGGPSVFLFPPRPjCOTLMI SRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNARTKPREEQFNSTYRWSVLWLHQDWLNGKEYKC^CVSN^CGL
GSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDiAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 3945)
T-OOlJ/HH-Fc-VHH
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSESKYGPPCPSCPESgCYGPPCPPCPAPEFE
GGPSyFLFPPKPKDTLMISRTPEVTCWVDV5QEDPE¥QFNW¥VDGVEVHNAKTKPREEQFNST¥RWSVLTVI.HQ DWLNGKEYKCKVSNKGLGSSIEICTISKAKGQPREPQWTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMREALHNHYTQKSLSLSLGKGGGGSGGGGSEyQLyES GGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTSSWDTVKNTVYLQMN
SLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 3947)
T-001JL2N88D-FC-VHH
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQaEEELKPLEEVLNLAQSKNFHLR PRDUSDINViVLELKGSETTFMCEYADETATIVEFLNRWITFCQSilSTLTGGGGSGGGGSGGGGS.GGGGSWTmCPPC PAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVICFNWYVDGVEVHNAKTKPREEQYNSTYRWSV LWLHQDWLNGKEYKaCVSNICALPAPIEKTISKAKGQPREPQYYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNyFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEV
QLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNTVY LQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 3949)
T-OO1„ VHH-rigidJinker-VHH
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQLVE SGgGLV^GGSLB^CMSGS|FRADAMGWHRQWGl<PBEFyAG|RSPGFn^¥AEAyKGRFriSWPTyKNiyYLSM NSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 3950)
T-007_2x¥HH-Fc
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK NTGYLEMNSLKVEDTAVYYCAATPSGKAY5YWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSL RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLEMNSLKVEDTAVYY GAATPSGKAYSYV¥GQGTQVT'/SSGGGGSESKYGPPCPSCPESK¥GPPCPPCPAPEFEGGPS¥FLFPPKPKDTLMISRT PE¥TCW¥D¥SQEDPEVQFNW¥yDG¥E¥HNAKTKPREEQFNST¥RV¥S¥LT¥LHQDWLNGKE¥KCR¥SNKGLGSS
IEKTISKAKGQPREPQ¥YTLPPSQEEMTKNQ¥SLTCL¥KGF¥PSDIA¥EWESNGQPENN¥KTTPP¥LDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 3951)
T-007_VHH-Fc-VHH
EVQLVESGGGLVQAGGSLRL5CAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTSSRDNAK NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSESKYGPPCP5CPESKYGPPCPPCPAPEFEG GPS¥FLFPPKPKDTLMISRTPEVTCW¥D¥SQEDPE¥QFN¥¥WDG¥£¥HNAKTKPREEQFNST¥RWS¥LT¥LHQD WLNGK£¥KCK¥SNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ¥SITCL¥KGFYPSDIAV£WESNGQP£N NYKTTPPYLDSDGSFFLYSRLTVDKSRWQEGNVFSCSYMHEALHNFIYTQKSLSLSLGKGGGGSGGGGSEyQLVESG
GGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINW5NGRTNYADSVKGRFTISRDNAKNTGYLEMN SLKVEDTAVYYCAATPSGKAYSYWGQGTQYTVSS (SEQ ID NO: 39S3)
T-007JL2N88D-Fc-¥HH
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ.CLEEEU(PLEEVLNLAQ$KNFHLR
PRD1/SD/A/WVLE£^GSE7TFMCEYADETAT/VEFL/VRVV/TFCQ5//STLTGGGGS.GGGGSGGGGSGGGGSDCTHTCPPC PAPEAAGAPSyFLFPPKPKDTLMISRTPE¥TC¥¥¥D¥SHEDPE¥KFNWY¥DG¥E¥HNAKTI(PREEQYNST¥R¥¥S¥ LWLHQDWLNGICEYK£K¥SNICALPAPIEKTISKAKGQPREPQ¥¥TLPPSREEMTICNQ¥SLTCLVKGFYPSDIA¥EWE§ NGQPENN¥KTTPP¥LDSDG§FFL¥SKLT¥DKSRWQQGN¥F§CS¥MHEALHNH¥TQKSLSLSPGGGGGSGGGGSEV QLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNT
GYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS (SEQ ID NO: 3955)
T-007_ VHH-rigidJinker-VHH
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTSSRDNAK NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQLVES
GGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAASNWSNGRTNYADSVKGRFTISRDNAKNTGYLEM NSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS (SEQ ID NO: 3956)
T"014__VHH-Fc-VHH
EVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRFTISRDNAKKAV YLQMNSLKPEDTAVYYCAAAPTGRAFTYWGQGTQVTVSSGGGGSESjCYGPPCPSCPESjCYGPPCPPCPAPEFEGGPS VFLFPPRPKDHMISRTPEVTCVWWSQEDPEVQFNWWDGVEVHNAKTKPREEQFNSTYRWSVLWLHQDWL NGKEYKa<¥SNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ¥SLTCL¥KGF¥P§DIA¥EWESNGQPENN¥ KTrPP¥LDSDGSFFL¥SRlTyDK$RWQEGN¥FSCS¥MHEALFINHYTQKSLSLSLGICGGGGSGGGGSEyQLyESGGG
LVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRFTISRDNAKKAVYLQMNSLKPE DTAVYYCAAAPTGRAFTYWGQGTQVTVSS (SEQ ID NO: 3957)
T-003„2xVHH-Fc
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDSAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGG mSCMSmEDDLWromQAPGKGL^^ CAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEFEGGPSyFLFPPKPKDT LMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNACTKPREEQFNSTYRWSVLTVEHQDWLNGKEYKCICVSN KGLGSSIEKTISKAKGQPREPQVYnPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYCTTPPVLDSTCS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 3959)
T-003„VHH-Fc-VHH
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSSYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEF EGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLH QDWLNGKEYKaCVSNKGLGSSiECTISKAKGQPREPQVYTLPPSQEEIMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLTVDK5RWQEGNVFSC5VMHEALHNHYTQK5LSLSLGKGGGGSGGGGSEVQLVE SGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSiYSYGPNTYYADSVKGRFTiSTDSAKNTLYLQMN SLKPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSS (SEQ ID NO: 3961)
T-003JL2N88D-FC-VHH
APTSSSTKKTQLQLEHLLLDLQMILNGiNNYKNPKLTRMLTFKFYMPKKATELKHLQaEEELKPLEEVLNLAQSKNFHLR PRD£/SD/WWV£E£/CGSETTFMCEYADETAT/VEF£NRVV/TFCQS//STLTGG.GGSGG.GG.SGGG,GSGGGGSDCTHTCPPC PAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV LTVLHQDWLNGKEYKOCVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEV
QLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFnSTDSAKNTLYL QMNSLKPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSS (SEQ ID NO: 3963)
T-003__ VHH-rigidJinker-VHH
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQLV ESGGGLVQPGGSLRLSCAASGFTFDDIAIViTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTiSTDSAKNTLYLQM NSLKPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSS (SEQ ID NO: 3964)
Example 9. TNFR2 agomsm by tetravalerst VHH-Fc fusion increases suppressive furscttors and stabilizes the phenotype of Treg by preventing effector differentiation ot wtro
[0989]TNFR2 agonism induces Treg proliferation and activation. It was hypothesized that TNFR2 stimulation will also increase Treg stability based on the phenotype of TNFR2 knockout mice in which Treg are unstable and convert into effector cells. To test this hypothesis, multivalent anti-TNFR2 VHH proteins with agonist activity on TNFR2 were assessed for their impact on Treg proliferation, activation, immune suppressive function and stability compared to IL-2 muteins.
[0990]Human naive Treg treated with anti-TNFR2 VHH or IL-2 mutein were evaluated to detect effects on proliferation, activation, suppressive function and stability in inflammatory conditions. Specifically, tetravaient-Fc agonists (T-007_2xVHH-Fc and T-003_2xVHH-Fc) were characterized in vitro for their ability to expand and activate Treg in the presence of anti-CD3 and IL-2 stimulation. The stability of human naive Treg (CD4+CD25+CD45RA+) sorted from healthy donor PBMC by magnetic beads was investigated upon stimulation with tetravalent VHH for 5 days followed by 11 to 12 days of inflammatory cytokine challenge (11-21, 11-23, IL-lb +/- TGFb) and IL-17A or I FNy intracellular staining after PMA/ionomycin restimulation. Suppressive function was measured by assessing Treg ability to inhibit the proliferation of naive CD4 responder cells. Similarly, the effect of TNFR2 agonist T-037 on proinflammatory cytokine production was evaluated from human PBMC treated with 5 nM TNFR2 V- body or CD28 agonist for 5 days in the presence of anti-CD3. Treg proliferation was measured by assessing percent of proliferating Treg (CD4+FOXP3+) by cell tracer dilution by flow cytometry and cytokine production was measured in the supernatant by homogeneous time resolved fluorescence (HTRF) assay (Figures 44A and 448).
[0991] Figures 31 and 33A-33B show that tetravalent TNFR2 VHH-Fc antibodies induced the expansion of naive human Treg and up-regulated biomarkers of enhanced immunosuppressive activity such as CCR8 and HLA-DR and stability marker EZH2. In line with these results, activation of TNFR2 increased the ability of naive human Treg to suppress the proliferation of CD4 responder cells (Figure 35). Furthermore, stimulation of naive human Treg with VHH-Fc prevented the loss of F0XP3 and their differentiation into I1-17A- or IFNy-secreting cells when cultivated in the presence of inflammatory cytokines (Figures 32A, 328 and 34A-34C). TNFR2 agonist expanded Treg (CD4+FOXP3+) without inducing proinflammatory cytokines compared to CD28 agonist (Figure 44).
[0992]Agonistic TNFR2 VHH induced the expansion of naive human Treg, increased the expression of activation markers and stimulated suppressive effects against CD4-r T effector cells. TNFR2 VHH constructs, but not IL-2 muteins, increased human Treg stability in the presence of proinflammatory cytokines based on sustained presence of FOXP3 bright cells and prevention of conversion of Treg into IL-17A- or IFNy-secreting cells. These data demonstrate that agonism of TNFR2 by a tetravalent VHH-Fc fusion antibody expands human Treg, increases their suppressive function and stabilize their phenotype under inflammatory conditions in vitro.
Example 10. TNFR2 agonism by tetravaleat VHH-Fc fasten and expands and activates Treg cell te v/vo
[0993]The effects of anti-TNFR2 VHH agonists on spleen lymphocyte population in human TNFR2 knock- in mice were assessed by flow cytometry 5 days after a single intravenous injection (2.5 mg/kg) of tetravalent VHH-Fc antibody T-007__2xVHH-Fc, or control VHH, or mAb into human TNFR2 knock-in mice. [0994]Upon injection in human TNFR2 knock-in mice, tetravalent VHH-Fc antibody T-007__2xVHH-Fc led to a 3-fold increase in the percentage of F0XP3+ cell among CD4+ cells in the spleen, while no increase of CD4+FOXP3- and CD8+FOXP3- conventional T cells and only a minor impact on overall splenic immune cell composition was observed (Figures 36A-36B and 38). Treatment led to Treg activation as shown by the upregulation of ICOS and ICAM-1 (Figure 37). Importantly, injection was well tolerated and did not induce weight loss or changes in systemic levels of inflammatory cytokines (TNF, IL-6, IFNy, IL-5) but increased serum level of IL-10, a cytokine with demonstrated immune-regulatory functions (Figure 39). TNFR2 agonists, but not IL-2 muteins, increased human Treg stability in the presence of proinflammatory cytokines based on sustained high expression of FOXP3 and prevention of conversion of Treg into IL-17A- or IFNy-secreting cells (Figures 34A-34C).
[0995]These data demonstrated that in vsvo} a single injection is safe in mice and leads to Treg expansion and activation. Anti-TNFR2 VHH agonistic proteins are effective at expanding Tregs that have a more stable immunosuppressive capacity and phenotype (preventing pathogenic conversion to Teff) compared to IL-2 muteins and may provide a durable and disease modifying therapy for multiple autoimmune diseases.
Example 11. Camelid immunization with
[0996]Three alpacas were immunized by four subcutaneous injections with recombinant human CD25 (223-2a/CF, R&D Systems) and complete/incompiete Freund's or Gerbu FAMA adjuvant using standard protocols to elicit a humoral immune response that included the generation of antigen-specific conventional and heavy-chain only (VHH) antibodies.
[0997jBefore the first and after the third injection, serum was prepared from blood samples. Antibody induction was monitored by comparing antigen-specific antibody titers in the sera before and after immunization by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well Maxisorp plates were coated with human CD25 (223-2a/CF, R&D Systems) blocked and incubated with diluted serum samples. CD25-specific antibodies were bound by alkaline phosphatase-conjugated goat anti-alpaca IgG (H+L) (Jackson ImmunoResearch, Cat. No. 128-055-160) and detected using p-Nitrophenyl Phosphate.
Example 12, Phage library construction for generatmg anti-CD25 VHH libraries
[0998]Four to ten days after the fourth injection, in accordance with procedures described in Example 1, blood samples were collected, and four to six days after the fourth injection a bone marrow sample was aspirated. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood or bone marrow following density gradient purification with Ficoll-PaqueTM Plus. Total RNA was extracted from freshly isolated PBMCs.
[0999]To generate anti-CD25 VHH immune libraries, total RNA was reverse transcribed to cDNA using random hexamer primers. Conventional and heavy chain IgH cDNA fragments were amplified by polymerase chain reaction (PCR) using primers annealing to the IgH leader sequence region and the CH2 region. The resulting amplicons represented the VHH and VH cDNAs, respectively. The VHH fragment was isolated and used as template for a nested PCR to introduce appropriate endonuclease recognition sites for cloning into the pQ81 phagemid in frame with gene III. Libraries were transformed into electrocompetent E. coli TGI cells. In total, six libraries were built, with 95.5% to 100% VHH insert frequency and maximum library sizes between 4.2x10s and 2.4xl09. Phage for phage display was prepared following standard protocols.
[lOOOJBinders to human and mouse CD25 were enriched from VHH immune libraries by two rounds of phage display. The general panning strategy is illustrated in Figure 45, using the panning substrates listed Table ?. For the Table below: PBS, phosphate buffered saline; Cat., catalog; MW, molecular weight; Calc., Calculated; Seq., Sequence; N-term., N-terminal; aa, amino acid.
Table 7. Panning substrates for anti-CD25 VHH
[1001]For the first panning round, libraries originating from the first harvested blood sample and the first harvested bone marrow sample of the same animal were pooled in equal parts (at the phage level), resulting in three pooled input libraries per antigen. Each library was panned under four conditions (two antigen concentrations and two ways of antigen immobilization) with human CD25, resulting in 12 panning reactions. For the second rounds of panning, six output samples (enriched libraries) from the first round were chosen and served as input libraries for the second round. Preferentially, the enriched libraries from the higher panning substrate concentration were chosen to preserve maximum diversity. Pannings of the second round were performed with three antigen concentrations of human and mouse antigen resulting in 36 conditions. This panning regimen was implemented to identify binders that crossreacted with human and mouse CD25. Antigen concentration in the second panning round was reduced by a factor of 10 and 100 to favor the retention of strong binders. High affinity CD25 bindings were enabled to drive cell specificity.
[1002]Phages were produced according to QVQ Holding B.V. (Q.VQ) standard operating procedures (SOPs) and phage titers were determined to ensure at least 10-fold excess over the maximum diversity of the libraries. Panning substrates were commercially purchased (see Table 7). The panning substrates were immobilized either by direct coating on enzyme-linked immunoassay (ELISA) plates or by binding of biotinylated antigen on neutravidin-coated ELISA plate. Glycerol stocks were prepared from all outputs and are stored at -80°C.
[1003]Panning outputs were analyzed by random clone picking/periplasmic extract (PE)-ELISA/Sanger sequencing (QVQ) and next-generation sequencing (NGS; Genewiz/PipeBio).
[1004]For random colony picking, rescued outputs of the first and second panning rounds were plated out and 460 random single clones (equal numbers of colonies from each condition) were selected to create masierplates (96-well format). From the masterplates, expression cultures in deep-well plates were inoculated to produce periplasmic extracts containing monoclonal VHH. Periplasmic extracts were used to determine binding of individual VHHs to human, mouse and cynomolgus antigen by ELISA. For conditions where the panning substrate was biotinylated and captured by neutravidin, background binders were identified by ELISA with neutravidin. All masterplates were sequenced by the Sanger method.
[1005]For NGS analysis, minipreps from input libraries and outputs after the first and second rounds of panning were prepared, amplified by PCR and sequenced by NGS.
Example 13. Mext-generatfon sequencmg of V-body candidates
[1006]Following two rounds of panning, phages were eluted and corresponding phagemid DNA was extracted. Identification of initial anti-CD25 V-body candidates was performed in a parallelized fashion, employing a random colony picking, as well as a next-generation sequencing (NGS) approach, as orthogonal techniques to yield a particularly diverse set of initial candidates. Prior to NGS techniques, random colony picking was the prevalent method for initial hit identification, which involved transformation of a phagemid pool (from a panning elution) and selection of individual bacterial colonies to isolate single clones. Following this approach, 460 single colonies were randomly picked from the 12 samples of the second panning round (Figure 4S). Then, individual clones were expressed and subject to ELISA screening against the target antigen to select for antigen binding V-bodies, which were further functionally characterized. [1007] AH panning eluates were sequenced using NGS. In brief, the entire VHH region was PCR-amplified from isolated phagemid pools by primers annealing to universal phagemid sequences 5' and 3' of the VHH-encoding region. In a second step, the generated amplicons were fused to sequencing-compatible and sample-specific barcodes. By fusing unique barcodes, it was possible to multiplex hundreds of different samples. Following the preparation of 33 samples, an Illumina NovaSeq 6000 with an SP flowcell was employed for sequencing, yielding 2S0 base pair (bp) reads from each direction and a total of '“600 million reads. To account for differences in the number of expected unique sequences in the library, and both panning rounds, each library was sequenced with a total of 20 million reads, compared to the first and second round of panning with 2 million reads each. This strategy allowed for covering sufficient sequence space in the libraries, as well as in the panning eluates. A spike-in of 30% of a standard PhiX reference genome control into the sequencing reaction helped to provide a technical quality control for assessing sequencing accuracy. The MGS raw data contained multiplexed sequencing reads, which were de~multiplexed based on the sample-specific barcodes. The de-multiplexed data containing unmerged sequencing reads were then processed by employing an NGS analysis platform, in brief, forward and reverse sequence pairs were merged by their overlapping sequence, thereby generating a full VHH sequence from two half sequences (Figure 47). The framework regions, CDRs, and sequence-specific liabilities were then annotated for the merged V-body sequences.
[100S]Based on CDR3 identity, anti-CD25 V-body sequences were clustered, allowing for a detailed analysis of V-body enrichment during phage display, sequence diversity, CDR3 length distribution and cluster abundance. Identified V-bodies can be classified into eight distinct clusters, as follows: Group A, Group B, Group C, Group D, Group E, Group F, Group G, and Group H. The following Table 8-1 to Table 8-24 display the amino acid frequency distribution at each amino acid (AA) position (IMGT) for CDR1, CDR2 and CDR3 for the eight clusters. Table 9 provides the sequence identifiers of amino acid sequences of the complementarity determining regions (CDR1, CDR2 and CDR3), amino acid and DMA sequences of the full-length anti-CD25 VHH domain for the identified anti-CD25 V-bodies.
Table 8-1. antl-CMS CDR1 ammo acid frequency distributism for Group A
Table 8-2. antl-CD25 CDR2 ammo acid frequency distribution for Group A Table §-3. anti-CD25 CDR3 ammo acid frequency distribution for Group A
Table 8-4. ants-CD25 CDR1 amino acid frequency distribution for Group B Table 8-5. anti-CM5 CDK2 ammo acid frequency distribution for Group B
Table 8-8. ants-CD25 CDR3 amino acid frequency distribution for Group B Table 8-7. anti-CM5 CDR1 ammo acid frequency distribution for Group C
Table 3-8. antl-CD25 CDR2 amino acid frequency distributism for Group C Table 8-9, anti-CM5 CDR3 amino acid frequency distribution for Group C
Table $-10. anti-CD25 CDR1 amino acid frequency distribution for Group D Table 8-11. anti-®25 CDR2 amine acid frequency distribution for Group D
Table 3-12. anti-CD25 CDR3 amino acid frequency distribution for Group D
3163S7184v9
Table §-13. anti-CD25 CDR1 amino acid frequency distribution for Group E
Table 3-14. anti-CD25 CDR2 amino acid frequency distribution for Group E Table B-15. anti-CD25 CDR3 amino acid frequency distribution for Group E
316357184v9
Table 8-16. anti-CD25 CDR1 ammo acid frequency distribution for Group F
Table 8-17. anti-CD25 CDR2 amino acid frequency distribution for Group F Table B-18. anti-CD25 CDR3 amino acid frequency distribution for Group F
3163S7134v9
Table 8-W. anti-®25 CDR1 amine acid frequency distribution for Group G
Tabie S-20. anti-CD25 CDR2 amino acid frequency distribution for Group G Table 8-21. anti-®25 CDR3 amine acid frequency distribution for Group G
Tabie S-22. anti-CD25 CDR1 amino acid frequency distribution for Group H Table 8-23. anti-®25 CDR2 amine acid frequency distribution for Group H
Table 3-24. anti-CD25 CDR3 amino acid frequency distribution for Group HJ 0
3163S7184v9
Table 9, Sequence Identifiers for anti-CD2S V-bodles Identified from banning
Example 14. Flow cytometry binding of
[1009]To measure the binding of V-bodies to cell-displayed CD25 from human, cynomalgus or mouse, HEK293 cells were transfected with plasmids encoding for respective antigens. After 48 to 72 hours, binding was measured by incubation of His-tagged V-bodies with cells at various fixed concentrations, followed by washing and detection with Alexa488 fluorophore-iabeiled anti-His antibodies.
[1010]For generation of data depicted in Figure 4§, HEK293T cells were transiently transfected with a plasmid encoding human CD25 (hCD25; hCD25_pcDNA3.4.dna). After 48 hours, HEK293T cells were harvested and incubated with 100 nM purified His-tagged (myc-his tag) anti-CD25 VHHs. VHH binding was then detected using an Alexa488-labeled a nti-His tag antibody and measured by flow cytometry OQue).
(lOll)For generation of data depicted in Figure 49, HEK293T cells were transiently transfected with a plasmid encoding cynomolgus CD25 (cCD25; cCD25„pcDNA3.4.dna) (top panel) or mouse CD25 (mCD25; mCD25_pcDNA3.4.dna) (bottom panel). After 48 hours, HEK293T cells were harvested and incubated with 100 nM purified His-tagged (myc-his tag) VHHs. Anti-CD25 VHH binding was then detected using an Alexa488-labeled anti-His tag antibody and measured by flow cytometry (IQue).
[1012]Figure§ 5GA-50B show testing of human CD25 V-body binding across a range of concentrations for C-004 and C-006. Anti-CD25 V-bodies were tested at the following molar concentrations: 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM, and 0.390625 nM. For generation of data depicted in Figures 50A-50B, HEK293T cells were transiently transfected with a plasmid encoding human CD25 (hCD25; hCD25_pcDNA3.4.dna). After 48 hours, HEK293T cells were harvested and incubated with increasing molar concentrations of purified His-tagged (myc-his tag) VHHs, including a control VHH against an irrelevant antigen. VHH binding was then detected using an Alexa488-labeled anti-His tag antibody and measured by flow cytometry (IQue). The bar histogram in Figure 50A shows the percentage of Alexa488 positive cells for C-004 and C-006. The bar histogram in Figure SOB shows the mean fluorescent intensity of Alexa488 positive cells for C-004 and C-006.
[1013]For generation of data depicted in Figure 58, HEK293T cells were transiently transfected with a plasmid encoding human CD25 (hCD25; hCD25_pcDNA3.4.dna) or cynomolgus CD25 (cCD25; cCD25_pcDNA3.4.dna). After 48 hours, HEK293T cells were harvested and incubated with 100 nM purified His-tagged (myc-his tag) VHHs. VHH binding was then detected using an Alexa488-labeled anti- His tag antibody and measured by flow cytometry (IQue). The bar histogram in Figure 58 shows the mean fluorescent intensity of Alexa488 positive cells for C-007, C-008, C-009, and C-010.
Example 15. Surface plasmon resonance binding affinities of anti-CD25 V-bodies
[1014]Binding affinities of the anti-CD25 V-bodies to their respective target was determined by surface plasmon resonance (SPR) using a Caterra ISA instrument. A schematic diagram depicting the experimental setup of the present Example is shown in Figure 51. Affinity purified V-bodies were covalently crosslinked onto an ISA HC200M chip using EDC/Sulfo NHS. The interaction with human, cynomolgus, and mouse CD25 (extracellular domain) (V-body coupling concentration: 3 pM) was measured under physiological conditions (Running Buffer: HBST- 50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 % (w/v) BSA, 0.05% (v/v) Tween20, 25°C) using eight different antigen concentrations (3-fold serial dilutions, starting from 200 nM). Resulting sensorgrams (Figures 52A-52C) were analyzed and equilibrium-binding affinities (KDS) were calculated using Carterra's data analysis software. For data processing, high or low Ag concentration curves were excluded based on affinity or curve fit. Figure 53 shows a summary of binding affinities of two anti-CD25 V-bodies: C-004 and C-006. Data corresponding to an anti~CD25 IgG control condition are also shown. The interaction with human, cynomolgus, and mouse CD25 (extracellular domain) (V-body coupling concentration: 0.2 pM) was also separately measured under physiological conditions (Running Buffer: HBST- 50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 % (w/v) BSA, 0.05% (v/v) Tween20, 25°C) using eight different antigen concentrations (3-fold serial dilutions, starting at from 500 nM) for V-body candidate anti-CD25 clone C-004, and applying an inverse setup in single channel mode. Resulting sensorgrams (see, e.g., Figure 53A) were analyzed and equilibrium-binding affinities (KDS) were calculated using Carterra's data analysis software.
[1015]For generation of sensorgrams displayed in Figures 5SA-5SC, affinity purified V-bodies were covalently crosslinked onto an ISA HC30M chip using EDC/Sulfo NHS. The interaction with human, cynomolgus, and mouse CD25 (extracellular domain) (V-body coupling concentration: 1 pM) was measured under physiological conditions (Running Buffer: HBST- 50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 % (w/v) BSA, 0.05% (v/v) Tween20, 25”C) using nine different antigen concentrations of human CD25 (2-fold serial dilutions, starting from 500 nM), ten different antigen concentrations of cynomolgus CD25 (2-fold serial dilutions, starting from 1000 nM) and eight different antigen concentrations of mouse CD25 (2-fold serial dilutions, starting from 600 nM). Resulting sensorgrams were analyzed and equilibrium-binding affinities (Kos) were calculated using Carterra's data analysis software (Figures 56A- 56C). For data processing, high or low Ag concentration curves were excluded based on affinity or curve fit.
[1016]For generation of sensorgrams displayed in Figures 59A-59C, affinity purified V-bodies were covalently crosslinked onto an ISA HC30M chip using EDC/Sulfo NHS. The interaction with human, cynomolgus, and mouse CD25 (extracellular domain) (V-body coupling concentration: 1 pM) was measured under physiological conditions (Running Buffer: HBST- 50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 % (w/v) BSA, 0.05% (v/v) Tween20, 25°C) using twelve different antigen concentrations of human CD25 (2-fold serial dilutions, starting from 1000 nM), twelve different antigen concentrations of cynomolgus CD25 (2-fold serial dilutions, starting from 1000 nM) and twelve different antigen concentrations of mouse CD25 (2-fold serial dilutions, starting from 1000 nM). Resulting sensorgrams were analyzed and equilibrium-binding affinities (fes) were calculated using Carterra's data analysis software (Figures 59A-59C). For data processing, high or low Ag concentration curves were excluded based on affinity or curve fit.
Example 16. CD25 IL-2 competition
[1017]To investigate whether humanized anti-CD25 V-bodies targeted the epitope recognized by IL-2, CD25 expressing HEK cells (clone 25) were incubated with or without recombinant IL-2 (100 nM) prior to (pre-) and/or during (co-) incubation with His-tagged CD25-specific V-bodies. After washing, anti-CD25 V-body binding was detected by labelled anti-His antibody. Binding inhibition in the presence of IL-2 indicated binding of CD25 V-bodies and IL-2 to an overlapping epitope. Results for C-004 and C-006 V- bodies are shown in Figure 54A and Figure 548, respectively, C-OOlhul, C-002Hul, and C~003hul were identified as non-competitive binders (Figures 55A-55B).
[1013]Each panel of the IL-12 competition data displayed in Figures 57A-57C represents a sensorgram overlay plot for a single V-body captured onto a discrete spot. The sensorgrams display I L2-Fc competition: association of human CD25-extracellular domain (ECD) to the V-body followed either by additional binding by IL2-Fc, indicating an unoccupied epitope (non-overlapping epitopes), or no IL2-Fc binding, indicating epitope blocking (overlapping epitopes), and a buffer control, association and dissociation of human CD25-ECD in the absence of IL2-Fc. Human CD25-ECD was injected (500 nM) under physiological conditions (50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 % (w/v) BSA, 0,05% (v/v) Tween20, 25°C) followed by human IL2-Fc (1000 nM). V-body C-008Hul was identified as a ligand competitive binder. C-OlOHul and C-009Hul were identified as non-competitive binders.
Example 17. bispecific V-bodies are able to bind to Treg
[1019]Rgures 61A-618 show that a TNFR2/CD25 bispecific antibody is able to bind to Tregs. Healthy donor PBMC were incubated with mono- (TNFR2 or CD25) or bispecific (TNFR2 and CD25) VHHs and stained for CD4, FOXP3, CD8 and CD14. VHH binding was detected after washing with a secondary fluorescently-labelled anti VHH antibody. Plots show binding to Tregs on the y-axis (mean fluorescent intensity) from 2 different donors for molecules that are monospecific for TNFR2 or bispecific for TNFR2 and CD25. Binding is shown for various monospecific or bispecific shown on the X-axis. The design of the molecule is described on top of the data point, from N to C term. T, Tl, T2: TNFR2-specific VHH, Fc: Fc fragment, CD, GDI, CD2 : CD25-specific VHH.
Example 1g. TNFR2/CD25 bispedfic antibodies are able to bind to Treg
[1020]Figures 62A-62B show that different anti-TNFR2/anti-CD25 multispecific antibodies are able to bind to Tregs. Healthy donor PBMC were incubated with mono or bispecific VHHs and stained for CD4, FOXP3, CD8 and CD14. VHH binding was detected after washing with a secondary fluorescently-labelled anti VHH antibody. Histograms show bispecific binding on CD4*FOXP3 Treg, CD4+FOXP3- CD4 effector, CD8 effector and CD14+ monocytes. Insertion of a CD25 VHH at the N- or C-terminus of the Fc portion drastically increases binding to Tregs.
Example 19. are able to activate reporter ceils
[1021]Flgures 63A-638 shows that a TNFR2/CD25 bispecific antibody is able to activate a reporter cell. Figure S3A shows the results of TNFR2 agonism tested at 3 different dilutions on HEX NF-kB -Luciferase reporter ceils expressing TNFR2. Y axis shows maximum agonism/reporter activity in relative luminescence unit (R.L.U.). Antibody formats are described above the respective data points. Figure 638 shows the results of TNFR2 agonism on HEK NF-kB-Luciferase reporter cells expressing TNFR2 only (Clone 18) or both TNFR2 and CD25 (Subclone 48 derived from clone 18). Monospecific and bispecific VHH were tested on TNFR2+ HEK and TNFR2-S-CD25+ HEK. Plot show equal TNFR2 agonism by TNFR2 VHH on both cells. In contrast, bispecific VHHs induced stronger agonism on reporter expressing CD25 (clone 48) compared to cells not expressing CD25 (clone 18).
[1022]Figure$ 64A-64B show that Tregs can be activated by mono- and bispecific tetravalent antibodies. Figure 64A shows the results of total CD4 T cells isolated from PBMC from healthy donors stimulated with anti-CD3/CD28 beads and IL-2 in the presence or absence of mono or bispecific VHHs. Mono and bispecific VHHs led to increased activation as measured by HLA-DR upregulation in Treg after 5 days of stimulation. Figure 64B shows the results of total CD4 T ceils isolated from PBMC from healthy donors stimulated with anti-CD3/CD28 beads and IL-2 in the presence or absence of mono or bispecific VHHs, Mono and bispecific VHHs led to increase percentage of F0XP3* cells among CD4 after 5 days of stimulation.
[1023]Amino acid sequences of exemplary multivalent anti-TNFR2 antigen-binding domain constructs described in herein are provided below. In the sequences, VHH sequences are indicated with a straight underline (e.g., VHH), Fc regions are indicated with bold letters (e.g., Fc), linker sequences are indicated with a wavy underline (e.g., Linker), and hinge regions are indicated with both a wavy underline and bold letters (e.g., hinge).
TC-001
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSEVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDN VKNTVYLQMNSLKAEPSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTI SSDNVKNTVYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGS GSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVK GRFTISSDNVKNTVYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 6401)
TC-002
EVQLVESGGGLVQAGGSLSVSCAASGRTFSWNGMGWFRQAPGEEREFVAAISQSGGRTRYSDSVKGRFTISRDNAKN
MVYLQMNSLKPEDTAVYYCAASDFLLATTISAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGG
GSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGSiVSTNGMGWHRQVPGKGRELVAGiRSDGFTNYADSVKGRFTIS
SDNVKNTVYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG
SGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFThiYADSVKG
RFTISSDNVKNTVYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYA
DSVKGRFTISSDNVKNTVYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGSI VSTN G M GWH RQVPG KGRELVAG I RSDG F TNYADSVKGRFTISSDNVKNTVYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 6402)
TC-003
EVQLVESGGGLVQAGGSLSVSCAASGRTFSWNGMGWFRQAPGEEREFVAAISQSGGRTRYSDSVKGRFTISRDNAICN MVYLQMNSLKPEDTAWCAASDFLLATriSAYDYWGQGTQVWSSGGGGSGGGGSGGGGSGGGGSGGGGSGGG
GSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFT
ISRDNAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG
GGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKG RFTiSRDNAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADS
VKGRFTISRDNAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAiNWSNGRTNY ADSVKGRFTISRDNAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS (SEQ ID NO: 6403)
TC-004
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK
NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG
GGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRD
NAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAASNWSNGRTNYADSVKGRFTI SRDNAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKG
RFTISRDNAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS (SEQ ID NO: 6404)
TC-005
EVQLVESGGGLVQAGGSLSVSCAASGRTFSWNGMGWFRQAPGEEREFVAAISQSGGRTRYSDSVKGRFTISRDNAICN MVYLQMNSLKPEDTAVYYCAASDFLLATnSAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGG
GSGGGGSEVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASiRRTGGSTSYADSVKGRFTIS
RDNAKKAVYLQMNSLKPEDTAVYYCAAAPTGRAFTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSEVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASiRRTGGSTSYADSVKGRFT! SRDNAKKAmOMl^UmT^^ GGGSGGGGSEVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRF TISRDNAKKAVYLQMNSLKPEDTAVYYCAAAPTGRAFTYWGQ,GTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSEVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGR
FTISRDNAKKAVYLQMNSLKPEDTAVYYCAAAPTGRAFTYWGQGTQVTVSS (SEQ ID NO: 6405)
TC-006
EVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRFTISRDNAKKAV YLQMNSLKPEDTAmCAAAPTGRAFIYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SEVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRFTISRDNAKKA VYLQMNSLKPEDTAVYYCAAAPTGRAFTYWGQGTQVTVSSGGGGSGG.GGSGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRFTSSRDNAKK AVYLQMNSLKPEDTAVYYCAAAPTGRAFTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG
GGSEVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASiRRTGGSTSYADSVKGRFTISRDNA
KKAVYLQMNSLKPEDTAVYYCAAAPTGRAFTYWGQGTQVTVSS (SEQ ID NO: 6406)
TC-007
EVQLVESGGGLVQAGGSLSVSCAASGRTFSWNGMGWFRQAPGEEREFVAASSQSGGRTRYSDSVKGRFTISRDNAKN MVYLQMNSLKPEDTAVYYCAASDFLLATTgSAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGG
GSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYAD5VKGRFTIS TDSAKNTLYLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGG5GGGG5GGGG5GG GGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSSYSYGPNTYYADS VKGRFTISTDSAKNTLYLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSEVQIVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPN
TYYADSVKGRFTgSTDSAKNTLYLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSI YSYGPNTYYADSVKGRFTISTOSAKNTLYLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSS (SEQ ID NO: 6407)
TC-008
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAmCAADSDLSWWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTD SAKNTLYLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG
SGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSiYSYGPNTYYADSVKG RFTISTDSAKNTLYLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYY ADSVKGRFTISTDSAKNTLYLQMNSLKPEDTAWYC^ADSDLSTVVVGPHDYWGQGTQVTVSS (SEQ ID NO: 6408)
TC-009
EVQLLESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTSSTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGG
SLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSiYSYGPNTYYADSVKGRmSTDSAKNTLYLQMNSLRPEDTAVYY
CAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDIA
MTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYYCAADSDLSTVWGPHD WGQGTQVIVSSGGGGSFSKYGPPCPPCPAPEFEGGPSVFLFPPKPRDTLMISRTPEVTCWVDVSQFDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK (SEQ ID NO: 6409)
TC-010
EyflLLESGGGLW.^^^
YLQMNSLRPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSSGGGG5GGGGSGGGG5EVQLLESGGGLVQPGG
SLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYY CAADSDLSTWVGPHDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLIVjgSRTPEVTCy WDVSQEDPEVQFNWWDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS^
AKGQPREPQyYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPyLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6410)
TC-011
EVQLVESGGGLVQPGGSLRVSCAASGSIFRADAMGWHRQAPGKPREFVAGIRSDGFTNYADSVKGRFTISWDTVKNT VYLQMNSLRPEDTAVYYCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSL RLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYYCA
ADSDLSTWVGPHDYWGQGTQVTVSSGGGGSES^YGPKPPCPAPEFEGGPSVFLFPPKPjCDTLMjSRTPEVTCVW D¥SQEDPE¥QFNWY¥DGyE¥HNARTKPREEQFNSTYRWS¥LTVLHQDWLNGKEYKCKySNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLWDKSR
WQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6411)
TC-012
EVQLLESGGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNAKNT LYLQMNSLRPEDTAVYYCARVRGTPYEYGYRGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLS CAASGRTFSSLFMGWFRQAPGKEREFVASIRYPGLITNYADSAKGRFTISRDSAKNTVYLQMNSLRPEDTGVYYCAAAP
TGRAFNYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPE VQFNWYVTCVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISICAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSMSFFLYSRLTVD^SRWQEGNVFSC
SVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6412)
TC-013
^VQllESGGGLyQPGG^BLSCAASGFiniDlAMIWyRQAPGKGLEWySSIYSY^I^nYYADSyKGRFJlSBSAKNK YLQMNSLRPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGG SLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYY
CAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSESRYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVrCV
WO¥SQEDPEyQFNWWDG¥EVHNAKTKPREEQFNST¥RWS¥LTVLHQDWLNGREYKCKVSNRGLPSSIERTKK
AKGQPREPQVYTLPPSQEEMTKNQVSLTCLyRGFYPSDIAyEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTaKSLSLSLGKGGGGSEVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWAR QAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKN7VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQ
GTQVTVSS (SEQ ID NO: 6413)
TC-014
EVQLVESGGGLVQPGGSLRVSCAASGSIFRADAMGWHRQAPGKPREFVAGIRSDGFTNYADSVKGRFTISWDTVKNT VYLQMNSLRPEDTAVYYCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSEYrQLLESGGGLVQPGGSL RLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYYCA
ADSDLSTWVGPHDYWGQ.GTQVTVSSGGGG5ESKYGPPCPPCPAPEFEGGPSVFLFPPICPKDTLMISRTPEVTCVW DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF^STYRWSVLWLHQDWLNS^EY^CKVSNKGLPSSIEKTISICAK GQPREPQV¥TLPPSQEEMTKNQVSLTCL¥KGF¥PSDIAVEWESNGQPENN¥KTTPPVLDSDGSFFL¥SRLTVDKSR WQEGNVFSCSVNjHEALHNHYTQKSLSLSLGKGGGGSEVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQ APGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQG TQVTVSS (SEQ ID NO: 6414)
TC-015
EVQLLESGGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNAKNT LYLQMNSLRPEDTAVYYCARVRGTPYEYGYRGQGTQV7VSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLS CAASGRTFSSLFMGWFRQAPGKFREFVASIRYPGLITNYADSAKGRFTISRDSAKNTVYLQMNSLRPEDTGVYYCAAAP TGRAFNYVYGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKFKDYLNRSRTPEVTCWVDVSQEDPE VQFNWYVDG¥EVHNARTKPREEQFNST¥RWS¥ETVLHQDWLNGKE¥KCKVSNKGEPSSIECTISKAKGQPREPQV YYLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQjCSLSLSLGKGGGGSEVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVS GIYSDGSGTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSS (SEQ ID NO: 6415)
TC-016
EVQLLESGGGLVQPGGSLRLSCAASG FTFDDI AMTWVRQAPG KG IE WVSSIYSYG PNTYYADSVKG RFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGG SLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVS5IYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYY CAADSDLST/''/VGPHDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWENGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSETCLVRGFYPSDIAVEWESNGQPENNYKTTPPV1DSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSEVQLLESGGGLVQPGGSLRVSCAASGRTFSWNGMGWF RQAPGKEREFVAASSQSGGRTRYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAASDFLLATTISAYDYWGQ GTQVTVSS (SEQ ID NO: 6416)
TC-017
EVQLVESGGGLVQPGGSLRVSCAASGSSFRADAMGWHRQAPGKPREFVAGIRSDGFTNYADSVKGRFTISWDTVKNT VYLQMNSLRPEDTAVYYCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSL RLSCAASGFTFDDSAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYYCA ADSDLST'VVVGPFIDYWGaGTQVrVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVS^KGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGN VFSCSVMH EALH NHYTQKSLSLSLG KGG.G iGSEVQLLESGGGLVQPGGSLRVSCAASGRTFSWNGMGWFR QAPGKEREFVAAiSQSGGRTRYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAASDFLLATTISAYDYWGQGT QVTVSS (SEQ ID NO: 6417)
TC-018
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPP KPKDTLMISRTPE¥TCVWDVSQEDPEVQFNW¥VDGVEVHNAKTRPREEQFNST¥RV¥SVLTVLHQDWLNGKE¥K CKVSNKGLPSSIEKTISKAKGQPREFQVmPPSQEEMTKNQVSLTaWGFYPSDIAVEWESNGQFENNYm'PPVL DSTCSFFLYSRLWDKSRWaEGNVFSCSVMHEALHNHYTQKSlSLSLGRGGGGSEVQLLESGGGLVQPGGSLRLSCA ASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYYCAADSD LSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVR QAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYYCAADSDLSTWVGPHDYWGQG TQVTVSS (SEQ ID NO: 6418)
TC-019
EVaLESGG^^
VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGFSVFLFPP KPKDTLMISRTPEVTCVWDVSQEDPEVQFNWWDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK'n'PPVL DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNNYTQKSLSLSLGKGGGGSFVQLVESGGGLVQ.PGGSLRVSC AASGSIFRADAMGWHRQAPGKPREFVAGIRSDGFTNYADSVKGRFTISWDTVKNTVYLQMNSLRPEDTAVYYCYYQS LSSPNYGQVFWGQGTQVTVSSGGGGSGGGG5GG6GSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQ APGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYYCAADSDLSTWVGPHDYWGQGT
QVTVSS (SEQ ID NO: 6419)
TC-022
EVQLLESGGGLVQPGGSLRLSCAASG FTFDDI AMTWVRQAPG KG LE WVSSIYSYG PNTYYADSVKG RFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGgPSVFLFPPK PKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVTCVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKn'PPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH¥TQKSlSLSLGKGGGGSEyQll.ESGGGLyQPGGSLRLSCAA SGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYYCAADSDLS TVWGPHDYWGQGTQVTVSS (SEQ ID NO: 6420)
TC-023
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPG GSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVY YCAADSDL5TWVGPHDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTC WVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSSEKTSS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCIVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFI.YSRI.TVDK SRWQEGN VFSCSVMH EALH N HYTQKSLSLSLG KGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVR
QAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYYCAADSDLSTVWGPHDYWGQG TQVTVSS (SEQ ID NQ: 6421)
TC-024
EVQLLESGGGLVQPGGSLRVSCAASGRTFSWNGMGWFRQAPGKEREFVAASSQSGGRTRYADSVKGRFTISRDNAKN TVYLQMNSLRPEDTAVYYCAASDFLLATTSSAYDYWGQGTQVTVSSGGGGSGGGGSGGGG5EVQLLESGGGLVQPG GSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVY YCAADSDLSrYVVGPHDYV/GQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVrC VWDVSQEDPEVQFNWWDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQFEMTKNQVSLTaVKGRFSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMH EALH N HYTQKSLSLSLG KGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVR
QAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYYCAADSDLSTVWGPHDYWGQG TQVTVSS (SEQ ID NO: 6422)
TC-025 EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPP §(PKDTL^iSRTPEVTCVWDVSQEDPEVQFNW¥VDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKE¥K CK¥SNKGLPS5IEKTISKAKGQPREPQ¥YTLPPSQEEMTKNQ¥SLTCL¥KGFYPSDIA¥EWESNGQPENNYKTTPPVl. DSTCSFFLYSRLWDjCSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSEVQLLESGGGLVQPGGSLRLSCA
ASGFNFSMYSMSW¥RQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARVR GTPYEYGYRG QGTQVTVSS (SEQ ID NO: 6423)
TC-026
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSBKYGPPCPPCPAPEFEGGPSVFLFPP KPKOTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGyEVHNAKTKPREEQFNSTYRWSyiTVLHQDWLNGKEYK CKySNKGLPSSI£KTISKARGQPREPQ¥¥TLPPSQEEMTKNQ¥SLTCLyKGFYPSDIA¥EWESNGQPENNYKTTPP¥L DSDGSFFlYSRLWDjCSRWQEGNVFSCSVMHEALMNHYTQKSLSLSLGKGGGGSEVQLVESGGGLVQPGGSLRLSCA
ASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNTWYLQMNSLRPEDTAVYYCSVLG RDM MTYWGQGTQVTVSS (SEQ ID NO: 6424)
TC-027
EVQLLESGGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTSSRDNAKNT LYLQMNSLRPEDTAVYYCARVRGTPYEYGYRGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDT EMISRTPE¥TCW¥D¥SQ£DPEVQFNWYVDG¥E¥^NAKTRPREEQFNSTYRWS¥LTVLHQDWLNGKEYKCKVSN KGLPSSIEKQSKAKGQPREPQVYTLPPSQEEMTRNQySLTCiyRGFYPSDJAyEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWaEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSEVQLLESGGGLAQPGGSLRLSCAASGFTF
SNYAMSWARQAPGKGLEWVSGiYSDGSGTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAKGRNSGSYY PWDDYWGQGTQVTVSS (SEQ ID NO: 6425)
TC-028
EVQLVESGGGLVQPGGSLRLSCAASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT WYLQMNSLRPEDTAVYYCSVLGRDMMTYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKD TLMffiRTP£VTC¥VVD¥SQEDPE¥QFNWY¥DGV£VHNAKTKPREEQFNST¥R¥VS¥LT¥EHQDWLNGRE¥KCK¥S NKGLPSSERTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLyKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWaEGNVFSCSVMHEALHNHYTQKSLS^LGKGGGGSEVQLLESGGGLAQPGGSLRLSCAASG
FTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAKGRNSG SYYPWDDYWGQGTQVTVSS (SEQ ID NO: 6426)
TC-029
EVQLLESGGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNAKNT LYLQMNSLRPEDTAVYYCARVRGTPYEYGYRGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSQEDPEVQFNWWDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCICVSN KGLPSSIEKTISKAKGQPREPQWTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYm’PPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSyMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6427)
TC-030
EVQLVESGGGLVQPGGSLRLSCAASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT WYLQMNSLRPEDTAVYYCSVLGRDMMTYWGQGTQVT¥SSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKD TLMISRTPEVTCy¥¥D¥§QEDPEyQFNWY¥DGVEVHNAKTKPREEQFN5TYRWSVLT¥l.HQDWLNGKEYKaC¥S NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ¥SLTCLVKGF¥PSDIAVEWESNGQPENN¥KTTPPVLDSD GSFFL¥SRLT¥DKSRWQEGNVFS€S¥MREALHNHYTQKSi.SI.SI.GK (SEQ ID NO: 6428)
TC-031
EV!^LESGGGLWG^
TVYLQMNSLRPEDTAVYYCAASDFLLATTISAYDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPP KP^DTLMISRTPEVTCVWWSQEDPEVQFNWWDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYK CKySN£GLPSSI£KTISKAKGQPREPQ¥¥KPPSQE£MTKNQVSLTt¥KGFYPSDIAVEWE5NGQFENNYKTrPPVL DSDGSFFLYSRLTVDjCSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSEVQLLESGGGLVQPGGSLRLSCA ASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARVR
GTPYEYGYRG QGTQVTVSS (SEQ ID NO: 6429)
TC-032
E¥QLLESGGGLVQPGGSLRVSCAASGRTFSWNGMGWFRQAPGKEREF¥AAISQSGGRTRYADS¥KGRFTISRDNAKN TVYLQMNSLRPEDTAVYYCAASDFLLATTSSAYDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPP KPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTIS^CAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKn'PPVL DSDGSFFLYSRLWDKSRWQEGNVFSCSVMHEALHNHYTQKSLSISLGKGGGGSE¥QLVESGGGLVQPGGSLRLSCA
ASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNTWYLQMNSLRPEDTAVYYCSVLG RDMMTYWGQGTQVTVSS (SEQ ID NO: 6430)
TC-033
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTSSRDNAKNT VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQPGGSLRVSCAASGSIFRADAMGWHRQAPGKPREFVAGIRSDGFTNYADSVKGRFTISW DTVKNTVYLQMNSLRPEDTAVYYCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGS EVQLVESGGGLVQPGGSLRVSCAASGSSFRADAMGWHRQAPGKPREFVAGIRSDGFTNYADSVKGRFTISWDTVKNT VYLQMNSLRPEDTAVYYCYYQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 6431)
TC-034
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG
SSGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDiAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTIST DSAKNTLYLQMNSLRPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGG GSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKN
TLYLQMNSLRPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSS (SEQ ID NO: 6432)
TC-035
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQPGGSLRVSCAASGSIFRADAMGWHRQAPGKPREFVAGIRSDGFTNYADSVKGRFTISW DTVKNTVYLQMNSLRPEDTAVYYCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGS EVQLLESGGGLVQPGGSLRLSCAASG FTFDDI AMTWVRQAPG KG LE WVSSIYSYG PNTYYADSVKG RFTISTDSAKNTL
YLQMNSLRPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSS (SEQ ID NO: 6433)
TC-036 EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISR DNAKNTLYLQMNSLRPEDTAVYYCARVRGTPYEYGYRGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEy QLLESGGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNAKNTLY LQMNSLRPEDTAVYYCARVRGTPYEYGYRGQGTQVTVSS (SEQ ID NO: 6434)
TC-037
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTjSRDNAKNT VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISR DNAKNTWYLQMNSLRPEDTAVYYCSVLGRDMMTYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEV QLVESGGGLVQPGGSLRLSCAASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNTW YLQMNSLRPEDTAVYYCSVLGRDMMTYWGQGTQVTVSS (SEQ ID NO: 6435)
TC-038
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSASDTGGSTRYADSVKGRFTISR DNAKNTLYLQMNSLRPEDTAVYYCARVRGTPYEYGYRGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEV QLLESGGGLVQPGGSLRLSCAASGRTFSSLFMGWFRQAPGKEREFVASIRYPGLITNYADSAKGRFTISRDSAKNTVYLQ MNSLRPEDTGVYYCAAAPTGRAFNYWGQGTQVTVSS (SEQ ID NO: 6436)
TC-039
EVQLLESGGGLVQPGGSLRVSCAASGRTFSWNGMGWFRQAPGKEREFVAAISQSGGRTRYADSVKGRFTISRDNAKN TVYLQMNSLRPEDTAVYYCAASDFLLATTISAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQPGGSLRVSCAASGSIFRADAMGWHRQAPGKPREFVAGIRSDGFTNYADSVICGRFTSSW DTVKNTVYLQMNSLRPEDTAVYYCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGS EVQLVESGGGLVQPGGSLRVSCAASGSSFRADAMGWHRQAPGKPREFVAGIRSDGFTNYADSVKGRFTISWDTVKNT VYLQMNSLRPEDTAVYYCYYQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 6437)
TC-040
EVQLLESGGGLVQPGGSLRVSCAASGRTFSWNGMGWFRQAPGKEREFVAAISQSGGRTRYADSVKGRFTISRDNAKN TVYLQMNSLRPEDTAVYYCAASDFLLATTISAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTIST DSAKNTLYLQMNSLRPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGG G5EVQLLESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKN TLYLQMNSLRPEDTAWYCAADSDLSTVWGPHDYWGQGTQVTVSS (SEQ ID NO: 6438)
TC-041
EVQLLESGGGLVQPGGSLRVSCAASGRTFSWNGMGWFRQAPGKEREFVAAISQSGGRTRYADSVKGRFTISRDNAKN WYLQMNSLRPEDTAWCAASDFLLATTISAYDYWGQGTQVWSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISR DNAKNTLYLQMNSLRPEDTAVYYCARVRGTPYEYGYRGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEV QLLESGGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNAKNTLY LQMNSLRPEDTAVYYCARVRGTPYEYGYRGQGTQVTVSS (SEQ ID NO: 6439)
TC-042 EVQLLESGGGLVQPGGSLRVSCAASGRTFSWNGMGWFRQAPGKEREFVAAISQSGGRTRYADSVKGRFTISRDNAKN TVYLQMNSLRPEDTAVYYCAASDFLL41Q1SAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISR DNAKNTWYLQMNSLRPEDTAVYYCSVLGRDMMTYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEy QLVESGGGLVQPGGSLRLSCAASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNTW YLQMNSLRPEDTAVYYCSVLGRDMMTYWGQGTQVTVSS (SEQ ID NO: 6440)
TC-043
EVQLVESGGGLVQPGGSLRLSCAASGRRFSTLiMGWYRQAPGKQRELVATiERGGTPTYADSVKGRFTISRDNAKNTVY
LQMNSLRPEDTAVYYCKTLRYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGGLVQPGGSLRVSCAASGSSFRADAMGWHRQAPGKPREFVAGIRSDGFTNYADSVKGRFTISWDTVKNTVYLQMN SLRPEDTAVYYCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQLVESGGGLV
QPGGSLRVSCAASGSIFRADAMGWHRQAPGKPREFVAGIRSDGFTNYADSVKGRFTISWDTVKNTVYLQMNSLRPED
TAVYYCYYQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 5441)
TC-044
EVQLVESGGGLVQPGGSLRLSCAASGRRFSTLIMGWYRQAPGKQRELVATIERGGTPTYADSVKGRFTISRDNAKNTVY LQMNSLRPEDTAVYYCKTLRYWGQGTQVTV5SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLES GGGLVQPGGSLRLSCAASGFTFDDSAMTWVRQAPGKGLEWVSSSYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNS LRPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQLLESGGGL VQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPE
DTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSS (SEQ ID NO: 6442)
TC-045
EVQLVESGGGLVQPGGSLRLSCAASGRRFSTLIMGWYRQAPGKQRELVATIERGGTPTYADSVKGRFTISRDNAKNTVY LQMNSLRPEDTAVYYCKTLRYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLES
GGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTiSRDNAKNTLYLQMN
SLRPEDTAVYYCARVRGTPYEYGYRGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQLLESGGGLVQPG GSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAV YYCARVRGTPYEYGYRGQGTQVTVSS (SEQ ID NO: 6443)
TC-046
EVQLVESGGGLVQPGGSLRLSCAASGRRFSTLIMGWYRQAPGKQRELVATIERGGTPTYADSVKGRFTISRDNAKNTVY LQMNSLRPEDTAVYYCKTLRYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGGLVQPGGSLRLSCAASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNTWYLQM NSLRPEDTAVYYCSVLGRDMMTYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQLVESGGGLVQP GGSLRLSCAASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNTWYLQMNSLRPEDT AVYYCSVLGRDMMTYWGQGTQVTVSS (SEQ ID NO: 6444)
TC-047
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQL LESGGGLVQPGGSLRVSCAASGRTFSWNGMGWFRQAPGKEREFVAAISQSGGRTRYADSVKGRFTISRDNAKNTVYL QMNSLRPEDTAVYYCAASDFLLATTISAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSEVQLLESGGGLVQPGGSLRLSCAASGFNFSMYSIMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNA KNTLYLQMNSLRPEDTAVYYCARVRGTPYEYGYRGQGTQVTVSS (SEQ ID NO: 6453)
TC-048 EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQL LESGGGLVQPGGSLRVSCAASGRTFSWNGMGWFRQAPGKEREFVAAISQSGGRTRYADSVKGRFTISRDNAKNTVYL QMNSLRPEDTAVWCAASDFLLATFiSAYDYWGQGTQm'SSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG
GGSEVQLVESGGGLVQPGGSLRLSCAASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTiSRDNA KNTWYLQMNSLRPEDTAVYYCSVLGRDMMTYWGQGTQVTVSS (SEQ ID NO: 6454)
TC-049
EVQLLESGGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNAKNT LYLQMNSLRPEDTAVYYCARVRGTPYEYGYRGQGTQV7VSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL5 CAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARV RGTPYEYGYRGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDP EVQF^WYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKaCVSNKGLPSSIEKTIS^AKGQPREPQ WTLPPSQEEMTKNQVSLiaVKGFYPSmAVEWESNGQPENNYKTWVLDSDGSFFLYSRLTWKSRWQEGWFS CSVMHEALHNHYTQ&SLSLSLGK (SEQ ID NO: 6455)
TC-050
EVQLVESGGGLVQPGGSLRLSCAASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT WYLQMNSLRPEDTAVYYCSVLGRDMMTYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRL SCAASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNTWYLQMNSLRPEDTAVYYCS VLGRDMMTYWGQGTO.VTv'SSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQE DPEVQFNWYVDGVEVFINAKTKPREEQFNSTYRWSVL.TVI.HQDWLNGREYKCKVSNKGEPSSIECTISKAKGQPRE
PQVYTLPPSQEEMTRNQVSLYaVICGFYPSDIAVEWESNGQPENNYKTTPPyLDSOGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NG: 6456)
TC-051
EVQLLESGGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNAKNT LYLQMNSLRPEDTAVYYCARVRGTPYEYGYRGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLS CAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA ATPSGKAYSYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQE DPEVQFNWYVDGVEVHIYAKTKPREEQFNSTYRWSVLTVI.HQDWLIYGKEYKCKVSNKGLPSSIECTISKAKGQPRE
PQWTLPPSQEEMTKNQVSLiaVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6457)
TC-052
EVQLVESGGGLVQPGGSLRVSCAASGSIFRADAMGWHRQAPGKPREFVAGIRSDGFTNYADSVKGRFTISWDTVKNT VYLQMNSLRPEDTAWYCYYQ5LSSPNYGQVFWGQGTQVWSSGGGG5GGGGSGGGGSEVQLLESGGG1-VQPGGSL RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVY YCAATPSGKAYSYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLIYjlSRTPEVTCWVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKOCVSNICGLPSSIERTIS^AKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAyEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6458)
TC-053
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT
VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSPAPAPAPAPGGGGSEVQLVESGGGLVQA GGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNTVYLQMNSLKAEDSG VYFCYYQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 6459)
TC-054
EyQWESGGGWOafiGSlMSCAASGSiy^^
VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS LRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNTVYLQMNSLKAEDSGVYFC YYQAESSPNYGQTFWGQGTQVTVSSGGGGSESICYGPPCPSCIPESKYGPPCPPCPAPEFEGGPSVFLFPPSCP^OTLMIS RTPEVTCVWDVSQEDPEVQFNWWDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWENGKEYRCKVSNKGL
GSSIEKTISKAKGQPREPQWTLPPSQEEMTKNQVSLTCiyKGFYPSDIAVEWESNGQPENNYKTTPFVLDSDGSFFL YSRLTVDRSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6460)
TC-055
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES
GGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGSRSDGFTNYADSVKGRFTISSDNVKNTVYLQMNS LKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEFEGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWWDGVEVHNACTKPREEQFNSTYRWSVLTVLHQDWLNGICE YKCKVSNRGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTWKSRWQEGNVFSCSVMHEAEHNHYTQKSLSLSLGK (SEQ ID NO: 6461)
TC-056
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEFE
GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWWDGVEVNNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLGSSIEKT^ICAKGQPREPQWTLPPSQEEMTICNQVSLTaVKGFYPSDIAVEWESNGQPE NNYKTrPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ^SLSLSLGKGGGGSGGGGSEyQLyES GGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNTVYLQMNS
LKAEDSGWFCYYQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 6462)
TC-057
£VQlVESGGGLAQrcGSLRI3CAASGFIfSNYAMSWARQAreKGLOVySG[VWGSGIYYADSVl<GRFnSRDNAI<N TVYLQMNSLKPEDTALYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEYT'CVWDVSQEDPEVQFNWWDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYK CKVSNKGLGSSIEKnSKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYm"PPVL DSDGSFFL¥SRLTODjCSRWaEGNVFSCSVMHEALHNH¥TQ.SCSLSLSLGKGGGGSGGGGSEVQLVESGGGLVQAGGS LRLSCAASGSSVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNTVYLQMNSLKAEDSGVYFC
YYQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 6463)
TC-058
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT
VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQLVE
SGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNTVYLQMN SLKAEDSGWFmQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 6464)
TC-059 EVQLVESGGGLVQAGGSLRLSCAASGSSVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPAPAPAPAPGGG GSEVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVK NTVYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 6465)
TC-060
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGSRSDGFTNYADSVKGRFTISSDNVKNTVYLQMNS LKAEDSGVYFCYYQAESSPNYGQTFWGQGTQV’TVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQA
GGSLRLSCAASGSiVSTNGMGWHRQVPGKGRELVAGiRSDGFTNYADSVKGRFTISSDNVKNTVYLQMNSLKAEDSG
VYFCYYQALSSPNYGQTFWGQGTQV3VSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLS CAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNTVYLQMNSLKAEDSGVYFCYYQ ALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 6466)
TC-061
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG
GGGSEVQLVESGGGLVQAGGSLRLSCAASGSiVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTiSSDN
VKNTVYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGSiVSTNGMGWHRQVPGKGRELVAGIRSDGnNYADSVKGRFTI SSDNVKNTVYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGSiVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVK
GRRISSDNVKNTWLQMNSLKAEDSGWFCYYQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 6467)
TC-062
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG
GGGSEVQLVESGGGLVQAGGSLRLSCAASGSiVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTiSSDN
VKNTVYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSPAPAPAPAPGGGGSEVQLVESGGG LVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNTVYLQMNSLKA EDSGWFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE SGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNTVYLQMN
SLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 6468)
TC-063
EVQLVESGGGLVQAGGSLRLSCAASGSiVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT
VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSEVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDN VKNTVYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEV
QLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNWYL
QMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVK
NTWLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 6469)
TC-064 EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT
VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGSGSGGGGSG
GGGSEVQLVESGGGLVQAGGSLRLSCAASGSjVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTiSSDN VKNTVYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPAPAPAPA PGGGGSEVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTSSS DNVKNWLQMNSLKAEDSGWFCWQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGSGSGGGGSGGGGS GGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGR
FTISSDNVKNTVYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSS (SEQ ID NO: 6470)
TC-065
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSPAPAPAPAPGGGGSEVQLVESG GGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTSSRDNAKNTVYLQMNS LKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSS (SEQ ID NO: 6471)
TC-066
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTiSRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLA QPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPE DTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEFEGGPSVFL FPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVITVLHQDWENGK
EYK£KVSNKGLGS5IEKT§$KAKGQPREPQV¥TLPPSQEEMYKNQV5LTCEVRGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALRNHYTQKSLSLSEGK (SEQ ID NO: 6472)
TC-067
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKN TVYLQIMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTSSRDNAKNT VYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCP APEFFGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWWDGVEVHNAKTKPREEQFNSTYRVVSVL mHQDWL^GKEYRCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCiyKGFYPSDIAVEWES NGQPENN¥KTTPPVLDSDGSFFLYSRLTVDICSRWQEGNVFSCSVN?HEALHNHYTQKSLSLSLGK (SEQ ID NO: 6473)
TC-068
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSD'TYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALVYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPC PAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSV LTVlNQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQWFLPPSQEEMTKNQVSLTaVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMFIEALHNHYTQKSESLSLGRGGGGSGGGGSE
VQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKNT VYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSS (SEQ ID NO: 6474)
TC-069
EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVI.TVLHQDWLNGKEYK CK¥SNKGLGSSIEKBSKAKGQPREPQ¥¥TLPPSQEEMTKNQVSLTCI.VICGF¥PSDIAVEWESNGQPENN¥KTTPPVL DSDGSFFLYSRLWDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEyQLVESGGGLA^GGS LRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGSSDDGSDTYYADSVKGRFTISRDNAKN'rVYLQMNSLKPEDTALYY CAKDAGSWGTGPFGYEYDYWGQGTQVTVSS (SEQ ID NO: 6475)
TC-070
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGS EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSS (SEQ ID NO: 6476)
TC-071
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPAPAPAP APGGGGSEVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGSSDDGSDTYYADSVKGRFTI SRDNAKNTVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSS (SEQ ID NO: 6477)
TC-072
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSD7YYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGSSDDGSDTYYADSVKGRFTISRDNAKNT VYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEV QLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKNTV YLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQ LVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDIYYADSVKGRFTISRDNAKNTVYL QMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSS (SEQ ID NO: 6478)
TC-073
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRONAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGR FT!SRDNMK[VYLfiMNSU<PEPIALn£A|©AG^raiGPFGYEYDYWGQGinV]V^GGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSEVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSD TYYADSVKGRFTiSRDNAKNTWLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEW VSGISDDGSDTYYADSVKGRniSRDNAKNTVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVS
5 (SEQ ID NO: 6479)
TC-074
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGR FTiSRDNAKNTVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSPAPAPAPAPGGGG SEVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAK NTVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSEVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKG RFTSSRDNAKNTVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSS (SEQ ID NO: 6480) TC-075
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARaAPGKGLEWVSGISDDGSDTYYADSVKGR FTISRDNAKNTVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVIVSSGGGGSPAPAPAPAPAPAP
APAPGGGGSEVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGR FTISRDNAKNTVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSEVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSD TYYADSVKGRFTiSRDNAKNTVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSS (SEQ ID NO:
6481)
TC-076
P^LVESGGGLAQPGGSLBLSCAASGFIfSRYAMSWARQ^GKGLEWySGISDpgSPIYYADSyKGBFnSRDNAJ^
TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGG-GSGGGGSG GGGSGGGGSEVQLVESGGGLAQ.PGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGR FTISRDNAKNTVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSPAPAPAPAPAPAP APAPAPAPAPAPGGGGSEVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTY
YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVS GISDDGSDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSS (SEQ ID NO: 6482)
TC-077
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSPAPAPAPAPGGGGSEVQLVESGGGLVQA GGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNTVYLQMNSLKPEDTA VYTCYYQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 6483)
TC-078
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT
VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS LRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNTVYLQMNSLKPEDTAVYT CYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSESKYGPPCPSCPESiCYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLIVjg
SRTPEVTCWVDVSQEDPEVQFNWWDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGL GSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSmAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSC3VMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6484)
TC-079
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGG LVQAGGSLRVSCAASGSI FRADAM G WH RQVPGKPREFVAG I RSDG FTNYAEAVKG RFTISWDTVKNTVYLQM N SLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSESjCYGPPCPSCPESSCYGPPCPPCPAPEFEGGPSVFL
FPPKPKDTLMISRTPEVT£WVD¥SQEDPEVQFNWWDG¥E¥HNAKYKPREEQFNSTYRWS¥LTVLHQDWENGK EYKCKVSNRGLGSSOTISKAKGQPREPQWRPPSQEEMTKNQVSLTaWGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6485)
TC-080 EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSESKYGPPCPSCPESICYGPPCPPCPAPEFE GGPS¥FEFPPKPKDTEMISRTPEVTC¥WDVSQEDPEVQFNWWDGVEVH^AKTKPREEQFNST¥RWSVLTVI.HQ DWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVICGFYPSDIAVEWESNGQPE NWKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGICGG.GGSGGGGSEVQLyES GGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNTVYLQMN
SLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 6486)
TC-081
EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSES^YGPPCPPCPAPEFEGGPSVFLFPP KPKOTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGyEVHNAKTKPREEQFNSTYRWSyiTVLHQDWLNGKEYK CKySNRGLGSSI£KTISKAKGQPREPQ¥¥TLPPSQEEMTKNQVSLTCL¥KGF¥PSDIAVEWESNGQPENN¥KTTPP¥L DSDGSFFEYSRLWDICSRWQEGNVFSCSVMHEALHNHYTQKSLSLSEGKGGGGSGGGGSEVQLVESGGGLVQAGGS LRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNTVYLQMNSLKPEDTAVYT
CYYQSLSSPNYGQVFWGQGTQVTVSS {SEQ ID NO: 6487)
TC-082
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQLVE SGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNTVYLQM
NSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 6488)
TC-083
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPAPAPAPAPGGG GSEVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVK
NTVYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 6489)
TC-084
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT WQJYlNS[J<PEmW^^
GGG LVQAGGSLRVSCAASGSI FRADAM G WH RQVPGKPREFVAG I RSDG FTNYAEAVKG RFTISWDTVKNTVYLQM N SLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQ AGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNTVYLQMNSLKPEDT AVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLR VSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTSSWDTVKNIVYLQMNSLKPEDTAVYTCY YQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 6490)
TC-085
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGSGGSGGGGSGGGGSGGGGSG GGGSEVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDT VKNTVYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSEVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTS
SWDWKNTVYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGfiSGGGGSGGGGSGGG GSGGGGSGGGGSEVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVK
GRFTISWDWKNTVYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 6491)
TC-086
EyflLyESGGGiyQAGGSLRySCAM.GSiFRAPAM.GWH
VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSEVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDT
VKNTVYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSPAPAPAPAPGGGGSEVQLVESGGG LVQAGGSLRVSCAASGSSFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNTVYLQMNSLKP EDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE
SGGGLVQAGGSLRVSCAASGSiFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNTVYLQM NSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVIVSS (SEQ ID NO: 6492)
TC-087
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT yYLQMNSLKPjm
GGGSEVQLVESGGGLVQAGGSLRVSCAASGSiFRADAMGWHRQVPGKPREFVAGiRSDGnNYAEAVKGRFTISWDT VKNTVYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEV
QLVESGGGLVQAGGSLRVSCAASGSSFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNTVY LQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTSSWDTVK NTVYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 6493)
TC-088
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT
VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSEVQLVESGGGLVQAGGSLRVSCAASGSiFRADAMGWHRQVPGKPREFVAGiRSDGFTNYAEAVKGRFTiSWDT VKNTVYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPAPAPAPA
PGGGGSEVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISW
DTVKNTVYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGS
GGGGSGGGGSEVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGR FTISWDTVKNTVYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSS (SEQ ID NO: 6494)
TC-089
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTSSRDNAK
NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSPAPAPAPAPGGGGSEVQLVESGGGLVQAG
G5LRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLEMNSLKVEDTA VYYCAATPSGKAYSYWGQGTQVTVSS (SEQ ID NO: 6495)
TC-090
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSL
RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLEMNSLKVEDTAVYY CAATPSGKAYSYWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSQEDPEVQFNWWDGVEVHNAiCTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNICGEGSS EKTISKAKGQPREPQWTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN¥ICTTPPVi.DSDGSFFLYSR
LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6496) TC-091
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK
NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG GGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLEMN SLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEFEGGPSVFLFPP
KPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYK CKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWQEGNVFSCSyMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6497)
TC-092
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSESICYGPPGPSCPESKYgPPCPPCPAPEFEG
GPSVFLFPPKPKOTLMISRTPEVTCVWDVSQEDPEVQFNWWDGVEVHNAKTKPREEQFN3TYRWSVLTVLHQD
WLNGKEYKCRVSNRGLGSSIEKTISKAKGQPREPQVYTEPPSQEEMTKNQVSITCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFlYSRLTVDKSRWQEGNVFSCSVMHEALHNRYTQKSLSLSLGKGGGGSGGGGSEyQLyESG GGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLEMN
SLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS (SEQ ID NO: 6498)
TC-093
EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKN
TVYLQMNSLKPEDTALYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFE5GPSVFLFPP KPKDTLMISRTPEVTCVWWSQEDPEVQFNWYVTCVEVHNAKTICPREEQFNSTYRWSVLTVLHQDWLNGKEYK CKVSNKGLGSSIEKTISKAKGQPREPQVYTEPPSQFEMTKNQVSI.TCEVKGFYP.SDIAVEWESNGQPENNYKTTPPVI. D5DGSFFmRLTVDK5RWQEGNVFSCSVMHEALHNN¥TQKSLS^LGKGGGGSGGGGSEyQLyESG.GGI:VQAGLGS LRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLEMNSLKVEDTAVY
YCAATPSGKAYSYWGQGTQVTVSS (SEQ ID NO: 6499)
TC-094
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQLVES GGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLEM
NSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTV5S (SEQ ID NO: 6500)
TC-095
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPAPAPAPAPGGGG SEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK
NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS (SEQ ID NO: 6501)
TC-096
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK
NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG
GGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLEMN
SLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGG SLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAASNWSNGRTNYADSVKGRFTISRDNAKNTGYLEMNSLKVEDTAV YYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAAS GRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLEMNSLKVEDTAVYYCAATPS GKAYSYWGQGTQVTVSS (SEQ ID NO: 6502)
TC-097
EyflLyESGGGiyQAG^^^^
NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGG5GGGGSGGGGSGGGGSGGGGSGG GGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFnSRD NAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGG5GGGG5GGGGSGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTI SRDNAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSG
GGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKG RFTISRDNAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS (SEQ ID NO: 6503)
TC-098
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK NTGYLEMNSLKVEDT^
GGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRD NAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSPAPAPAPAPGGGGSEVQLVESGGGLV QAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLEMNSLKV EDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGG GLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLEMNS
LKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS (SEQ ID NO: 6504)
TC-099
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGIMGWFRQAPGKDSEFVAAINWSNGRTNYADSVICGRFTISRDNAK NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG
GGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRD NAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQL
VESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGY LEMNSLICVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKN TGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS (SEQ ID NO: 6505)
TC-100
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTSSRDNAK NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG5GG
GGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFnSRD NAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPAPAPAPAPG GGG5EVQLVESGGGLVQAGGSLRLSCAASG RTFSDYG M G WFRQAPG KDSEFVAAI N WSNG RTN YADSVKG RFTISR DNAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFT
ISRDNAKNTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS (SEQ ID NO: 6506)
TC-101
EVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRFTISRDNAKKAV YLQMNSLKPEDTAVYYCAAAPTGRAFTYWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEFEGGPS VFLFPPKPRDTLMI5RTPEVTCVWDVSQEDPEVQFNWWDGVEVMNAKTKPREEQFNSTYRWSVLTVLHQDWL NGKE¥KO<VSNKGLGSSIEKTISKAKGQPREPQV¥TLPPSQEEMTKNQVSLTCL¥KGF¥PSDIAVEWESNGQPENN¥ KTTPP¥LDSDGSFFLYSRI.T¥DK5RWQEGN¥FSCSVMHEALNNH¥TQKSl5LSi.Gi<GGGGSGGGGSEVQL.yESGGG LVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRFTISRDNAKKAVYLQMNSLKPE DTAVYYCAAAPTGRAFTYWGQGTQVTVSS (SEQ iD NO: 6507)
TC-102
EVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRFTiSRDNAKKAV YLQMNSLKPEDTAmCAAAPTGRAFIYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SEVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRFTISRDNAKKA VYLQMNSLKPEDTAVYYCAAAPTGRAFJYWGQGTQVWSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRFTISRDNAKK
AVYLQMNSLKPEDTAVYYCAAAPTGRAFTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSEVQLVESGGGLVEAGGSLRLSCAASGRTFGSYUVIGWFRQAPGREQEFLASiRRTGGSTSYADSVKGRFTiSRDNA KKAVYLQMNSLKPEDTAVYYCAAAPTGRAFTYWGQGTQVTVSS (SEQ iD NO: 6508)
TC-103
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSPAPAPAPAPGGGGSEVQLVESGGGLV QPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLKPEDT AVYYCAADSDLSTVWGPHDYWGQGTQVTVSS (SEQ ID NO: 6509)
TC-104
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSiYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGG SLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSSYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLKPEDTAVYY CAADSDLSTWVGPHDYWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEFEGGPSVFLFPP^PKDT LMISRTPEVTCVVVWSQEDPEVQFNWYVDGVEVRNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KGLGSSOTISKAKGQPREPQWTLPPSQEEMTKNQVSLTCLVRGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFL¥SRLTVDKSRWQEG^VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6510)
TC-105
£VQlVESGGGLyQPGGSlMSCAASGFITO2IAM]^yR^PGi<GLEWySSlYSYGPHTYYADSyi<GRFIlSIDSAKMl
YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLV ESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQM NSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSESKYGPPCPSCPESRYGPPCPPCPAPEFEGGP SVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVETVE^QDWL NGKEYKCiCVSN^GLGSSiEKTISICAKGQPREPQVYnPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSRLTVDiCSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 6511)
TC-106
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSESKYGPPCPSCPESKYGPPCPPCPAPEF EGGPS¥FLFPPKPKDTLMISRTPE¥TCVWDVSQEDPEVQFNWWDGVE¥HNAKTKPREEQFNSTYRV¥SVLTVLH QDWLNGKEYKCKVSNKGLGSSIEKQSKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENN¥RTTPP¥LDSDGSFFL¥SRLTVDKSRWQEGNVFS€SyMHEAi.HNH¥TQKSLSLSLSKGGGGSGGGGSEVQLVE
SGGGLVQPGGSLRLSCAASGFTFDDiAMTWVRQAPGKGLEWVSSiYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMN SLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQ.VTVSS (SEQ ID NO: 6512) TC-107
EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKGRNSGSYYPWDDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSQEDPEVQFNWWDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYK CK¥SNKGLGSSIEKTISKAKGQPREPQ¥YTLPP5QEEMTKNQVSLTCL¥KGF¥PSDIA¥EWESNGQPENNYI<TTPPVL. DSDGSFFLYSRLTVDICSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVQLVESGGGLVQPGGS
LRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSSYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLKPEDTAVYYC AADSDLSTVWGPHDYWGQGTQVTVSS (SEQ ID NO: 6513)
TC-108
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGGSEVQLV ESGGGL^^GGSLMSCAASGFTfDDIAMlWygQAPGKGL^WySSlYSWPNIYYADSyKGREnHPSMinLYLSM NSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSS (SEQ ID NO: 6514)
TC-109
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSS.GGGGSPAPAPAPAP.APAPAPAPAPA.PAPAP.GG GGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSA KNTLYLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSS (SEQ ID NO: 6515)
TC-110
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL
YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLV ESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQM N5LKPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG5EVQLVESGG GLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLKP EDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQP
GGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLKPEDTAV YYCAADSDLSTWVGPHDYWGQGTQV7VSS (SEQ ID NO: 6516)
TC-111
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAmCAADSDLSWWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTD SAKNTLYLQMNSLKPEDTAVYYCAADSDLSTVVVGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKG
RFTSSTDSAKNTLYLQMNSLKPEDTAVYYCAADSDLS'TVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSEVaVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYY ADSVKGRFTISTDSAKNTLYLQMNSLKPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSS (SEQ ID NO: 6517)
TC-112
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTD SAKNTLYLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSPAPAPAPAPGGGGSEVQLVES GGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNS LKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLY LQMNSLKPEDTAVYYCAADSDLSTVVVGPHDYWGQGTQVTVSS (SEQ ID NO: 6518)
TC-113
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGG5GGGGSGGGGSGGGGSGGGGS GGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDiAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTD SAKNTLYLQMNSLKPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPGGGG SEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNT LYLQMNSLKPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTIST DSAKNTLYLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSS (SEQ ID NO: 6519)
TC-114
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQ.VTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGG5EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTD SAKNTLYLQMNSLKPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSSGGGGSPAPAPAPAPAPAPAPAPAPAP APAPGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRF TISTDSAKNTLYLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYAD SVKGRFTISTDSAKNTLYLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSS (SEQ ID NO: 6520) Example 20. Evahatiori of the effect of anti-TNFRZ VHH-Fc fusion on Treg functicm m wVo [1024]Human TNFR2 knock-in mice were injected with the constructs as indicated in Figures 42A-42B at 3 mg/kg intravenously. After 5 days, percentage of Treg (FOXP3+CD4+) among CD45+ cell as well as F0XP3, ICAM-1, ICOS expression (MFI, mean fluorescent intensity) on Treg was measured by flow cytometry. Data as depicted in Figures 42A and 428 show Treg expansion in the spleen as well as increased expression of F0XP3, linked to Treg stability and function, and Treg activation shown by upregulation of ICAM-1 and ICOS, Administration of anti-TNFR2 VHH agonists to mice increased Treg by three-fold in blood and tissues without increasing conventional Teff or NK cells as it induced a higher level of F0XP3 and surface markers (F0XP3, ICAM-1, OX-40, ICOS, and CCR8), The effects of anti-TNFR2 VHH agonists on spleen lymphocyte population or on blood cells (eosinophils) in human TNFR2 knock-in mice were assessed after a single intravenous injection (3 mg/kg) of tetravalent VHH-Fc antibody T-037, or control VHH, or IL-2 N88D into human TNFR2 knock-in mice. Similarly, the effect of anti~TNFR2 VHH agonist was assessed on serum cytokines 1 day after the single injection of tetravalent VHH-Fc antibody T-037, control VHH or IL-2 N88D (Figures 41A-41E). Further, anti-TNFR2 VHH agonists expand Treg across tissues (Figures 40A-4OB). Example 21, Evateattosi of the effect of ants-TMFR2 VHH-R fasten on arthritis /n viv® [1025]At day 0, human TNFR2 knock-in mice were injected with anti-collagen antibodies as well construct T-037 at 3 mg/kg intravenously or control VHH. At day 3, mice were challenged with lipopolysaccharide (LPS). Treg cells were measured at day 6 and at day 7, arthritis score (total number of affected digits) and paw volume were measured. The same experiment was repeated with T-043 construct. Data as depicted in Figure 43B and 43C show reduction of arthritis, measured by paw volume and arthritis score, in a model of collagen-antibody induced arthritis upon treatment with TNFR2 agonist T-037 and T-043,
[1026]Amino acid sequences of exemplary fusion protein constructs described in Examples 11 and 12 as well as additional engineered constructs are provided below. In the sequences, VHH sequences are indicated with a straight underline (e.g., VHH;, Fc regions are indicated with bold letters (e.g., hlgG4~ SPLE or higGl LALAPA), N-terminal modification are indicated with both bold and a straight underline (e.g., N-term modification), C-terminal modification are indicated with both italic and a wavy underline (e.g,, C-tem modification} linker sequences are indicated with a wavy underline (e.g,, Linker), and hinge regions are indicated with both a wavy underline and bold letters (e.g., hinge).
T-037
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTG.VTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL
SCAASGRTFSDYGMGWFRQAPGKDSEFVAASNWSNGRTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC
AATPSGKAYSYWGQGTQVWSSGGGGSESR¥GPP£PP€PAPEF£GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQ
EDPEVQF^WYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGgEYKCKVSNICGLPSSIEKTISICAKGQPR EPQV¥TLPPSQEEMTKNQVSLTCLVKGF¥PSDIAVEWESNGQPENN¥m"PPVLDSDGSFFLYSRLWDKSRWQEG
NVFSCSVMHEALHNHYTQKSL5L3LGK (SEQ ID NO: 7055)
T-038
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL
SCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTSSRDNAKNTGYLQMNSLRVEDTAVYYC
AATPSGKAYSYWGQGTQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPIKDTLMISRTPEVTCVWDVSHE
DPEVKFNWYVTCVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLhlGKEYKCKVSNKALAAPIEICTISICAKGQPRE
PQVYTLPPSREEMTKNQVSLT£LVKGF¥PSDIAVEWESNGQPENN¥KTTPPVLDSDGSFFl¥SKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 7056)
T-039
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKN TCYbQMNSU)MiOT
SCAASG RTFSDYG MG WFRQAPG KDSEFVAAI N WSN G RTNYADSVKG RFTISRDN AKNTGYLQM NSLRVEDTAVYYC ATTPSGKA¥S¥WGQGTQVWSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE¥TCVWD¥SHED PEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGREYKCKVSNKALAAPIEKTISKAKGQPREP QV¥TLPPSREEMTKNQVSLTCLVKGF¥PSDIAVEWESNGQPENN¥CTTPP¥LDSDGSFFLYSKLT¥DKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 7057)
T-040
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT VYLQMNSLRPEDTAVYYCVASPTGRAFTYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLS CAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCVAS PTGRAFTYWGQGTQ¥rVSSGGGGSESR¥GPP£PP£PAPEFEGGPS¥FLFPPKPKDTLI¥PSRTPEVTC¥VVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIERTIS^AKGQPREPQ ¥YTLPPSQEEMTKNQ¥SLTaVKGF¥PSDIA¥EWESNGQPENN¥KTTPPVLDSDGSFFL¥SRLTVDKSRWQEGNVFS CSVMHEALHNHYTQ^SLSLSLGK (SEQ ID NO: 7058)
T-041
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT VYLQMNSLRPEDTAVYYCVASPTGRAFTYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLS CAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCVAS PTGRAFTYWGQG'TQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPE ¥KFNWYVDG¥EVHNAKTKPREEQ¥NSTYRVVSVLT¥LHQDWLNGKE¥KCK¥SNKALAAPIEKTISKAKGQPREPQ V¥TLPPSR£ENITKNQVSLTCLVKGF¥PSaiA¥EWESNGQP£NN¥KTTPPVLDSDGSFFL¥SKLT¥DKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7059)
T-042
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGKTNYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGKTNYADSVKGRFTSSRDNAKNTGYLQMNSLRVEDTAVYYC AATPSGKAYSYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSO EDPEVQFNWYVDGVEVFIFIAKTRPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAICGQPR EPQyYTLPPSQEEMTRNQVSLTCLVRGF¥PSDIAVEWESNGQPENN¥iaTPPVLDSDGSFFLYSRLT¥DKSRWQEG
NVFSCSVMREALHNHYTQKSLSLSLGK fSEQ ID NO: 7060)
T-043
EVQLLESGGGLVQPGGSLRLSCAASG FTFDDI AMTWVRQAPG KG IE WVSSIYSYG PNTYYADSVKG RFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGG SLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYY CAADSDLSTVTVGPHDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYSCCKVSNKGLPSSIEKTISK AKGQPREPQy¥nPPSQEEMTKNQVSLTCLVKGF¥PSDIAV£W£SNGQP£NN¥RTTPP¥LOSDGSFFL¥SRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NG: 7061)
T-044
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTSSRDNAKN TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AATPSGKAYSYWGQGTQVTVS5GGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMSSRTPEVTCVWDySQ EDPEVQFNWYVDGVEVHNARTRPREEQFNSTYRWSVLWLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQ¥YTLPPSQEEMTKNQVSLTCL¥KGFYPSDIA¥EWESNGQPENNYKrrPP¥LDSDGSFFL¥SRLT¥DKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGIC (SEQ ID NO: 7062)
T-045
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVPAGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLR
LSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYY CAATPSGKAYSYWGQGTQVTVPAGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMiSRTPEVTCWVDVS
QEDPE¥QFNWY¥DGVE¥HNAKTKPREEQFNSTYRWS¥LTVLHQDWLNGKEYKaC¥SNKGLPSS?£KTISKAKGQP REPQV¥TLPPSQE£MTKNQ¥SLTCL¥KGF¥PSDIA¥EWESNGQPENN¥KTTPPyLDSDGSFFLYSRLTVDKSRWQEG NVFSCS¥MHEALHNHYTQKSLSLSLGK (SEQ. ID NO: 7063)
T-046
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVPAGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYY CAATPSGKAYSYWGQGTQVTVPAGGGGSPKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMSSRTPEVTCVWDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYICCICVSNICALAAPIEKT8SKARGQPR EPQVYTLPPSREEMTKNQVSLTaVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSTCSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7064)
T-047
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTiSRDDAKKT VYLQMNSLRPEDTAVYYCVASPTGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCVA
SPTGRAFTYWGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKP^DTLMISRTPEVTCVWDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYICaCVSNICALAAPIEKTiSKAKGQPREPQ
V¥TLPPSREEMTKNQV5LTCL¥KGF¥PSDIA¥EWESNGQPENNYKTTPP¥LDSDGSFFL¥SKLT¥DKSRWQQGN¥FS CSVWHEALHNHYWSLSESPGK (SEQ ID NO: 7065)
T-048
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT VYLQMNSLRPEDTAVYYCVASPTGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCVA
SPTGRAFIYWGQGTQVTVpAGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPjCPjCDTLMiSRTPEVTCVWDVSQED PEVQFNW¥¥OGVE¥HNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKySNKGLPSSIEKTISRAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTrPPVmSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7066)
T-049
DVQLVESGGGLVQPGGSLRLSCAASGSIFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT WYLQMNSLRPEDTAVYYCSVLGRDLVTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLVESGGGLVQPGGSLRLS CAASGSIFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNTWYLQMNSLRPEDTAVYYCSVL
GRDLVTYWGQGTQWMGGG.GSDITHICPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVrCWVDVSHEDPEV KFNW¥yDGVEyHNACTKPREEQ¥NST¥RWSVLTVLHQQWLNGKEYK€KVSNRALAAPIECTISKAKGQPR£PQV¥ TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYICTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7255)
T-050
DVQLVESGGGLVQPGGSLRLSCAASGSIFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT WYLQMNSLRPEDTAVYYCSVLGRDRVTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLVESGGGLVQPGGSLRL SCAASGSIFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNTWYLQMNSLRPEDTAVYYCSV LGRDRVTYWGQ.G7QVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPE
VKFNWYVTCVEVHNACTKPREEQYNST¥RWSVLTVLHQDWLNGKEYKCKVSNKALAAP§EKTISKAICGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKnVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7256)
T-051
DVQLVESGGGLVQPGGSLRLSCAASGSIFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT WYLQMN5LRPEDTAVYYCSVLGRDIVTYWGQ,GTQVTVPAGGGGSGGGGSGGGGSDVQLVESGGGLVQPGGSLRLS CAASGSIFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNTWYLQMNSLRPEDTAVYYCSVL
GRDIVTYWGQGTQVTVPAGGGGSDKTHICPPCPAPEAAGGPSVFLFPPKPICDTLMISRTPEVTCVWDVSHEDPEV KFNWYVDGVEVHNACTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNRALAAPIECTISKAKGQPREPQVY nPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7257)
T-052
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAAAPSGKAYSYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYY CAAAPSGKAYSYWGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKOTLMISRTPEVTCWVDVSH
EDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYRCICVSNKALAAPIEKTISKA^GQPR EPQVYTLPPSREEMTKMQVSLTCLVKGFYPSDIAVEWESNGQPEMNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALH^HYWSLSLSPGK (SEQ ID NO: 7258)
T-053
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATASGKAYSYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYY CAATASGKAYSYWGQGTQVTVPAGGGGSDOHTCPPCPAPEAAGGPSVFLFPPKPKDTLMgSRTPEVTCVWDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTaVKGFYPSDIAVEWESNGQPENNYRTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7259)
T-054
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPAGKAYSYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKDSEFVAASNWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYY CAATPAGKAYSYWGQGTQVTVPAGGGGSPnHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSH
EDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGICEYRaCVSNKALAAPIEKTISICA^GQPR EPQV¥TLPPSREEMTKNQVSLTCLVKGF¥PSDIAVEWESNGQPENN¥KTTPPVI.DSDGSFFL¥SKLT¥»KSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7260)
T-055
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSAKAYSYWGQGTQVTVPAGGGGSGGGGSGGGgSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYY CAATPSAKAYSYWGQGTQVTVPAGGGGSDRTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEyTCWVDySH EDPEVKFNW¥VDG¥EVRNAKTKPREEQ¥NSTYRWSVLT¥LHQDWLNGKEYKCICySNKALAAPIEKTISKAKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK {SEQ ID NQ: 7261)
T-056
DVQLLESGGGLVQPGGSi-RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPSGAAYSYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYY CAATPSGAAYSYWGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMgSRTPEVTCVWDVSH
EDPEVKFNW¥VDG¥E¥HIYAKTKPRE£QYNSI¥R¥¥SVLTVLHQDWLNGKE¥KCKVSNKALAAPIEKTISKAICGQPR EPQV¥TLPPSRE£MTKNQVSLTCLVKGF¥PSDIA¥EWESNGQPENN¥KTTPPVLDSDGSFFL¥SKLTVDICSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7262)
T-057
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPSGKAASYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYY CAATPSGKAASYWGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSH
EDPEVKFNWWDGVEVHNAKTKPREEQV^STYRWSVLTVLHQDWLNGICEYICCICVS^KALAAPIEKTISKA^GQPR EPQVYTLPPSREEMTKNQVSLTCLVKGF¥PSDIAVEWESNGQPEMN¥KTTPPVLDSDGSFFL¥SKLTVDKSRWQQG NVFSCSVMREALHNHYTQKSLSLSPGK (SEQ ID NO: 7263)
T-058
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPSGKAYAYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYY CAATPSGKAYAYVv'GQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVS^
EDPEVKFNWYVDGVEVHIYAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYRCKVSNKALAAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTaVKGFYPSDIAVEWESNGQPEMNY^TTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMNEALHNHYTQKSLSLSPGK (SEQ ID NO: 7264)
T-059
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPSGKAYSAWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYY CAATPSGKAYSAWGQGTQVTVPAGGQSSPKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSH
EDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGICEYICaCVSNKALAAPIEKTISKA^GQPR EPQV¥TLPPSREEMTKNQVSLTCLVKGF¥PSDIAVEWESNGQPENN¥KTTPPVLDSDGSFFL¥SKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7265)
T-060
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAK NTGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVPAGGGG5GGGGSGGGGSDVQLLESGGGLVQPGGSL RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVY YCAATPSGKAYSYVYGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVS HEDPEVKFNWYVTCVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKFISKAKGQP REPQV¥TLPPSRE£MTKNQVSLTCLVKGF¥PSDIAV£WESNGQPENNYKTTPPVLDSDGSFFL¥SKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7067)
T-0S1
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAK NTGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSL RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVY YCAATPSGKAYSYWGQ.GTQVTVPAGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDV SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQWTLPPSQEEMTKNQVSLTCLVICGFYPSDIAVEWESNGQPEN^YCTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNH¥TQKSLSLSLGK (S£Q ID NO: 7068)
T-062
EVQLLESGGGLVQPGGSLRLSCAASG FTFDDI AMTWVRQAPG KG LE WVSSIYSYG PNTYYADSVKG RFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGG SLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTSSTDSAKNTLYLQMNSLRPEDTAVYY G4ADSDLSTVTVGPHDYWGQGTQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMgSRTPEVT£W VDVSHEDPEVKFNWYyTCVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKA KGQPREPQVYnPPSREEMTK^QVSLTCLVKGFYPSD^AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCS¥MH£ALHNM¥TQKSLSLSPGK (SEQ ID NO: 7069)
T-063
EVQLLESGGGLVQPGGSLRLSCAASG RTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AATPSGKAYSYWGQGTQVTVSSGGGGSPKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHE DPEVKFNWYVTCVEVHNAKT^CPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPRE PQWTLPPSREEMTKNQVSLTCLVRGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDRSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGSC (SEQ ID NO: 7070)
T-064
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT VYLQMNSLRPEDTAVYYCVASPTGRAFTYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRLS CAASGRTFGSYTMGWFRQAPGKEREFLASSRWTGGSTSYADSVKGRFTISRDDAKKTWLQMNSLRPEDTAVYYCVAS PTGRAFTYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDP EVQFMWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNICGLPSSIEKnS^AKGQPREPQ ¥¥TLPPSQEEMTKNQVSLTCEVKGF¥PSDIAVEWESNGQPENN¥KTTPP¥I.DSDGSFFL¥SRLTVDKSRWQEGNVFS CSVNIHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7071)
T-065
DVQLLESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLLESGGGLVQPGG SLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYY
CAADSDLSTVTVGPHDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMjSRTPEVTCV ¥VDVSQEDPEVQFNWWDG¥EVI-iNAKTKPREEQFNSTYRWS¥I.TVLHQDWLNGK£¥KaCVSNKGLPSSI£KTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK {SEQ ID NO: 7072)
T-066
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAK NTGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSL
RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVY YCAATPSGKAYSYWGQ.GTQVTVSSGGGGSESRYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCWVDVS
QEDPE¥QFNWYVDG¥EVHNAKTKPREEQFNSTYRWS¥LTVLHQDWLNGKEYKC§C¥SN§(GLPSSIEK-nSKAKGQP REPQ¥¥TLPPSQEEMTKNQ¥SLTCL¥KGF¥PSDIA¥EWESNGQPENN¥KTTPP¥LDSDGSFFL¥SRLT¥DKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK fSEQ ID NO: 7073)
T-067
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT VYLQMNSLRPEDTAVYYCVASPTGRAFTYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRLS CAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCVAS PTGRAFTYWGQGTQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPgCDTLMSSRTPEVTCVWDVSHEDPE yKFNW¥VDGVEVHNAKTKPREEQ¥NST¥R¥yS¥LTVLHQDWLNGKE¥KCK¥SNKALAA?EKTISKA§CGQPREPQ V¥TLPPSR££M7KNQVSLTCLyKGFYPSDIAVEW£SNGQPENN¥KTTPPVLDSDGSFFL¥SKLTVDKSRWQQGN¥FS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7074)
T-068
DVQLLESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLLESGGGLVQPGG SLRLSCAASGFTFDDSAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYY CAADSDLSTVTVGPHDYWGQGTQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPRPKDTLMISRTPEVTCW
VDVSREDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLiyLHQDWLNGKEYKCKVSNRALAAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTaVKGFYPSDIAVEWESNGQPENWKTTPPVLDSDGSFFLYSKLWDICSR WQQG NVFSCSVMHEALHNFIYTQKSLSLSPGK (SEQ ID NO: 7075)
T-069
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAK NTGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQELESGGGLVQPGGSL
RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVY YCAATPSGKAYSYWGQGTQVTVSSGGGGSPKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMjSRTPEVTCWVDVSH
EDPEVKFNWWDGVEVHNAKTICPREEQYNSTYRWSVLTVLHQDWLNGICEYKCKVSNKALAAPIEKTISRA^GQPR EPQVYTLPPSREEMTKNQVSLTCLVKGF¥PSDIAVEWESNGQPENN¥KTTPPVI.DSDGSFFL¥SKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7076)
T-070
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT VYLQMNSLRPEDTAVYYCVASPTGRAFTYWGQGTQVTVPAGGGGSGGGG5GGGGSEVQLLESGGGLVQPGGSLRLS CAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCVAS PTGRAFTYWGQGTQVTVPAGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDniVRSRTPEVTCVWDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCICVSNICGLPSSIEKTISKAKGQPREPQ V¥TLPPSQEEMTKNQVSLTCLVKGF¥PSDIAVEWESNGQPENN¥KTTPPVLDSDGSFFL¥SRLTVDKSRWQEGNVFS
CSVMHEALHNHYTQKSLSLSLGK (SEQ ID NQ: 7077)
T-071
EVQLLESGGGLVQPGGSLRLSCAASG FTFDDI AMTWVRQAPG KG LE WVSSIYSYG PNTYYADSVKG RFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVPAGGGGSGGGGSGGGGSEVQLLESGGGLVQPGG SLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYY CAADSDLSTVTVGPHDYWGQGTQVTVPAGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPICDTLMISRTPEVTCV WDVSQEDPEVQFNWYVTCVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7078)
T-072
EVQLLESGGGLVQPGGSLRLSCAASG RTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT VYLQMNSLRPEDTAVYYCVASPTGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLS CAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCVAS PTGFtAFnWGQGTQyryPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPICPICDTLMISRTPEVTCVWDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQ V¥TLPPSR£EMTKNQVSLTCLVKGF¥PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL¥SKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7079)
T-073
EVQLLESGGGLVQPGGSLRLSCAASG FTFDDI AMTWVRQAPG KG LE WVSSIYSYG PNTYYADSVKG RFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVPAGGGGSGGGGSGGGGSEVQLLESGGGLVQPGG SLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYY CAADSDLSTVTVGPHDYWGQGTQVTVPAGGGGSDICTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCW VDVSREDPEVKFIYWYVDGVEVFINAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNICALAAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTaVKGFYPSDIAVEWESNGQPE^WK'nPPVLDSDGSFFLYSICLWDICSR WQQGNVFSCSVMHEALHNRYTQKSLSLSPGK (SEQ ID NO: 7080)
T-074
DVQLLESGGGLVQPGGSLRLSCAASGFTFDDSAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPG GSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVY YCAADSDLSTVTVGPHDYWGQGTQVTVPAGGGGSESKYSPPCPPCPAPEFEGGPSVFLFPPSCPKDTLMSSRTPEVTC VWDVSQEDPEVQFNWYVDGVEVHNAKTICPREEQFNSTYRWSVLTVLHQDWLNGKEYKCSCVSNKGLPSSIEKTIS KAKGQPREPQWTI.PPSQEEMTKNQVSLTCI.VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRI.TVDK SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7081)
T-075
DVQLLESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVPAgGGGSGGGGSGGGGSDVQLLESGGGLVQPG GSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVY YCAADSDLSTVTVGPHDYWGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV
WDVSHEDPEVKFNWYVTCVEVHNAKTKPREEQYNSTYRWSVLTVI.HQDWLIMGKEYKCKVSNKALAAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTGLVKGFYPSDIAVEWESNGQPENNYRTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7082)
T-076
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT VYLQMNSLRPEDTAVYYCVASPTGRAFTYWGQGTQVTVAGGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCVA
SPTGRAFr/WGQGTQVTy.4GGGGGSESKYGPPCPPCPAPEFEGGPS¥FLFPPRPKDTLMiSRTPEVTCWVDVSQED PEVQFNWYVDGVEyHNAKTKPREEQFNS'TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP Q¥¥TLPPSQEEMTKNQ¥SLTCLVKGFYPSDIA¥£WESNGQPENNYKTTPP¥LDSDGSFFLYSRLT¥DKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLG^ (SEQ ID NO: 7083)
T-077
DVQLLESGGGLVQPGGSLRLSCAASGFFFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVAGGGGGSGGGGSGGGGSDVQLLESGGGLVQPG GSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVY YCAADSDI.STVTVGPHDYWGQGTQVTVAGGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTC
WVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDiAVEWESNGQPE^NYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMNEAEHNHYTQKSLSLSLGK (SEQ ID NO: 7084)
T-078
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAK NTGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVAgGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSL RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVY YCAATPSGKAYSYWGQ.GTQVTVAGGGGGSESK¥GPPCPPCPAPEFEGGPS¥FLFPPKPKDTLMISRTPEVTC¥VyD¥
SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQyYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKFTPpyLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7085)
T-079
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTSSRDNAKN TGYLQMNSLRVEDTAVYYCAAAPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AAAPSGKAYSYWGQGTQVrVSSGGGGSESKYGPPCPPCPAPEFEGGPSyFLFPPKPKDTLMISRTPEVTCWVDVSG
EDPEVQFNWYVDGVEVRNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKARGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NyFSCSVMHEALHNHYTQKSLSLSLGK (SEO. ID NO: 3930)
T-OSO
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAAAPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTSSRDNAKNTGYLQMNSLRVEDTAVYYC AAAPSGKAYSYWGQ.GTQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTEMISRTPEVTCWVDVSHE
DPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLWLHQDWLNGKEYKCKVSNKA1AAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCL¥KGF¥PSDIAVEWESNGQP£NN¥KTTPP¥LDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3931)
T-081
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAK NTGYLQMNSLRVEDTAVYYCAAAPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSPVQLL£SGGGi-VQPGGSL RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVY
YCAAAPSGKAYSYWGQGTQVTVSSGGGG.SESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCWVDV SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEIGnSKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVICGFYPSDiAVEWESNGQPENNYCTTPPVLDSDGSFFLYSRLTVDKSRWQE
GNVFSCSVMHEALHNH¥TQKSLSLSLGK (SEQ ID NO: 3932)
T-082
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAK
NTGYLQMNSLRVEDTAVYYCAAAPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSL RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVY YCAAAPSGKAYSYWGQGTQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVtTVLHaDWLNGKEYKCKVSNKALAAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTaVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG^ (SEQ ID NO: 396S)
T-083
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAAAPSGKAYSYWGQGTQVTVPAGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYY CAAAPSGKAYSYWGQGTQVTVPAGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDyS
QEDPEVQFNWWDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKOCVSNICGLPSSIEICTISKAKGQP REPQWFLPPSQEEMTKNQVSLTaVKGFYPSDIAVEWESNGQPENNYKTTPPyLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK CSEQ ID NO: 3966)
T-084
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTSSRDNAKN
TGYLQMNSLRVEDTAVYYCAAAPSGKAYSYWGQGTQVTVPAGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKDSEFVAASNWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYY CAAAPSGKAYSYWGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSyFLFPPKPKDTLMSSRTPEVTCYVVDVSH
EDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGICEYICaCVSNKALAAPIEKTISICA^GQPR EPQV¥TLPPSREEMTKNQVSLTCLVKGF¥PSDIA¥EWESNGQPENN¥KTTPPVI.DSDGSFFL¥SKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3967)
T-0S5
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAK NTGYLQMNSLRVEDTAVYYCAAAPSGKAYSYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSL RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVY YCA,AA.PSGKAYSYWGQGTQVr/PAGGGGSESK¥GPPCPPCPAPEFEGGPSVFLFPPICPKDTL.MISRTPEVTC¥WDV
SQEDPEVQFNWYVDGVEVHNACTKPREEQFNSTYRWSVLTVLHQDWLNGKEYICCKVSNKGLPSSIEKTISICAICGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYCTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 3968)
T-086
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAK NTGYLQMNSLRVEDTAVYYCAAAPSGKAYSYWGQGTQVTVAGGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGS LRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVY YCAAAPSGKAYSYWGQGTQVTVAGGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDV
SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQWTLPPSQEEMTKNQVSLTCLVICGFYPSDIAVEWESNGQPENNYCTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCS¥MHEALHNH¥TQKSLSLSLGK (SEQ ID NO: 7278)
T-087
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL
SCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA ATPTGKAYTYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMjSRTPEVTCVWDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLWLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQ¥SLTCLVKGF¥PSDIA¥EWESNGQPENNYKTTPPVLDSDGSFFL¥SRLTVDKSRWQEGN
VFSCSVMHEALNNFIYTQKSLSLSLGK (SEQ ID NO: 7297)
T-088
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAICN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL
SCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA A'TPTGKAYIYWGQGTQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNG^EYKCKVSNKALAAP^EKTISKAKGQPREP QYYTFPPSREEMTKNQVSLTCIVKGFYPSDIAVEWESNGQPENNYICTTPPVLDSDGSFFLYSKLTVD^SRWQQGNV
FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7298)
T-089
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATSNWSNGRTTYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR
LSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AATPTGKAYTYWGQGTQ.VTVSSGGGGSESK¥GPPCPP€PAPEFEGGPSVFLFPP8(PKDTLMISRTPEVTC¥WD¥Sa EDPEVQFNWYVDGVEVFINAKTRPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKARGQPR EPQ¥¥TLPPSQEEMTKNQVSLTCL¥KGF¥PSDIAVEWESNGQPENN¥KTT?P¥I.DSDGSFFL¥SRI.T¥Di(SRWQEG NyFSCSYMHEALHNHYTQKSLSLSLGK (SEQ. ID NO: 7299)
T-090
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTFYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQII.ESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AATPTGKAYTYWGQGTQVTVSSGGGGSDOHTCPPCPAPEAAGGPSVFLFPP^PICDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLWLHQDWLNGKEYKCKVSNKA1AAPIEKTISKAKGQPRE PQ¥¥TLPPSREEMTKNQ¥SLTCL¥KGF¥PSDIA¥EWESNGQPENN¥KTTPP¥LDSDGSFFL¥SKLT¥DKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7300)
T-091
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVPAGGGGSGGGGSGGGGSB/QLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AATPTGKAYTYWGQGTQVTVpAGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPRDTLMjSRTPEVTCWVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNICGLPSSIEKTISICAKGQPR EPQ¥¥TLPPSQE£MTKNQ¥SLTCL¥KGF¥PSDIA¥EWESNGQPENN¥KTFPP¥LDSDGSFFL¥SRLT¥DKSRWQ£G
NVFSCSVMHEALH^HYTQKSLSLSLGK fSEQ ID NO: 7301)
T-092
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVPAGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AATPTGKAYTYWGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPP^PICDTLMISRTPEVTCVWDVSHE DPEVKFNWY¥DG¥EVHNAKTKPREEQYNST¥RWSVLTVLHQDWLNGKE¥K€KVSNKALAAPIEKTISKAKGaPRE PQVYTLPPSREEMTKNQVSLTCLVKGF¥PSDIAVEWESNGQPENN¥KTrPP¥LDSDGSFFL¥SKLT¥DKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7302)
T-093
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTSSRDNAKN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGR'nYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AATPTGKAYTYWGQGTQVTVPAGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCWVDysa EDPEVQFNWYVDGVEVHNAKTKPREEQFIMSTYRWSVLTVLHQDWLNG^EYKCKVSNICGLPSSIE^TISKAKGQPR £PQ¥¥TLPP5QEEMTKNQySLTCL¥KGFYPSDIA¥EWESNGQPENNYI<TEPmDSDGSFFL¥SRLT¥DKSRWQ£G
NVFSCSVMHEALH^HYTQKSLSLSLGK fSEQ ID NO: 7303)
T-094
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATSNWSNGRTTYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAWYC AATPTGKAYTYWGQGTQVTVPAGGGGSPjCTHTCPPCPAPEAAGGPSVFLFPPKPICDTLNjISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNfCALAAPIEKTISRAICGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE5NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7304)
T-095
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATSNWSNGRTrYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVAGGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AATPTGKAYTYWGQGTQVTVAGGGGGSE6KYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQWTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7305)
T-096
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNAR'FTYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNARTTYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA ATPTGKAYTYWGQ.GTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPRDTLMISRTPEVTCWVDVSGE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLWLHQDWLNGKEYKCKVSNKGLPSSIEKTISKARGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDiAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDRSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7306)
T-097
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNARTFYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNARTTYADSVKGRFTiSRDNAKNTGYLQMNSLRVEDTAVYYCA ATPTGKAYTYWGQGTQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPgCPKDTLMISRTPEVTCWVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTBKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAYEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGR (SEQ ID NO: 7307)
T-098
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNARTTYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQi-LESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNARTTYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AATPTGKAYTYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNG^EYKCKVSN^CGLPSSIE^TISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK CSEQ ID NO: 7308)
T-099
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNARTTYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNARTTYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AATPTGKAYTYWGQGTQVTVSSGGGGSDOHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAICGQPRE
EPQV¥TLPPSQEEMTKIYQVSLTCL.¥KGF¥PSDIAVEWESNGQPENN¥KTTPP¥LDS[)GSFFL¥SRI.TVDi(SRWQ£G NVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7314)
T-105
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA GTLSGKAYTYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMSSRTPEVTCVWDVSQE
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSSEKTISKAKGQPRE PQWTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7315)
T-106
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA
GTLSGKAYmVGQGTQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKOCVSNKALAAPIEKT8SKAKGQPREPQ WTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7316)
T-107
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA GTLSGKAYTYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMSSRTPEVTCWVDVSQE
DPEVQFNWYVTCVEVHNAKTKPREEQFNSTYRWSVLWLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALNN^YTQKSLSLSEGK (SEQ ID NO: 7317)
T-108
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLI-ESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA GTLSGKAYTYWGQGTQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKP^CDTLMISRTPEVTCWVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEY^CCKVSNKALAAPIEKTISKAICGQPREPQ WTLPPSREEMTKNQVSETCLVICGFYPSaiAVEWESNGQPENNYKTTPPVLDSDGSFFLYSICLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7318)
T-109
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVPAgGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL
SCAASGRTFSDYGMGWFRQAPGKEHEFVASSNWSNGRTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA GTLSGKAYTYVVGQGTQYTVPAGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPRPKDTLMSSRTPEVTCWVDVSQ.E DPEVQFNWYVTCVEVHNAKTKPREEQFNSTYRWSVLTV8.HQDWLNGKEYKCICVSNKGLPSS8EKTISKAICGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK5RWQEGN ¥FSCS¥MHEALHNH¥TQKSLSLSLGK (SEQ ID NO: 7319)
T-110
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVPAGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA GTLSGKAYTYWGQGTQVTVPAGGGGSOKTHTCPPCPAPEAAGGPSVFLFPPKPKOTLMISRTPEVTCWVDVSHED
PEVKF^WYVDGVEVHNAKTICPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISICAKGQPREP QVYTLPPSREEMYKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7320)
T-lll
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTgSRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AGTLSGKAYTYWGQGTQVTVPAGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKP^DTLMISRTPEVTCVWDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNICGLPSSIEKTISICAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYm'PPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALH^HYTQKSLSLSLGK fSEQ ID NO: 7321)
T-112
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AGTLSGKAYTYWGQGTQVTVPAGGGGSPKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKaCVSNKALAAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTrPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7322)
T-113
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVAGGGGGSGGGGSGGGGSOVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEHEFVASSNWSNGRTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AGTLSGKAYTYWGQGTQVTVAGGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPICDTLMISRTPEVTCVWDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGICEYKCKVSNICGLPSSIEICTISKAKGQPR FPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNH¥TQICSLSLSLGK (SEQ ID NO: 7323)
T-114
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL
SCAASGRTFSDYGMGWFRQAPGKEHEFVASSNWSNARTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA GTLSGKAYTYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMSSRTPEVTCWVDVSQE DPEVQFNWWDGVEVHNAKTKPREEQFFJSTYRWSyLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAICGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7324)
T-115
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA
GTLSGKAYTYWGQGTQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLIYRSRTPEVTCWVDVSIHEDP EVKFNW¥VDGVE¥HNAKTKPREEQYNST¥RWS¥LT¥LHQDWLNGKEYKCKVSNKALAAPIEKT8SKAKGQPREPQ V¥TLPPSR£EM7KNQVSLTCLVKGFYPSDIA¥EWESNGQPENM¥KTTPPVLDSDGSFFL¥SKnVDKSRWQQGN¥FS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7325)
T-116
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA GTLSGKAYTYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMjSRTPEVTCVWDVSQE
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLWLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDICSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7326)
T-117
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL SCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA
GTLSGKAYTYWGQGTQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKOTLMISRTPEVTCWVDVSHEDP EVKFNWYVDGVEVHNAICFKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7327)
T-118
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVPAGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL
SCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA GTLSGKAYTYWGQGTQVTVPAGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPICDTLMjSRTPEVTCVWDVSQE DPEVQFNWYVDGVEVHNA^TKPREEQFNSTYRWSVLWLHQDWLNG^EYKC^VSN^GLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYRTTPPVLDSDGSFFLYSRLTVSKSRWQEGN
VFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 7328)
T-119
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVPAGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL
SCAASGRTFSDYGMGWFRQAPGKEHEFVASSNWSNARTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYCA GTLSGKAYTYWGQGTQVTVPAGGGGSPKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLIVjlSRTPEVTCWVDVSHED PEVKFNWY¥DG¥EVHNAKTKPREEQYNST¥RWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTBKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGF¥PSDIAVEWESNGQPENN¥ICrFPPVLDSDGSFFLYSKLT¥DKSRWQQGN¥ FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7329)
T-120
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC
AGTLSGKAYTYVy'GQGTQVTVPAGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHKHYTQKSLSLSLGK (SEQ ID NO: 7330)
T-121
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKNTGYLQMNSLRVEDTAVYYC AGTLSGKAYTYWGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHE DPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLWLHQDWLNGKEYKCKVSNKALAAPiEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7331)
T-122
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASiNWSNARTSYADSAKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAGTLSGKAYTYWGQGTQVTVAGGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTiSRDNAKNTGYLQMNSLRVEDTAVYYC AGTLSGKAYTYWGQGTQVTVAGGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMSSRTPEVTCWVDVSQ
EDPEVQFNWYVDGVEVHNAKTRPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
NVFSCSVMREALHNHYTQKSLSLSLGK fSEQ ID NO: 7332)
T-030 (tetravalent igG4 SPLE [N/C term modifications, CDR2 G/A integrin binding site mutation, CDR3
T/S ALK-2 mutation); T-007hul CDR3 T/S)
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAK
NTGYLQMNSLRVEDTAVYYCAASPSGiCAYSYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSL
RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKNTGYLQMNSLRVEDTAVY
YCAASPSGKAYSYWGQGTQVTVPAGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMiSRTPEVTCVWDV
SQEDPEVQFNWYVTCVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSSEKTISKAKGQ PREPQWTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE5NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGR fSEQ ID NO: 10372)
T-031 (tetravalent IgGl LALAPA [N/C term modifications, CDR2 G/A integrin binding site mutation, CDR3 T/S ALK-2 mutation]; T-007hul CDR3 T/S)
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAK NTGYLQMNSLRVEDTAVYYCAASPSGKAYSYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSL
RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAiNWSNARTNYADSVKGRFTiSRDNAKNTGYLQMNSLRVEDTAVY YCAASPSGKAYSYWGQGTQ¥TVPAGGGGSDKTHTCPPCPAPEAAGGPSyFLFPPKPICDTLMISRTPEVT£VWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLWLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQ¥SLTCL¥KGF¥PSDIA¥EWESNGQPENNYKTTPPVLDSDGSFFLYSKLiyDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10373)
T-032
DVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTIFSYSSNTYYADSVKGRFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLLESGGGLVQPGG SLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTIFSYSSNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVYY CAADSDLSTVTVGPHDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCV
WDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISR AKGQPREPQ¥¥TLPPSQEEMTKNQ¥SLTCLVKGF¥PSDIA¥£WESNGQPENN¥KTTPP¥LDSDGSFFLYSRLT¥DKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKfSEQ ID NO: 10376)
T-033
DVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTIFSYSSNTYYADSVKGRFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSDVQLLESGGGLVQPGG SLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTIFSYSSNIYYADSVKGRFTjSTDSAKNTLYLQMNSLRPEDTAVYY
CAADSDLS'TVTVGPMDYVYGQGTQ.VTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMSSRTPEVTCW VD¥SHEDPEVKFNWY¥DGVE¥HNAKTKPREEQYNST¥RWSVLTVLHQDWLNGKE¥KCKVSNKALAAPIERTISKA KGQPREPQVYTLPPSREEMTK^QVSLTaVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLWDICSR WQQG WFSCSVMHEALMNNYTQKSLSLSPGK (SEQ ID NO: 10377)
T-034
DVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTIFSYSSNTYYADSVKGRFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQELESGGGLVQPG GSLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTIFSYSSNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVY YCAADSDLSTVTVGPHDYWGQGTQVTVPAGGGGSESKYSPPCPPCPAPEFEGGPSVFLFPPKPKDTLMjSRTPEVTC
VWDVSQEDPEVQFNWYVDGVEVHNAKTICPREEQFNSTYRWSVLTVLHQDWLNGKEYKC^VSNKGLPSSIEKTIS KARGQPREPQVYTLPPSQEEMTRNQVSLTaVKGFYPSDIAVEWESNGQPENNYKTFPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMFIEAEHNHYTQKSLSLSLGK (SEQ ID NO: 10378)
T-035
DVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTIFSYSSNTYYADSVKGRFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPG GSLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTIFSYSSNTYYADSVKGRFTISTDSAKNTLYLQMNSLRPEDTAVY YCAADSDISTVTVGPHDYWGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV
WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY^STYRWSVLTVLHQDWLNGICEYICaCVSNICALAAPIEKTISK AKGQPREPQVYTLPPSREEMTIKNQVSLTCLVKGFYPSDIAVEWESNGQPENWKTTPPVLDSDGSFFLYSKITVDRS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 10379) Example 22. Surface plasmon resonance binding affinities of addittonal anti-CD25 V-bodies [1027]For generation of sensorgrams displayed in Figures 65-69, affinity purified V-bodies were covalently crosslinked onto an ISA HC30M chip using EDC/Sulfo NHS. The Interaction with human, cynomolgus, and mouse CD25 (extracellular domain) (V-body coupling concentration: 1 pM) was measured under physiological conditions (Running Buffer: HBST- 50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 % (w/v) BSA, 0.05% (v/v) Tween20, 25°C) using 8 different antigen concentrations of human CD25 (3- fold serial dilutions, starting from 500 nM), 8 different antigen concentrations of cynomolgus CD25 (3- fold serial dilutions, starting from 500 nM) and 8 different antigen concentrations of mouse CD25 (3-fold serial dilutions, starting from 500 nM). Resulting sensorgrams were analyzed and equilibrium-binding affinities (KDs) were calculated using Carterra's data analysis software. For data processing, high or low Ag concentration curves were excluded based on affinity or curve fit.
Table 10. Sequence identifiers for tested humanized anti-CD25 VHH antibodies
Example 23. Surface plasmon resonance binding affinities of additional anti-TNFR2 V-bodses of duster D
[1028]Binding affinities of the V-bodies to human or cynomolgus TNFR2 was determined by surface plasmon resonance (SPR) using a Caterra LSA instrument, A schematic diagram depicting the experimental setup of the present Example is shown in Figure 8, Affinity purified V-bodies were covalently crosslinked onto an LSA HC30M or HC200M chip using EDC/Sulfo NHS. The interaction with human and cynomolgus TNFR2 (extracellular domain) (V-body coupling concentration: human, 1 pM; cynomolgus, 3-4 pM) was measured under physiological conditions (Running Buffer: HBST- 50 mM HEPES pH 7,4, 150 mM NaCI, 0.1 % (w/v) BSA, 0.05% (v/v) Tween20, 25°Q using twelve different antigen concentrations {2-fold serial dilutions, start at 1000 nM), Resulting sensorgrams (Figures 70A-7GF) were analyzed and equilibrium-binding affinities (KDS) were calculated using Carterra's data analysis software. [1029]Amino acid sequence of an exemplary fusion protein construct from cluster D is provided below. In the sequence, VHH sequences (T-009HulmSGRmN/C) are indicated with a straight underline (e.g,, VHH), the Fc region is indicated with bold letters (e.g., hlgGl LALAPA), N-terminal modification are indicated with both bold and a straight underline (e.g., N-term modifieation), C-terminal modification are indicated with both italic and a wavy underline {e.g., modification), linker sequences are indicated with a wavy underline (e.g., Linker), and the hinge region is indicated with both a wavy underline and bold letters (e.g., hinge).
T-036 DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLR LSCAASGRTFSD¥GMGWFRQAPGKEREFVATINWSSGRTT¥ADSVKGRFTISRDNAKNTG¥LQMNSLRVEDTAVY¥C AATPTGKAYTVWGQG'TQ.VTVPAG'GGGSDKTHTCPPCPAPEAAGGPSyFLFPPKPKDTLMISRTPEVTCWVDVSHE DPEVKFNWYVDGVE¥HNAKTKPREEQ¥NST¥RVVSVLWLHQDWLNGKE¥KCKVSNKALAAPIEKTIISKAKGQPRE PQV¥TLPPSREEMTKNQVSLTCLVKGF¥PSDIAVEWESNGQPENN¥KTTPPVLDSDGSFFL¥SKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGIC (SEQ ID NO: 10503)
Example 24. Surface plasmon resonance binding affinities of additional anti- clysters D and K obtained by aiamne scanning mutagenesis
[1030]Binding affinities of the V-bodies listed in Table 11 to human or cynomolgus TNFR2 was determined by surface plasmon resonance (SPR) using a Caterra LSA instrument. A schematic diagram depicting the experimental setup of the present Example is shown in Figure 8. Affinity purified V-bodies were covalently crosslinked onto an LSA HC30M or HC200M chip using EDC/Sulfo NHS. The interaction with human and cynomolgus TNFR2 (extracellular domain) (V-body coupling concentration: human, 1 pM; cynomolgus, 3-4 pM) was measured under physiological conditions (Running Buffer: HBST- 50 mM HEPES pH 7.4, ISO mM NaCI, 0.1 % (w/v) BSA, 0.05% (v/v) Tween20, 25“C) using twelve different antigen concentrations (2-fold serial dilutions, start at 1000 nM). Resulting sensorgrams (Figures 71A-71J) were analyzed and equilibrium-binding affinities (KDS) were calculated using Carterra's data analysis software. For data processing, high or low antigen concentration curves were excluded based on affinity or curve fit.
Table 11. Sequence identifiers for exemplary anti-TNFR2 VHH antibodies obtained by alanine scanning mutagenesis
Example 25. Aianine scanning mutagenesis (anti~CD25 V-bodfesl
[1031]A number of anti-CD25 VHH antibodies were generated by alanine scanning mutagenesis to fine tune the binding affinity with CD25. The list of VHH variants is provided in Tabie 12-1. In the "Antibody IDs" listed In the table, "A#" or "G#" indicates the residue at position in the CDR3 of the parental VHH antibody has been mutated to the amino acid residue alanine or glycine, respectively. "L8" indicates that the 8th position in the CDR3 of the parental VHH antibody has been mutated to the amino acid leucine.
Table 12-1. Sequence identifiers for exemplary anti-CD25 VHH antibodies obtained by alanine scanning mutagenesis
[10B2] Binds ng of His-tagged VHHs to CD25+ HEK cells was analyzed via flow cytometry (Figures 74A- 74D). Figures 74A-74B show monovalent VHH binding without anti-His antibody pre-incubation. To increase binding sensitivity, His-tagged VHHs were pre-incubated with anti-His antibody at a 2:1 ratio to form complexes of 2 VHHs to 1 anti-His antibody. Figures 74C-74D show bivalent VHH binding with anti- His antibody/VHH complex pre-incubation. Mutations of CDR3 residues in CD25-specific VHH decreases cell binding to CD25-J- cells. For example, A17 and A14 mutations result in decreased binding of ~10-fold and ~100-fold or more, respectively, compared to the parental C-010Hul.L8 VHH. Based on these data, the C-010Hul.L8 VHH with A14 mutation, the C-010Hul.L8 VHH with A17 mutation, and the parental C- 010Hul.L8 VHH were tested in the bispecific antibodies.
[1033]The list of VHH variants corresponding to the tested antibodies, comprising both a V5 tag (GSGKPIPNPLLGLDSTGGS, SEQ ID NO: 10789) and a His-tag (HHHHHH, SEQ ID NQ: 9425), Is provided In Table 12-2.
Table 12-2. Sequence Identifiers for exemplary anti-CD25 VHH antibodies tested
Example 26. Serfage plasmon resonance binding affinities o TMFR2 to Fc-tagged anti-TMFR2 V-bodies
[1034]Binding affinities of human or cynomolgus TNFR2 (extracellular domain) to selected Fc-tagged anti-TNFR2 V-bodies listed in Table 13 was determined by surface plasmon resonance (SPR) using a Caterra LSA instrument. Purified TNFR2 was covalently crosslinked onto an LSA HC30M chip using EDC/Sulfo NHS. The interaction with the Fc-tagged anti-TNFR2 V-bodies (TNFR2 coupling concentration: 75nM) was measured under physiological conditions (Running Buffer: HBST- 50 mM HEPES pH 7.4, 150 mM NaCI, 0.1 % (w/v) BSA, 0.05% (v/v) Tween20, 25”C) using twelve different antigen concentrations of TNFR2 (2-fold serial dilutions, starting from 1000 nM). Resulting sensorgrams (Figures 72A-72B) were analyzed and equilibrium-binding affinities (KEJS) were calculated using Carterra's data analysis software. For data processing, high or low antigen concentration curves were excluded based on affinity or curve fit. Example 27, Binding and agonistic activity of bivalent V-bodies on TNFR2 HEK NF-kB reporter cells
[1035]Binding was characterized by incubation of increasing concentrations of the V-bodies listed in Table 13 on TNFR2 HEK reporter cells, followed by washing and detection by an anti-human IgG secondary antibody. On-cell binding was measured by flow cytometry (Figure 73A).
[1036]Agonistic activity was characterized by measuring reporter activity via luminescence after overnight incubation of TNFR2 HEK NF-kB-luciferase reporter cells with the tested V-bodies (Figure 73B). Data demonstrate that decreased TNFR2 agonism can be engineered by introducing specific mutations in the CDR regions.
Table 13, Sequence identifiers for exemplary anti-TNFIR2 Fc-tagged bivalent V-bodies
[1037]The Fc-tagged bivalent V-bodies listed In Table 13 contain two copies of the respective monomer V-bodies as indicated. Amino acid sequences of exemplary Fc-tagged bivalent B-body constructs described in Table 13 are provided below. In the sequences, VHH sequences are indicated with a straight underline (e.g., VHH), Fc regions are indicated with bold letters (e.g., hlgGl LALAPA), N-terminal modification are indicated with both bold and a straight underline (e.g., N-term modification), C- terminal modification are indicated with both italic and a wavy underline (e.g., G:Certn wrfi/Tcatffin), linker sequences are indicated with a wavy underline (e.g., Linker), and hinge regions are indicated with both a wavy underline and bold letters (e.g., hinge). The monomer V-bodies were modified to replace Glu (E) with Asp (D) at the first position of the amino-terminus and to introduce the amino acid sequence VPAG (SEQ ID NO: 7267) at the carboxy-terminus for preparing the respective fusion protein constructs. The amino acid sequences of monomer V-bodies present within the fusion protein constructs are provided as SEQ ID Nos: 10720-10727 in the section titled 'List of Sequences'.
T-022
DY&LLESGGGLy^
VYLQMNSLRPEDTAVYYCVASPTGRAFTYWGQGTQVTVPAGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFNWWDGVEVHNACTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEIMNYRTTPPVLDSDGSFF LYSKLWDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGIC (SEQ ID NO: 10705)
T-023
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTiSRDDAKKT
VYLQMNSLRPEDTAVYYCVASPAGAAFTYWGQGTQVTVPAGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPi<DTLM
ISRTPEVTCVWDVSHEDPEVICFNWYVDGVEVHNACTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKaCVSNKA
LAAPIECTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLWGFYPSDIAyEWESNGQPENNYRTTPPVLDSDGSFF
LYSKLTVDICSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGIC (SEQ ID NO: 10707)
T-024
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTiSRDDAKKT
VYLQMNSLRPEDTAVYYCAASPAGRAFTYWGQGTQVTVPAGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLWLHQDWLNGKEYKCKVSNKA LAAPIECTISKAKGQPREPQVYTLPPSREEMTRNQVSLTCLWGFYPSOIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNNYTQKSLSLSPGK (SEQ ID NO: 10709)
T-025
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTiSRDDAKKT
VYLQMNSLRPEDTAVYYCVAAPAGRAFTYWGQGTQVTVPAGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKOTLM
ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLWLHQDWLNGKEYKCKVSNICA LAAPIECTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAyEWESNGQPENNYRTTPPVLDSDGSFF 1YSRLTVDKSRWQQGNVFSCSVMHEA1HNNYTQKSLSLSPGK (SEQ ID NO: 10711)
T-026
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTSSRDDAKKT
VYLQMNSLRPEDTAVYYCVASAAGRAFTYWGQGTQVTVPAGGGSPKTHTCPPCPAPEAAGGPSVFLFPPjCPRDTLM
ISRTPEVTCVWDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKaCVSNKA LAAPIEKTiSKAKGQPREPQVYTLPPSREEMTKNQVSLTCL¥KGFYPSDiAyEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNyFSCSWHEALHNRYTQKSLSLSPGK (SEQ ID NO: 10713)
T-027
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTiSRDDAKKT
VYLQMNSLRPEDTAVYYCVASPAARAFTYWGQGTQVTVPAGGGSDRTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM
ISRTPEVTCWVDVSHEDPEyKF^WYVDGVEVHNAKTKPREEQY^STYRWSVLWLMQDWLNGKEYKCmNKA LAAPiEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLyKGFYPSDIAyEWESNGQPENNYRTFPPVLDSDGSFF LYSKLTVDKSRWQQGNyFSCSVMHEALHNRYTQKSLSLSPGK (SEQ ID NO: 10715) T-028 DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT VYLQMNSLRPEDTAVYYCVASATGAAFTYWGQGTQVTVPAGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFMWYVDGVEVHNAKTKPREEQYNSTYRWSVETVLHQDWLNGKEYICCKVSNKA LAAPIECTISICAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDiAVEWESNGQPENNYICTTPPVLDSTCSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10717)
T-029 DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT VYLQMNSLRPEDTAVYYCAASATGRAFTYWGQGTQVTVPAGGGSDICTHTCPPCPAPEAAGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LAAPIEKTISICAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP5DIAVEWESNGQPENNYKTTPPVLOSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10719)
Example 28. Optimization of
[1033]Bispecific TNFR2/CD25 antibodies were optimized by fine-tuning the binding activity to TNFR2 and CD25. A number of TNFR2/CD25 antibodies were designed and tested for their ability to induce Treg cell activation and expansion, as well as their binding to TNFR2+ or CD25+ cell lines, Tregs, or monocytes, and their agonistic activities.
[1039]Treg cell activation was measured by incubating isolated Treg cells with anti-CD3 (100 ng/mL) and IL-2 (300 lU/mL) for 5 days in the presence of a TNFR2/CD25 bispecific V-body TC-115 or a TNFR2/control antibody comprising a control VHH with irrelevant specificity in place of the CD25 VHH. Treg cell proliferation was measured indirectly by Ki67 expression and activation by glucocorticoid- induced tumor necrosis factor receptor (GITR) upregulation. The TNFR2/CD25 bispecific V-body has increased potency on Treg cell activation compared to the TNFR2/control antibody (Figure 75).
[1040jTreg cell expansion was evaluated following the injection with a TNFR2/CD25 bispecific V-body TC-115into human TNFR2/human CD25 knock-in mice, compared to a TNFR2/control antibody comprising a control VHH with irrelevant specificity in place of the CD25 VHH. Percent Treg cells (CD4+ FOXP3+) of immune cells (CD45+) in blood was measured 5 days after subcutaneous injection of the antibodies. Injection in human TNFR2/human CD25 double knock-in mice with the TNFR2/CD25 bispecific V-body led to enhanced Treg expansion in vivo compared to the TNFR2/control antibody (Figure 76).
[1041] Binding affinity to TNFR2 was measured by incubating stable TNFR2+ HEK ceils with a dose range of bispecific TNFR2/CD25 antibodies or control antibodies comprising control VHH with irrelevant specificity in place of the TNFR2 and/or the CD25 VHH (Figures 77A-77B). After washing, binding to TNFR2 was detected with secondary anti-human IgG and analyzed by flow cytometry. Reducing the TNFR2 VHH affinity through specific CDR3 mutations decreases the TNFR2/CD25 bispecific antibodies cell binding to TNFR2 and increases the binding EC50 from 0.1 - 1 nM, to 1 - 10 nM, 10 - 100 nM, 100 nM - 1 pM, or above 1 pM. This permits lower binding to TNFR2* cells and increased binding to CD25+ cells.
[1042] Bind mg affinity to CD25 was measured by incubating stable CD25+/low TNFR2 HEK cells with a dose range of bispecific TNFR2/CD25 antibodies or control antibodies comprising control VHH with irrelevant specificity in place of the TNFR2 and/or the CD25 VHH (Figures 78A-78C). After washing, binding to CD25 was detected with secondary anti-human IgG and analyzed by flow cytometry. Reducing the CD25 VHH affinity through specific CDR3 mutations allows fine-tuning of TNFR2/CD25 bispecific antibodies cell binding to CD25 by increasing binding EC50 from 1 - 10 nM, to 10 - 100 nM, 100 nM - 1 pM, or above 1 pM. This permits engineering of desired avidity/binding to CD25+ target cells.
[1043]NF-kB luciferase reporter activity was measured in TNFR2+ HEK cells upon 16-20 hours of incubation with a dose range of bispecific TNFR2/CD25 antibodies or control antibodies comprising control VHH with irrelevant specificity in place of the TNFR2 and/or the CD25 VHH (Figures 79A-7SB ). Agonistic activity of TNFR2/CD25 bispecific antibodies (potency and Ema)!) can be fine-tuned by decreasing affinity for TNFR2 through CDR3 mutations. This increases the EC50 from < 0.01 nM, to an ECso of < 0.1 nM, an ECM of < 1 nM, an EC50 of < 10 nM, an EC50 of < 100 nM, or an EC50 of above 100 nM. Decreasing TNFR2 agonistic activity permits engineering of increased dependency on CD25 expression to achieve TNFR2 agonism.
[1044] The binding of bispecific TNFR2/CD25 antibodies to Treg cells or monocytes was measured by incubating bispecific TNFR2/CD25 antibodies or control antibodies comprising control VHH with irrelevant specificity in place of the TNFR2 and/or the CD25 VHH with Treg cells (CD4+ FOXP3+) or monocytes (CD14*) from human PBMC (Figures BQA-B0B). After washing, binding was detected by anti- IgG secondary antibody and analyzed by flow cytometry. Reducing avidity for TNFR2 allows targeting of bispecific TNFR2/CD25 antibodies selectively to the desired CD25+ TNFR2-r Treg cell target population vs. TNFR2+ monocytes. Engineering avidity for CD25 allows decreasing or increasing binding of bispecific TNFR2/CD25 antibodies to Treg cells.
[1045]Overall, the data suggest that TNFR2/CD25 bispecific V-bodies with low avidity for TNFR2 can bind to Treg cells, and induce Treg expansion and activation in blood m vivo. Both effects are increased by CD25 anchoring. Moreover, reducing the affinity to TNFR2 allows binding to be driven by the CD25 anchor to CD25+ TNFR2+ Treg cel Is, while reducing binding to monocytes.
[1046]Amino acid sequences of exemplary bispecific TNFR2/CD25 antibodies tested in this Example are provided below. In the sequences, VHH sequences are indicated with a straight underline (e.g., VHH), Fc regions are indicated with bold letters (e.g., hlgGl LALAPA), N-terminal modification are indicated with both bold and a straight underline (e.g., N-term modification), C-terminal modification are indicated with both italic and a wavy underline (e.g,, C-term modificatJQii], linker sequences are indicated with a wavy underline (e.g., Linker), and hinge regions are indicated with both a wavy underline and bold letters (e.g., hinge).
TC-115
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTiSRDDAKKT VYLQMNSLRPEDTAVYYCVASAAGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL SQ^SGRTFGH^
SAAGRAFTYWGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPiCOTLMiSRTPEVTCWVDVSHEDP EWFNWYVDGVEVHNAKTSCPRFEQYNSTYRWSVLTVLHQDWLNGREYKCKVSNRALAAPOTISKARGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGiCGGGSDVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVS GIYSDSSGTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCVKGRGSGSYYPFDDYWGQGTQVTVPAG ISEQ ID NO: 10792)
TC-115
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASiRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASPAGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASiRWTGGSTSYADSVKGRnjSRDDAKKTVYLQMNSLRPEDTAVYYCVA
SPAGRAFTYWGQGTQVTVPAGGGGSPiCTHTCPPCPAPEAAGGPSVFLFPPKPKOTLMiSRTPEVTCWVDVSHESP
EViCFNWYVDGVEVHNAKTiCPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTiSKAiCGQPREPQ
WTLPPSREEMTICNQVSLTCLVICGFYPSDiAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVIVjHEALHNH¥Ta^SLSLSPGKGGGGSDVQLLESGGGLVQPGGSLRL.SCAASGFTLDYYAIGWFRQAPGKEREGVL
SISSTDGRTYYADSVKGRFTISRDNPKNTVYLQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPLFLDEYDYWGQGTQ
VTVPAG (SEQ SD NO: 10793)
TC-117
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCAASATGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASiRWTGGSTSYADSVKGRFTiSRDDAKKTVYLQMNSLRPEDTAVYYCAA
SATGRAFTYWGQGTQVTVPAGGGGSPKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMiSRTPEVTCWVDVSHEDP
EVKFNWYVDGVEVHNAkTKPREEQYNSTYRWSVLTVLHQDWLNGKEYICCKVSNKALAAPiEKTiSKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSESGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQgSLSLSPGiCGGGGSDVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVL SSSSTDGRTYYADSVKGRFTSSRDNPKNTVYLQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPLFLDEYDYWGQGTQ
VTVPAG (SEQ jD NO: 10794)
TC-118
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASSRWTGGSTSYADSVKGRFTiSRDOAKKT
WLQMNSLRPEPTAVYYCAASPAGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTSSRDOAKICTVYLQMNSLRPEDTAVYYCAA
SPAGRAFTYWGQGTQVTVPAGGGGSPKTHTCPPCPAPEAAGGPSVFLFPPKPRDTLMjSRTPEVTCVWDVSHEDP
E¥KFNWVOG¥EVHfc(AKTKFREEQYNST¥RW5VLT¥LHQDWLNGKE¥KCK¥SNRALAAPOT!SKAKGQPREPQ
VYTLPPSREEMTK^QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVPORWQQGNVFS CSVMHEALHNHYTQjCSLSLSPGKGGGGSDVQLLESGGGLVQPGGSLRLSCAASGFTLDYYASGWFRQAPGKEREGVL ^SST^TYWJSWGR^^
VTVPAG (SEQ !D NO: 10795)
TC-119
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASjRWTGGSTSYADSVKGRFTiSRDPAKKT WLQMNSLRPEPTAVYYCVAAPAGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTjSRDPAKKTVYLQMNSLRPEDTAVYYCVA APAGRAFTYWGQGTQVTVPAGGGGSPKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMjSRTPEVTCWVDVSHEIDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAP^KTiSKAKGQPREPQ
¥YTLPPSRE£MTKNQVSLTCL¥KGFYPSD(A¥EWESNGQPENF(¥KTTPPVLDSOGSFFL¥SKn¥DKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGKGGGGSDVQLLESGGGLVQPGGSLRLSCAASGFTLDYYASGWFRQAPGKEREGVL SISSTDGRTYYADSVKGRFTISRDNPKNTVYLQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPLFLDEYDYWGQGTQ. VTVPAG (SEQ !D NO: 10796)
TC-120
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASjRWTGGSTSYADSVKGRFTjSRDDAKKT
VYLQMNSLRPEDTAVYYCVASAAGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCVA
SAAGRAFTYWGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGP5VFLFPPKPRDTLM(SRTPEVT£VWDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQ¥^STYRWSVLTVLHQDWLNGKEYKCKVS^KALAAPIE((T8SKAKGQPREPQ
¥¥TLPPSREEMTKNQVSETCLVKGFYPSP(AVEWESNGQPENNYKTrPPVLPSDG5FFLYSKLTVDRSRWQQGNVFS
CSVNfflEALHNHYTOKSLSLSPGKGGGGSPVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAjGWFRQAPGKEREGVL
5SSSTDGRTYYADSVKGRFTSSRDNPKNTVYLQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPLFLDEYDYWGQGTQ
VTVPAG (SEQ SD NO: 10797)
TC-121
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTiSRDDAKKT
VYLQMNSLRPEDTAVYYCVASASGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSOVQLLESGGGLVQPGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASjRWTGGSTSYADSVKGRFTjSRDDAKKTVYLQMNSLRPEDTAVYYCVA
SASGRAFTYWGQGTQVTVPAGGGGSPKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMSSRTPEVTCVWDVSHEDP EVRFNWYVDGVEVHNAKTICPREEQYMSTYRWSVLTVLHQDWLNGKEYKCKVSMKALAAPIEKTSSKAKGQPREPQ
¥YTLPPSREEMTK^Q¥SLTa.VKGFYPSO(AVEWESNGQPEN^YRTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSFSPGKGGGGSWQLlESGGGtVQ.PGGSLRLSCAASGFTLDYYAJGVVFRQAPGKEREGVL
SSSSTDGRTYYADSVKGRFTiSRDNPKNTVYLQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPLFLDEYDYWGQGTQ
VTVR4G (SEQ SD NO: 10798)
TC-122
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASjRWTGGSTSYADSVKGRFTiSRDDAKKT
VYLQMNSLRPEDTAVYYCVASPAGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQ.PGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTSSRDDAKICTVYLQMNSLRPEDTAVYYCVA
SPAGRAFTYWGQGTOVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKOTLMjSRTPEVTCWVDVSHEDP
EVRFNWYVDGVEVHNAKTKPREEQY^STYRWSVLTVLHQDWLNGKEYKOCVSNKALAAPIEKKSKAKGQPREPQ
VYnPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYRTTPPVLDSDGSFFLYSKLTVDICSRWQQGWFS
CSVMHEALHNH¥TQKSLSISPGKGGGGSDVQLLESGGGLVQPGGSLRLSCAASGFTLDYYA:GWFRQAPGKEREGVL
SS5STDGRTYYADSVKGRFTSSRDNPKNTVYLQLNSLRPEDTAVYYCAAKRLGPLVHRYSAEVLTPLFLDEYDYWGQGTQ
VTVPAG (SEQ SD NO: 10799)
TC-123
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASjRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCAASATGRAFTYWGQGTQVTVpAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTSSRDDAKKTVYLQMNSLRPEDTAVYYCAA
SATGRAFTYWGQGTQVTVPAGGGGSPnHTCPPCPAPEAAGGPSVFLFPPKPKDTLMjSRTPEVTCVWPVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQY^STYRWSVLTVLHQDWLNGREYKa<VS^KALAAPIEKTISKA§CGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSD^AVEWESNGQPE^NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG^VFS
CSVMHEALHNHYTQKSLSLSPGjCGGGGSDVQLLESGGGLVQPGGSLRLSCAASGFTLDYYASGWFRQAPGKEREGVL
SISSTDGRTYYADSVKGRFTISRDNPKNTVYLQLNSLRPEDTAVYYCAAICRLGPLVHRYSAEVLTPLFLDEYDYWGQGTQ.
VTVPAG (SEQ SD NO: 10800)
TC-124
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFT(SRDDAKKT
VYLQMNSLRPEDTAVYYCAASPAGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSOVQLLESGGGLVQPGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCAA
SPAGRAFTYWGQGTQVTVPAGGGGSPKTHTCPPCPAPEAAGGPSVFLFPPKPKDTEMiSRTPEVTCWVDVSHEDP
EVKFNWY¥OG¥EyH^AKTKPREEQYNSTYRWS¥LTVLHQDWLNGKEYKCKVSNKALAAP(EKTSSKAKGQPREPQ
VYTlPPSREEMTKNQ¥SETCLVRGFYPSD(AVEWESNGQPEN^YRTTPPVLDSPGSFFLYSKLTVORSRWQQGNyFS
CSVIVjHEALHNHYTaKSLSLSPGKGGGGSDVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVL
SjSSTDGRTYYADSVKGRFTSSRDNPKNTWLQLNSLRPEDTAVYYCAAKRLGPLVHRYSAEVLTPLFLDEYDYWGQGTQ
VTVPAG (SEQ !D NO: 10801)
TC-125 DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASjRWTGGSTSYADSVKGRFTjSRDDAKKT
VYLQMNSLRPEDTAVYYCVAAPAGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSPVQLLESGGGLVQPGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCVA
APAGRAFTYWGQGTQVTVPAGGGGSpjCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMSSRTPEVTCVWDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYMSTYRWSVLTVLHQDWLNGREYKaCVSNKALAAPIE^SKAKGQPREPQ
VYTLPPSREEMTK^QVSETCLViCGFYPSO^AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDRSRWQQGNVFS
CSVIVjHEALHNHYTQKSLSLSPGSCGGGGSDVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVL
SSSSTDGRTYYADSVKGRFTSSRDNPKNTVYLQLNSLRPEDTAVYYCAAKRLGPLVHRYSAEVLTPLFLDEYDYWGQGTQ
VTVPAG (SEO SD NO: 10802)
TC-126
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTiSRDDAKKT
VYLQMNSLRPEDTAVYYCVASAAGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASiRWTGGSTSYADSVKGRFTiSRDDAKKTVYLQMNSLRPEDTAVYYCVA
SAAGRAFTYWGQGTQVTVPAGGGGSPKTHTCPPCPAPEAAGGPSVFLFPPKPj(DTLMgSRTPEVTCWVDVSHEDP
EV^FNWWDGVEVHNAKTKPREEQYMSTYRWSVLTVLHQDWLNGKEYKCKVSMKALAAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYmPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQRSLSLSPGKGGGGSPVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVL
SSSSTDGRTYYADSVKGRFTSSRDNPKNTVYLQLNSLRPEDTAVYYCAAKRLGPLVHRYSAEVLTPLFLDEYDYWGQGTQ
VTVPAG (SEQ JD NO: 10803)
TC-127
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTiSRDDAKKT
VYLQMNSLRPEDTAVYYCVASASGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTSSRDDAKKTVYLQMNSLRPEDTAVYYCVA
SASGRAFTYWGQGTQVTV'pAGGGGSPKTHTCPPCPAPEAAgGPSVFLFPPKPKDTLMjSRTPEVTCVWDVSHEDP
E¥KF^WVDGVE¥HR§AKTKPREEQ¥^ST¥RWSVLT¥LMQOWL^GKEYKCKVS^KALAAPOTSSKAKGQPREPQ
V¥TLPPSREEMTK^QVSLTCL¥KGFYPSD§AVEWESNGQPEfMNYKTTPPVLDSDGSFFL¥SKLTVPK5RWQQSM¥FS
CSVMHEALHNHYTQKSLSLSPGKGGGGSDVQLLESGGGLVQPGGSLRLSCAASGFTLDYYASGWFRQAPGKEREGVL
SISSTDGRTYYADSVKGRFTISRDNPKNTVYLQLNSLRPEDTAVYYCAAKRLGPLVHRYSAEVLTPLFLDEYDYWGQGTQ
VTVPAG LSEQ SD NO: 10804)
TC-123
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTiSRDDAKKT VYLQMNSLRPEDTAVYYCVASPAGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSPVQLLESGGGLVQPGGSLRL SCAASGRTFGSYTMGWFRQAPGKEREFLASjRWTGGSTSYADSVKGRFTSSRDDAKKTVYLQMNSLRPEDTAVYYCVA SPAGRAFTYWGQGTQVTVpAGGGGSpjCTHTCPPCPAPEAAGGPSVFLFPPSCP^OTLMjSRTPEVTCVWDVSHEDP EVKFNWYVDGVEVMIMAKTKPREEQY^STYRWSVL'WLHQDWLNGiCEYKaCVS^iCALAAPIEKTSSICAKGQPREPQ VYTLPPSREEMTK^QVSLTCLVKGFYPSDIAVEWESNGQPE^NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVSVIHEALHNHYTOKSLSISPGKGGGGS^VQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVL SSSSTDGRTYYADSVKGRFTjSRDNPKNTVYLQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVATPLFLDEYDYWGQGTQ
VTVPAG (SEO jD NO: 10805) TC-129
DVQLLESGGGLVQPGGSLRLSCAA.SGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCAASATGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCAA
SATGRAFTYWGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMSSRTPEVTCVWDVSHEDP
EVKFNWYVDGVEVHNAKnCPREEQYMSTYRWSVLTVLHQDWLNGKEYKaCVSMKALAAPIEICTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAyEWESNGQPEN^YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGKGGGGSDVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVL
SSSSTDGRTYYADSVKGRFTISRDNPKNTVYLQ.LNSLRPEDTAVYYCAAKRLGPLVHRYSLEVATPLFLDEYDYWGQGTQ
VTVPAG t'SEQ ID NO: 10806)
TC-130
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASSRWTGGSTSYADSVKGRFTISRDDAKKT VYLQMNSLRPEDTAVYYCAASPAGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQ.PGGSLRL SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCAA
SPAGf^FTWGQGTQyWPAG&G^SD^TCPPXPAPEAAGGPSVFLFPPKPKDTLMSSRTPEWCVWDVSHEDP
EWFNWYVDGVEVH^AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPOTiSKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVICGFYPSDIAVEWESNGQPE^NYKTTPPVLDSDGSFFLYSKLTVDICSRWQQGNVFS £SVMHEALHNH¥TQI<SLSLSPGKGGGGSDVQLLESGGGLVQPGGSLRLSCAASGFTLDYYASGWFRQAPGKEREGVL SISSTDGRTYYADSVKGRFTISRDNPKNTVYLQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVATPLFLDEYDYWGQGTQ VTVPAG (SEQ ID NO: 10807)
TC-131
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTiSRDDAKKT VYLQMNSLRPEDTAVYYCVAAPAGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASiRWTGGSTSYADSVKGRRISRDDAKKTVYLQMNSLRPEDTAVYYCVA APAGRAFTYWGQGTQVTVPAGGGGSDjCTHTCPPCPAPEAAGGPSVFLFPPICPKDTLMISRTPEVTCVWDVSHEDP EVKFNW¥VDGVEVHR§AKTKPR£EQ¥NST¥RWSVLTVLHQDWL^GKEYKCRVS^RALAAP^EKTiSKAKGQPREPQ VYTLPPSREEIMTK^QVSLTCLVKGFYPSDIAVEWESNGQPEN^YRTTPPVLDSDGSFFLYSKLTVDKSRWQQGNyFS CSVMHEALH^HYTOKSLSLSPGKGGGGSDVQLLESGGGLVQPGGSLRLSCAASGFTLDYYASGWFRQAPGKEREGVL SjSSTDGRTYYADSVKGRFTjSRDNPKNTVYLQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVATPLFLDEYDYWGQGTQ
VTVPAG (SEQ ID NO: 10808)
TC-132
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASAAGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL
SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKTVYLQMNSLRPEDTAVYYCVA
SAAGRAFTYWGQGTQVTVPAGGGGSDKJHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDP
EVKFNWYVDGVEVHMAKTKPREEQY^STYRWSVLTVLHQDWLNGKEYICaCVSMKALAAPIEKTSSKAKGQPREPQ
VYTLPPSREEMTK^QVSLTCLVKGFYPSDIAVEWESNGQPEIM^YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVIVjHEALHNHYTaKSLSLSPGgCGGGGSDVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVL SISSTDGRTYYADSVKGRFTISRDNPKNTVYLQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVATPLFLDEYDYWGQGTQ
VTVPAG (SEQ ID NO: 10809)
TC-133
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASSRWTGGSTSYADSVKGRFTISRDDAKKT VYLQMNSLRPEDTAVYYCVASASGRAFTYWGQGTQVTVPAGGGGSGGGGSGGGGSDVQLLESGGGLVQPGGSLRL SCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKICTVYLQMNSLRPEDTAVYYCVA SASGRAFTYWGQGTQVTVPAGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMSSRTPE¥TCVWDVSHEIbP
EWFNWYVDGVEVHhIAKTKFREEQYNSTYRWSVLTVEHQDWLNGKEYKCkVSNkALAAPOTISKAKGQPREPQ VYTLPPSREEMTKIMQVSLTCLVKGFYPSDiAVEWESNGQPENNYK'n'PPVLDSDGSFFLYSKLTVDORWQQGNVFS CSVMHEALHNHYTQKSLSLSPGKGGGGSDVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVL
* * *
[1047]The present invention is not to be limited in scope by the specific embodiments described herein, indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fail within the scope of the appended claims.
[1048]AII patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.
List of Sequences
SEQ ID NO: 1 T-001. CDR1
GSIFRADA
SEQ ID NO: 2 T-OO1/ T-002. CDR2
IRSDGFT
SEQ ID NO: 3 T-OO1..CDR3
YYQSLSSPNYGQVF
SEQ ID NO: 4 T-OOlmfull-iength VHH (amino acid sequence)
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT
VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSS
SEQ ID NO: 5 T-002. CDR1
GSIVSTNG
SEQ ID NO: 6 T-002mCDR3
YYQALSSPNYGQTF
SEQ ID NO: 7 T-002_full-iength VHH (amino acid sequence)
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT
VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSS
SEQ ID NO: 8 T-003...CDR1
GFTFDDIA
SEQ ID NO: 9 T-003. CDR2
IYSYGPNT
SEQ ID NO: 10 T-OO3J2DR3; T-004.CDR3
AADSDLSTWVGPHDY
SEQ ID NO: 11 T-003__full-length VHH (amino acid sequence)
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL
YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSS
SEQ ID NO: 12 T-OO6_CDR1
GFTFSRYA
SEQ ID NO: 13 T-006_CDR2
ISDDGSDT
SEQ ID NO: 14 T-006„CDR3
AKDAGSWGTGPFGYEYDY SEQ ID NO: 15 T-006_full"length VHH (amino acid sequence)
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKN
TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSS
SEQ ID NO: 18 T-007.. CDR3
AATPSGKAYSY
SEQ ID NO: 19 T-007_full-length VHH (amino acid sequence)
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK
NTGYLEM NSLKVEDTAVYYCAATPSG KAYSYWGQGTQVTVSS
SEQ ID NO: 20 T-011...CDR1 GLTLDYYA
SEQ ID NO: 21 T-011„CDR2 ISTSDGST
SEQ ID NO: 22 T-011_ CDR3 ATPGPYTYCAPYGSSWSRGYDY
SEQ !D NO: 23 T-011_full-length VHH (amino acid sequence)
EVQLVESGGGLVQPGGSLRLSCAASGLTLDYYAIGWFRQAPGKEREGVSaSTSDGSTYYTDSVKGRFTISRDNAKNTVY
LQMNSLKPEDTAVYYCATPGPYTYCAPYGSSWSRGYDYWGQGTQVTVSS
SEQ ID NO: 24 T-012.. CDR1
GFTFSMYS
SEQ ID NO: 25 T-012„CDR2 iDTRGST
SEQ ID NO: 26 T-012„CDR3
ARVGGAPYEYNY
SEQ ID NO: 27 T-012_fulMength VHH (amino acid sequence)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSMYSMSWVRQAPGKGPEWVSAIDTRGSTRYADSVKGRFTISRDNAKNT
LYLQMDSLKPEDTALYYCARVGGAPYEYNYRGQGTQVTVSS
SEQ ID NO: 28 T-O13„CDR1
GFNFSMYS
SEQ ID NO: 29 T-013„CDR2 iDTGGST SEQ ID NO: 30 T-013_CDR3
ARVRGTPYEYGY
SEQ ID NO: 31 T-O13_full-length VHH (amino acid sequence)
EVQLVESGGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNAKNT
LYLQMDSLKPEDTALYYCARVRGTPYEYGYRGQGTQVTVSS
SEQ ID NO: 32 T-O14„CDR1
GRTFGSYT
SEQ ID NO: 33 T-014„CDR2
IRRTGGST
SEQ ID NO: 34 T-014 CDR3
AAAPTGRAFTY
SEQ ID NO: 35 T-014_fulMength VHH (amino acid sequence)
EVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRFTISRDNAKKAV
YLQM NSLKPEDTAVYYCAAAPTG RAFTYWGQGTQVTVSS
SEQ ID NO: 36 T-015J2DR1
GRTFSSLF
SEQ ID NO: 38 T-Q15.. CDR3
AAAPTGRAFNY
SEQ ID NO: 39 T-015_fulMength VHH (amino acid sequence)
EVQLVESGGGLVQAGGSLRLSCLASGRTFSSLFMGWFRQAPGKEREFVASIRYPGLITNYADSAKGRFIISRDSAKNTVY
LQM DSLKPEDTG LYSCAAAPTG RAFNYWGLGTQVTVSS
SEQ ID NO: 40 T-O17„CDR1
GASLSRNA
SEQ ID NO: 41 T-017JZDR2
IYDDGET
SEQ ID NO: 42 T-017_ CDR3
AGSAFDF
SEQ ID NO: 43 T-017_fulMength VHH (amino acid sequence)
EVQLVESGGGSVQPGGSLRLLCAVSGASLSRNAIIWVRQTPEKGLEWVSTIYDDGEIYYRDSVKGRFTISRDLAENTVHL
QMGNLQAEDTAVYYCAGSAFDFWGRGTQVTVSS SEQ ID NO: 44 T-O18„CDR1
GSTFRFPP
SEQ ID NO: 45 T-018_ CDR2
LTSGGST
SEQ ID NO: 46 T-018_CDR3
SVLGRDMMTY
SEQ ID NO: 47 T-018_full-length VHH (amino acid sequence)
EVQLVESGGGLVQAGGSLRLSCAASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT
WYLQMSSLRPEDTAVYYCSVLGRDMMTYWGQGTQVTVSS
SEQ ID NO: 48 T-001.full-length VHH (DNA sequence)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGAGTCTCCTGTGCAGCCTC
TGGAAGCATCTTCAGGGCCGATGCCATGGGCTGGCACCGCCAGGTTCCAGGGAAGCCGCGCGAGTTTGTCGCGG
GTATTCGTAGTGATGGATTTACCAACTATGCGGAGGCCGTGAAGGGCCGATTCACCATCTCCTGGGATACCGTCAA
GAACACGGTGTATCTGCAGATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATACTTGTTATTATCAATCCCTC
AGTAGTCCTAATTACGGTCAAGTCTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 49 T-002_full-length VHH (DNA sequence)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGAAGCATCGTCAGTACGAATGGCATGGGATGGCACCGCCAGGTTCCAGGGAAGGGCCGCGAGTTGGTCGCAG
GTATTCGTAGTGATGGATTTACAAACTATGCGGACTCCGTGAAGGGCCGATT'CACCATCTCCAGCGATAACGTCAA
GAACACGGTGTATCTGCAGATGAACAGCCTGAAAGCTGAGGACTCAGGCGTCTATfTTTGTTATTATCAAGCCCTC
AGTTCTCCTAATTACGGCCAAACCTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 50 T-003_ful!-iength VHH (DNA sequence)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGTTCTCTGAGACTCTCCTGTGCAGCCTCT
GGATTCACTTn'GATGATATTGCCATGACCTGGGTCCGACAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCCAGT
ATTTATAGTTACGGGCCAAACACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCACAGACAGCGCCA
AGAACACACTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTGCAGCAGATTCAG
ACCTAAGTACAGTAGTAGTTGGTCCCCATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 51 T-006„fuli-iength VHH (DNA sequence)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGCGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACCTTCAGTCGCTATGCCATGAGCTGGGCCCGCCAGGCTCCAGGAAAGGGGCTCGAATGGGTGTCCGG
TATTTCTGATGATGGCAGTGACACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC
AAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCACTGTATTACTGTGCAAAAGACGCG
GGGAGTTGGGGTACGGGTCCCTTTGGCTATGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 52 T-007_fuli-!ength VHH (DNA sequence)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTC
TGGACGCACCTTTAGTGACTATGGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGATAGTGAGTTTGTAGCGGC
GATTAACTGGAGTAATGGTCGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGC
CAAGAACACGGGGTATCTGGAAATGAACAGCCTGAAAGTTGAGGACACGGCCGTTTATTACTGTGCAGCAACCCC
CTCCGGAAAGGCGTATAGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA SEQ ID NO: 53 T-Oll_ful!-!ength VHH (DNA sequence)
GAAGTCCAATTAGTAGAGTCTGGTGGCGGTCTGGTCCAGCCTGGCGGTTCTTTGCGCCTCAGCTGCGCCGCATCC GGTTTAACCCTGGATTATTATGCAATAGGATGGTTTCGTCAAGCTCCGGGCAAAGAGCGGGAAGGCGTATCATGT
ATTTCAACATCCGATGGGTCTACTTACTACACCGACAGCGTTAAGGGACGCTTCACAATCTCGCGTGATAACGCTA AAAATACAGTTTATCTTCAGATGAATAGTCTGAAACCCGAAGACACTGCGGTGTACTATTGCGCGACGCCTGGCCC ATATACTTACTGTGCCCCGTATGGAAGCTCATGGAGTAGAGGTTATGATTATTGGGGGCAGGGGACGCAGGTGAC
CGTTTCGAGT
SEQ ID NO: 54 T-012 Juli-length VHH (DNA sequence)
GAAGTTCAGTTGGTCGAGTCGGGTGGGGGATTAGTACAGCCAGGGGGCTCTCTCCGCCTGTCATGTGCAGCATCG GGCTTTACTnTAGCATGTATAGTATGAGCTGGGTCCGACAGGCCCCCGGTAAAGGCCCGGAGTGGGTGTCTGCC ATTGATACACGTGGATCCACTCGGTATGCTGACAGTGTGAAAGGCCGTTTCACCATCTCCAGAGACAATGCAAAAA
ACACGTTATACCTTCAGATGGATAGCCTGAAGCCTGAAGATACCGCGCTGTACTATTGCGCTCGTGTTGGTGGAGC GCCGTATGAATACAATTATCGCGGTCAAGGTACACAAGTAACCGTTTCATCT
SEQ ID NO: 55 T-013 _ful!-iength VHH (DNA sequence)
GAAGTCCAACTGGTAGAGAGCGGTGGCGGACTGGTGCAACCGGGTGGTAGTTTACGCTTGTCTTGCGCAGCTTCC
GGATrCAATTTTTCTATGTATTCAATGTCATGGGTGCGGCAGGCGCCAGGCAAAGGGCCTGAATGGGTTTCGGCC
ATTGATACTGGCGGGTCTACACGTTATGCGGACAGTGTCAAAGGTCGTTn'ACCATCAGCAGAGACAACGCTAAG AATACACTCTATCTTCAGATGGATAGCTTAAAACCCGAGGATACCGCACTGTACTATTGTGCCCGTGTTCGAGGCA CGCCGTACGAATATGGATACCGCGGTCAGGGCACTCAGGTTACCGTATCCTCG
SEQ ID NO: 56 T-014 „ful!-iength VHH (DNA sequence)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGGAGGCTGGGGGTTCTCTGAGACTCTCCTGTGCAGCCTC TGGACGCACCTTCGGGAGCTATACCATGGGCTGGTTCCGCCAGGCTCCAGGAAGGGAGCAAGAGTTTTTAGCGA GTATTAGGCGGACTGGTGGTAGCACAAGTTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACG
CCAAGAAGGCGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGCCC CCACCGGGAGAGCGTTTACCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 57 T-O15„full-!ength VHH (DNA sequence)
GAGGTCCAATTGGTTGAATCTGGCGGTGGTn'AGTACAGGCAGGTGGATCCCTGAGACTCTCGTGTTTGGCCAGC GGCCGGACTnTAGTTCCCTGTTCATGGGGTGGTTTCGCCAGGCCCCCGGGAAGGAACGAGAGTTTGTCGCGAGT ATACGTTATCCTGGTCTGATTACAAATTACGCCGATAGCGCGAAAGGCCGTTn'ATCATTTCACGTGATTCTGCAAA
AAATACGGTGTATCTTCAGATGGACTCGCTTAAACCGGAAGATACTGGTCTGTACTCTTGCGCAGCGGCTCCAACC GGCCGCGCTTTCAACTATTGGGGATTAGGAACCCAAGTTACAGTGTCAAGC
SEQ ID NO: 58 T-017_ful!-length VHH (DNA sequence)
GAAGTGCAGCTGGTTGAGAGCGGTGGTGGATCGGTGCAGCCTGGCGGCTCTTTACGATTACTCTGCGCCGTTTCC
GGAGCGAGCTTGTCTCGCAATGCAATCATTTGGGTGAGACAGACACCGGAAAAGGGTCTGGAGTGGGTCAGTAC GATCTATGATGACGGCGAGACATAnATCGCGATTCAGTTAAAGGGCGTTTTACCATTTCCCGTGATCTTGCAGAA AATACGGTTCATCTGCAAATGGGGAACCTGCAGGCGGAAGACACTGCCGTATACTACTGTGCTGGTAGTGCTTTC
GATTTTTGGGGACGGGGCACCCAAGTCACTGTATCAAGC
SEQ ID NO: 59 T-018„ful!-!ength VHH (DNA sequence)
GAAGTTCAACTGGTGGAAAGTGGCGGTGGCTTGGTTCAGGCTGGGGGAAGTCTGCGTTTATCGTGTGCCGCTTCT
GGATCCACGTTTCGTTTTCCGCCTATGGGTTGGTATCGACAGGCACCCGGGAAGCAGAGAGAACAAGTCGCGCAG CTCACGTCAGGTGGTAGCACCAACTATGCCGACTCTGTGAAAGGCCGGTTCACTATTTCCCGTGATAATGCGAAAA
ATACATGGTATCTTCAAATGTCTTCATTACGCCCAGAGGATACTGCAGTGTACTATTGCAGCGTCCTGGGCCGCGA
TATGATGACATACTGGGGGCAGGGTACCCAGGTTACCGTATCGAGC
SEQ ID NO: 60 anti-TNFR2 CDR3 consensus (Y/F)YQ(S/A)LS(T/S)(P/A)N(Y/F)GQ(V/T)F
SEQ ID NO: 61 anti-TNFR2 CDR3 consensus AADSDL(S/R)TV(V/T) )(V/T)VGPHDY
SEQ ID NO: 62 anti-TNFR2 CDR3 consensus AKDAG(S/G)WG(T/R)GPFG(Y/F)(E/D)YDY
SEQ ID NO: 63 anti-TNFR2 CDR3 consensus AA(T/A)PSGKAY(T/S)Y
SEQ ID NO: 64 anti-TNFR2 CDR3 consensus
ATPGPY(T/S/M)YCAPYGSSWSRGYDY
SEQ ID NO: 65 anti-TNFR2 CDR3 consensus
ARV(R/G)G(T/S/A)PY(E/D)Y(N/G)Y
SEQ ID NO: 66 anti-TNFR2 CDR3 consensus (T/A/V)A(S/A)PTGRAF(T/N/A)Y
SEQ ID NO: 67 anti-TNFR2 CDR3 consensus
S(V/M)(V/L)GRDM(M/V)TY anti-TNFR2 CDR1 consensus
GSI(V/F)(R/S)(T/A)(N/D)(S/G/A)
SEQ ID NO: 69 anti-TNFR2 CDR1 consensus GFT(F/L)DD(I/Y)A
SEQ ID NO: 70 anti-TNFR2 CDR1 consensus GFTFS(S/R/G)YA
SEQ ID NO: 71 anti-TNFR2 CDR1 consensus
G(L/F)TLDY¥A
SEQ ID NO: 72 anti-TNFR2 CDR1 consensus GF(T/N)FSMYS
SEQ ID NO: 73 anti-TNFR2 CDR1 consensus
GRTF(G/R/S)(N/S)(Y/L)(T/F)
SEQ ID NO: 74 anti-TNFR2 CDR1 consensus GS(I/T)FRFPP
SEQ ID NO: 75 anti-TNFR2 CDR2 consensus
IRSDGF(T/I)
SEQ ID NO: 76 anti-TNFR2 CDR2 consensus l(Y/F)SY(S/G)(S/P)NT
SEQ ID NO: 77 anti-TNFR2 CDR2 consensus l(Y/S)(S/D)DGS(E/D)T
SEQ ID NO: 78 anti-TNFR2 CDR2 consensus l(S/N)(V/T)(S/G)DGST
SEQ ID NO: 79 anti-TNFR2 CDR2 consensus
IDT(R/G)GST
SEQ ID NO: 80 anti-TNFR2 CDR2 consensus
IR(W/R/Y)(T/P)G(G/L)(S/I)T
SEQ ID NO: 81 T-001_Humanized VHH
EVQLVESGGGLVQPGGSLRVSCAASGSIFRADAMGWHRQAPGKPREFVAGIRSDGFTNYADSVKGRFTISWDTVKNT
VYLQMNSLRPEDTAVYYCYYQSLSSPNYGQVFWGQGTQVTVSS
SEQ ID NO: 82 T-002„Humanized VHH
EVQLVESGGGLVQPGGSLRLSCAASGSIVSTNGMGWHRQAPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT
VYLQMNSLRAEDTGVYYCYYQALSSPNYGQTFWGQGTQVTVSS
SEQ ID NO: 83 T-003_Humanized VHH
EVQLLESGGGLVQPGGSLRLSCAASG FTFDDI AMTWVRQAPG KG IE WVSSIYSYG PNTYYADSVKG RFTISTDSAKNTL
YLQMNSLRPEDTAVYYCAADSDLSTVVVGPHDYWGQGTQVTVSS
SEQ ID NO: 84 T-006_Humanized VHH
EVQLLESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSD7YYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSS
SEQ ID NO: 85 T-007„Humanized VHH
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 86 T-011„Humanized VHH
EVQLLESGGGLVQPGGSLRLSCAASGLTLDYYAiGWFRQAPGKEREGVSCISTSDGSTYYADSVKGRFTISRDNAKNTVY
LQMNSLRPEDTAVYYCATPGPYTYCAPYGSSWSRGYDYWGQGTQVTVSS
SEQ ID NO: 87 T-012„Humanized VHH
EVQLLESGGGLVQ.PGGSLRLSCAASGFTFSMYSMSWVRQAPGKGPEWVSAIDTRGSTRYADSVi<GRFTISRDNAKNTL
YLQMNSLRPEDTAVYYCARVGGAPYEYNYRGQGTQVTVSS SEQ ID NO: 88 T-013 ^Humanized VHH
EVQLLESGGGLVQPGGSLRLSCAASGFNFSMYSMSWVRQAPGKGPEWVSAIDTGGSTRYADSVKGRFTISRDNAKNT
LYLQMNSLRPEDTAVYYCARVRGTPYEYGYRGQGTQVTVSS
SEQ ID NO: 89 T-014_Humanlzed VHH
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEQEFLASIRRTGGSTSYADSVKGRFTISRDNAKKTV YLQMNSLRPEDTAVYYCAAAPTGRAFTYWGQGTQVTVSS
SEQ ID NO: 90 T-015_Humanized VHH
EVQLLESGGGLVQPGGSLRLSCAASGRTFSSLFMGWFRQAPGKEREFVASIRYPGLITNYADSAKGRFTISRDSAKNTVY LQMNSLRPEDTGVYYCAAAPTGRAFNYWGQGTQVTVSS
SEQ ID NO: 91 T-017„Humanized VHH
EVQLVESGGGLVQPGGSLRLSCAVSGASLSRNAliWVRQAPGKGLEWVSTlYDDGETYYADSVKGRFTISRDLAKNTVYL QMNSLRAEDTAVYYCAGSAFDFWGQGTQVTVSS
SEQ ID NO: 92 T-018_ Humanized VHH
EVQLVESGGGLVQPGGSLRLSCAASGSTFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT WYLQM NSLRP EDTAVYYCSVLG RDM MTYWGQGTQVTVSS
SEQ ID NO: 3934 T-002~2xVHH-Fc (v2)
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS
LRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNTVYLQMNSLKAEDSGVYFC YYQAESSPNYGQTFWGQGTQ,VTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCWVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ.PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVM H EALH N H YTQKSLSLSLG K
SEQ ID NO: 3936 T-002mVHH-Fc-VHH (v2)
EVQLVESGGGLVQAGGSLRLSCAASGSIVSTNGMGWHRQVPGKGRELVAGIRSDGFTNYADSVKGRFTISSDNVKNT VYLQMNSLKAEDSGVYFCYYQALSSPNYGQTFWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPK
DTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAAS GSIVSTNGMGWHRQVPGKGRELVAGSRSDGFTNYADSVKGRFTISSDNVKNTVYLQMNSLKAEDSGVYFCYYQALSSP NYGQTFWGQGTQVTVSS
SEQ ID NO: 3940 T-OOS _2xVHH-Fc (v2)
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLA QPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPE
DTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLGSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYIOTPPVLDSDGSFFLYSRLTV DKSRWQEG N VFSCSVM H EALHN HYTQKSLSLSLG K SEQ ID NO: 3942 T-006„VHH-Fc-VHH (v2)
EVQLVESGGGLAQPGGSLRLSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCAKDAGSWGTGPFGYEYDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEY KCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVQLVESGGGLAQPGGSLR LSCAASGFTFSRYAMSWARQAPGKGLEWVSGISDDGSDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCA KDAGSWGTGPFGYEYDYWGQGTQVTVSS
SEQ ID NO: 3946 T-001„2xVHH-Fc (v2)
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGS LRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFnSWOTVKNTVYLQMNSLKPEDTAVYT CYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO: 3948 T-001 _VHH-Fc-VHH (v2)
EVQLVESGGGLVQAGGSLRVSCAASGSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNT VYLQMNSLKPEDTAVYTCYYQSLSSPNYGQVFWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPK DTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVQLVESGGGLVQAGGSLRVSCAAS GSIFRADAMGWHRQVPGKPREFVAGIRSDGFTNYAEAVKGRFTISWDTVKNTVYLQMNSLKPEDTAVYTCYYQSLSSP NYGQVFWGQGTQVTVSS
SEQ iD NO: 3952 T~007...2xVHH-Fc (v2)
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSL RLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLEMNSLKVEDTAVYY CAATPSGKAYSYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VM H EALH N H YTQKSLSLSLG K
SEQ ID NO: 3954 T-0G7 _VHH-Fc-VHH (v2)
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK NTGYLEMNSLKVEDTAVYYCAATPSGKAYSYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNK GLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASG RTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKNTGYLEMNSLKVEDTAVYYCAATPSGK AYSYWGQGTQVTVSS
SEQ ID NO: 3958 T-014 _VHH-Fc-VHH (v2) EVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRFTISRDNAKKAV YLQMNSLKPEDTAVYYCAAAPTGRAFTYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTL MiSRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGL GSSIEKTiSKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVQLVESGGGLVEAGGSLRLSCAASGRT FGSYTMGWFRQAPGREQEFLASIRRTGGSTSYADSVKGRFTISRDNAKKAVYLQMNSLKPEDTAVYYCAAAPTGRAFT
YWGQGTQVTVSS
SEQ ID NO: 3960 T-003„2xVHH-Fc (v2)
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGG SLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLKPEDTAVYY CAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCW VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE
GNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO: 3962 T-003mVHH-Fc-VHH (v2)
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSSGGGGSESKYGPPCPPCPAPEFEGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS'TYRWSVLTVLHQDWLNGKEYKCKVS NKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTFPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAA SGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTLYLQMNSLKPEDTAVYYCAADSDLS
TVWGPHDYWGQGTQVTVSS
SEQ ID NO: 4101 C-001, CDR1 sequence, amino acid sequence GRKFSTLI
SEQ ID NO: 4102 C-001, CDR2 sequence, amino acid sequence
IERDGTT
SEQ ID NO: 4103 C-001, CDR3 sequence, amino acid sequence
NALQY
SEQ ID NO: 4104 C-001, full-length VHH, non-humanized VHH, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRKFSTLIMAWYRQAPGKQRELVATIERDGTTTYADSVEGRFFISRDNAKNTVT LQMNNLEPEDSA7YYCNALQYWGQGTQVTVSS
SEQ ID NO: 4105 C-002, CDR1 sequence; C-002 Group B, CDR1 consensus, amino acid sequence GRSFSTLI
SEQ ID NO: 4106 C-002, CDR2 sequence, amino acid sequence
IERDGTP
SEQ ID NO: 4107 C-002, CDR3sequence, amino acid sequence NALRF SEQ ID NO: 4108 C-002, full-length VHH, non-humanized VHH, amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGRSFSTLIMAWYRQAPGEQRELVATIERDGTPTYTDSVKGRFFISRDNAKNTVT
LQMNNLKPEDTAIYYCNALRFWGQGTQVTVSS
SEQ ID NO: 4109 C-003, CDR1 sequence, amino acid sequence GRRFSTLI
SEQ ID NO: 4110 C-003, CDR2 sequence, amino acid sequence
IERGGTP
SEQ ID NO: 4111 C-003, CDR3 sequence, amino acid sequence
KTLRY
SEQ !D NO: 4112 C-003, full-length VHH, non-humanized VHH, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRRFSTLiMGWYRQAPGKQRELVATiERGGTPTYADSVKGRFilSRDNAKNTVT
LQMNNLKPDDTAIYYCKTLRYWGQGTQVTVSS
SEQ ID NO: 4113 C-004, CDR1 sequence, amino acid sequence
GFTFSNYA
SEQ ID NO: 4114 C-004_Group D, CDR2 consensus, CDR2 sequence, amino acid sequence IYSDGSGT
SEQ ID NO: 4115 C-004, CDR3 sequence, amino acid sequence
AKGRNSGSYYPWDDY
SEQ ID NO: 4116 C-004, full-length VHH, non-humanized VHH, amino acid sequence
EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTiSRDNAKN
TVYLQMNSLKPEDTALYYCAKGRNSGSYYPWDDYWGQGTQVTVSS
SEQ ID NO: 4117 C-006, CDR1 sequence, amino acid sequence
GRTFSWNG
SEQ ID NO: 4118 C-006_Group E, CDR2 consensus, CDR2 sequence, amino acid sequence iSQSGGRT
SEQ ID NO: 4119 C-006, CDR3 sequence, amino add sequence
AASDFLLATTISAYDY
SEQ ID NO: 4120 C-006, full-length VHH, non-humanized VHH, amino acid sequence
EVQLVESGGGLVQAGGSLSVSCAASGRTFSWNGMGWFRQAPGEEREFVAAISQSGGRTRYSDSVKGRFTISRDNAKN
MVYLQMNSLKPEDTAVYYCAASDFLLA'TTISAYDYWGQGTQVTVSS
SEQ ID NO: 4121 C-001, full-length VHH DNA, nucleotide sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGACGAAAATTCAGTACCCTTATTATGGCCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAC TATTGAGAGGGATGGTACGACAACCTATGCAGACTCCGTGGAGGGCCGATTCTTCATCTCCAGAGACAACGCCAA
GAACACGGTGACTCTGCAAATGAACAACCTGGAACCTGAGGACTCAGCCACCTA'n'ACTGTAATGCCCTCCAATAC
TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 4122 C'-002j full-length VHH DMA, nucleotide sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGCAGAAGCTTCAGTACCCTTATTATGGCCTGGTACCGCCAGGCTCCAGGGGAGCAGCGCGAGTTGGTCGCGAC
TATTGAGAGGGACGGTACGCCAACCTATACAGACTCCGTGAAGGGCCGATTCTTCATCTCCAGAGACAACGCCAA
GAACACGGTGACTCTGCAAATGAACAACCTGAAACCTGAGGACACAGCCATCTATTACTGTAATGCCCTCCGGTTC
TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 4123 C-003, full-length VHH DNA, nucleotide sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGCAGAAGGTTCAGTACCCTTATTATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAC
TATTGAGAGGGGTGGTACGCCAACCTATGCAGACTCCGTGAAGGGCCGATTTATCATCTCCAGAGACAACGCCAA
GAACACGGTGACTCTGCAAATGAACAACCTGAAACCTGATGACACAGCCATCTATTACTGTAAGACCCTCCGGTAC
TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 4124 C-004, full-length VHH DNA, nucleotide sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGCGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACCTTCAGTAACTATGCCATGAGCTGGGCCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTGTCCGG
TATTTATAGTGATGGTAGTGGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC
AAGAATACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCACTGTATTACTGTGCAAAAGGGAG
GAATAGTGGTAGTTACTATCCCTGGGATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 4125 C-006, full-length VHH DMA, nucleotide sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCGCTGAGCGTCTCCTGTGCAGCCTC
TGGACGCACCTTCAGTTGGAATGGTATGGGCTGGTTCCGCCAGGCTCCAGGAGAGGAGCGTGAGTn'GTAGCAG
CTATTAGTCAAAGTGGTGGGCGTACAAGGTACTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACG
CCAAGAATATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCCTCAGA
TTTCCTGTTAGCGACTACTATATCTGCATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 4126 C-001, humanized VHH, amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGRKFSTLIMAWYRQAPGKQRELVATIERDGTTTYADSVKGRFTiSRDNAKNTVY
LQMNSLRPEDTAVYYCNALQYWGQGTQVTVSS
SEQ ID NO: 4127 C-002, humanized VHH, amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGRSFSTLiMAWYRQAPGKQRELVATIERDGTPTYADSVKGRFTISRDNAKNIVY
LQMNSLRPEDTAVYYCNALRFWGQGTQVTVSS
SEQ ID NO: 4128 C-003, humanized VHH, amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGRRFSTLIMGWYRQAPGKQRELVATIERGGTPTYADSVKGRFTISRDNAKNTVY
EQMNSLRPEDTAVYYCKTLRYWGQGTQVTVSS
SEQ ID NO: 4129 C-004, humanized VHH, amino acid sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCAKGRNSGSYYPWDDYWGQGTQVTVSS SEQ ID NO: 4130 C-006, humanized VHH, amino acid sequence
EVQLLESGGGLVQPGGSLRVSCAASGRTFSWNGMGWFRQAPGKEREFVAAISQSGGRTRYADSVKGRFTISRDNAKN
TVYLQMNSLRPEDTAVYYCAASDFLLATTISAYDYWGQGTQVTVSS
SEQ ID NO: 4131 C-001 _Group A, CDR3 consensus, amino acid sequence NAL(G/L/P/Q/W)Y
SEQ ID NO: 4132 C-001_Group A, CDR1 consensus, amino acid sequence GR(S/K)FSTLI
SEQ ID NO: 4133 C-001_Group A, CDR2 consensus, amino acid sequence
(l/V)(D/E)R(D/G)GT(A/P/T)
SEQ ID NO: 4134 C-002_Group B, CDR3 consensus, amino add sequence NALR(D/H/N/F)
SEQ ID NO: 4135 C-002__Group B, CDR2 consensus, amino add sequence l(D/E)RDGT(T/P)
SEQ ID NO: 4136 C-003_Group C, CDR3 consensus, amino add sequence (K/S/T)TLRY
SEQ ID NO: 4137 C-003_Group C, CDR1 consensus, amino add sequence GR(K/R/S)FSTLi
SEQ ID NO: 4138 C-003j3roup C, CDR2 consensus, amino add sequence i(D/E)R(D/G)(D/G)T{P/T)
SEQ ID NO: 4139 C-004_Group D, CDR3 consensus, amino acid sequence
AKGR(H/N)SGS¥YPWD(D/E)Y
SEQ ID NO: 4140 C-004_Group D, CDR1 consensus, amino acid sequence GFTFS(N/S)YA
SEQ ID NO: 4141 C-006mGroup E, CDR3 consensus, amino add sequence
AA(S/T)(D/N/Y)FL(l/L)ATnS(A/G)YDY
SEQ ID NO: 4142 C-006mGroup E, CDR1 consensus, amino add sequence
GRTFS(S/W)(F/N/Y)G
SEQ ID NO: 6633 T-020_ CDRl; T-004.CDR1; T-004.VTV„CDRl
GFTLDDYA
SEQ ID NO: 6634 T-020_ CDR2; T-004...CDR2: T-004.VTV„CDR2
IFSYSSNT SEQ ID NO: 6635 T-020„CDR3
AVGDFEGELVLKGDY
SEQ ID NO: 6636 T-O2O_fulMength VHH (amino add sequence)
EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTiFSYSSNTYYADSVKGRFTiSTDNAKNT
LYLQM NSLKPEDTAVYYCAVG DFEG ELVLKG DYWGQGTQVTVSS
SEQ ID NO: 6637 T-O21___CDR1 GFTLDYYT
SEQ ID NO: 6638 T-021 __CDR2 ISSNDGSV
SEQ ID NO: 6639 T-021......CDR3 AADLGYLYVDYVRLHTHHFGS
SEQ ID NO: 6640 T-021___full-length VHH (amino add sequence)
EVQLVESGGGLVQSGGSLRLSCAASGFTLDYYTIGWFRQAPGKEREGVSYISSNDGSVYYADSVKGRFTIEKDSAKNTVY
LQMNSLKPEDTAVYYCAADLGYLYVDYVRLHTHHFGSWGQGTQVTVSS
SEQ ID NO: 6641 T-016 CDR1 GRTFGSYT
SEQ ID NO: 6642 T-016 CDR2 IRWTGGST
SEQ ID NO: 6643 T-016„CDR3 VASPTGRAFTY
SEQ ID NO: 6644 T-016_ful!~iength VHH (amino add sequence)
EVQLVESGGGLVEAGGSLRLSCAASGRTFGSYTMGWFRQAPGREREFLASIRWTGGSTSYADSVKGRFTISRDDAKKA
VYLQMNSLKPEDTAVYYCVASPTGRAFTYWGQGTQVTVSS
SEQ ID NO: 6645 T-020„fuil-!ength VHH (DNA sequence)
GAGGTGCAGCTTGTAGAGAGTGGCGGTGGACTGGTCCAGCCGGGTGGATCGTTGCGTCTCTCATGTGCTGCGTCT
GGTTTTACGCTGGATGATTATGCCATGTCATGGGTGCGCCAAGCTCCTGGTAAAGGCrTGGAATGGGTGAGCACT
ATTTTTAGCTATrCCAGTAATACCTATTACGCGGACTCTGTTAAGGGGCGGTTTACAATCTCCACTGATAACGCCAA
AAATACCTTATACCTGCAGATGAACTCGCTTAAACCAGAAGATACCGCAGTTTACTATTGCGCAGTTGGGGACTTC
GAAGGTGAACTGGTCTTAAAAGGCGATTATTGGGGACAGGGCACACAAGTAACGGTTAGTAGC
SEQ ID NO: 6646 T-021„full-!ength VHH (DNA sequence)
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTTTGGTGCAGTCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC TGGATTCACTTTGGATTATTATACCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATA TATTAGTAGTAACGATGGTAGTGTGTACTATGCAGACTCCGTGAAGGGCCGATTCACCATCGAGAAAGACAGTGC
CAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTACTGTGCAGCAGATCT CGGTTATCTGTACGTCGACTATGTCCGTCTTCACACGCATCACTTTGGTTCCTGGGGCCAGGGGACCCAGGTCACC GTCTCCTCA SEQ ID NO: 6647 T-016_full-!ength VHH {DNA sequence)
GAGGTACAGCTGGTGGAGAGTGGGGGCGGCTTAGTTGAAGCGGGCGGTAGTCTGCGACTCTCTTGCGCCGCATC
CGGGCGGACTTnGGTAGCTATACGATGGGCTGGTTCCGCCAAGCCCCTGGACGTGAACGGGAATTTTTGGCATC
GATTCGCTGGACAGGTGGTAGCACATCTTACGCTGACTCAGTCAAAGGAAGATTCACAATCTCACGTGATGATGC
GAAAAAGGCTGTATATCTTCAGATGAATAGCCTGAAACCAGAAGATACTGCAGTCTACTATTGTGTTGCGTCGCCG
ACGGGGCGTGCCTTTACCTATTGGGGCCAAGGTACCCAGGTGACCGTTTCCTCT
SEQ ID NO: 6648 T-020_Humanized VHH
EVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTIFSYSSNTYYADSVKGRFTISTDNAKNTL
YLQMNSLRPEDTAVYYCAVGDFEGELVLKGDYWGQGTQVTVSS
SEQ ID NO: 6649 T-021_Humanized VHH
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYTIGWFRQAPGKEREGVSYISSNDGSVYYADSVKGRFTISKDSAKNTVY
LQMNSLRPEDTAVYYCAADLGYLYVDYVRLHTHHFGSWGQ.GTQVTVSS
SEQ ID NO: 6650 T-016_ Human!zed VHH
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASiRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASPTGRAFTYWGQGTQVTVSS
SEQ ID NO: 7086 anti-TNFR2 CDR3 consensus
AAD(L/V)G(F/V/Y)L¥{A/T/V)DYV{P/R)LH(M/T)HHFGS
SEQ ID NO: 7087 anti-TNFR2 CDR2 consensus l(N/S)SNDG(S/T)(T/V)
SEQ ID NO: 7088 anti-TNFR2 CDR1 consensus
G(F/V)(S/T)LD(D/Y)(H/Y)T
SEQ ID NO: 7089 T-O19„CDR1
GSIFRFPP
SEQ ID NO: 7090 T-019_full-length VHH {amino add sequence)
EVQLVESGGGLVQAGGSLRLSCAASGSIFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT
WYLQMSSLRPEDTAVYYCSVLGRDMVIYWGQGTQVTVSS
SEQ ID NO: 7091 T-019„full-length VHH {DNA sequence)
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGACTGAGCTGCGCCGCCA
GCGGCAGCATCTTCAGATTCCCCCCCATGGGCTGGTACAGACAGGCCCCCGGCAAGCAGAGAGAGCAGGTGGCC
CAGCTGACCAGCGGCGGCAGCACCAACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAACGC
CAAGAACACCTGGTACCTGCAGATGAACAGCCTGAGACCCGAGGACACCGCCGTGTACTACTGCAGCGTGCTGGG CAGAGACATGGTGACCTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCTGA
SEQ ID NO: 7092 T-019_ Humanized VHH
EVQLVESGGGLVQPGGSLRLSCAASGSIFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT
WYLQM NSLRP EDTAVYYCSVLG RDM VTYWGQGTQVTVSS SEQ ID NO: 7093 T-005hul_CDR3; T~004.VTV„CDR3; T-004Hul.VTV_CDR3
AADSDLSTVTVGPHDY
SEQ ID NO: 7094 T-005hul_full-!ength VHH (DNA sequence)
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGACTGAGCTGCGCCGCCA
GCGGCTTCACCTTCGACGACATCGCCATGACCTGGGTGAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC
AGCATCTACAGCTACGGCCCCAACACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCACCGACAGC
GCCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGACCCGAGGACACCGCCGTGTACTACTGCGCCGCCGAC AGCGACCTGAGCACCGTGACCGTGGGCCCCCACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCTG A
SEQ ID NO: 7095 T-005hul_ Humanized VHH
EVQLLESGGGLVQPGGSLRLSCAASG FTFDDI AMTWVRQAPG KG LE WVSSIYSYG PNTYYADSVKG RFTISTDSAKNTL YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVSS
SEQ ID NO: 7096 T-008hul/ T-008hul.A3_CDR2
INWSNART
SEQ ID NO: 7097 T-008hul_fu!i-iength VHH (DNA sequence)
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGACTGAGCTGCGCCGCCA
GCGGCAGAACCTTCAGCGACTACGGCATGGGCTGGTTCAGACAGGCCCCCGGCAAGGACAGCGAGTTCGTGGCC
GCCATCAACTGGAGCAACGCCAGAACCAACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAA CGCCAAGAACACCGGCTACCTGCAGATGAACAGCCTGAGAGTGGAGGACACCGCCGTGTACTACTGCGCCGCCAC CCCCAGCGGCAAGGCCTACAGCTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCTGA
SEQ ID NO: 7098 T-008hul„Humanized VHH
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 7099 T-008hul.A3_CDR3
AAAPSGKAYSY
SEQ ID NO: 7100 T-008hul.A3„full-iength VHH (DNA sequence)
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGACTGAGCTGCGCCGCCA GCGGCAGAACCTTCAGCGACTACGGCATGGGCTGGTTCAGACAGGCCCCCGGCAAGGACAGCGAGTTCGTGGCC GCCATCAACTGGAGCAACGCCAGAACCAACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAA
CGCCAAGAACACCGGCTACCTGCAGATGAACAGCCTGAGAGTGGAGGACACCGCCGTGTACTACTGCGCCGCCGC CCCCAGCGGCAAGGCCTACAGCTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCTGA
SEQ ID NO: 7101 T-008hul.A3„Humanized VHH
EVQLLESGGGLVQPGGSLRLSCAASG RTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAAAPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 7222 T-003
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSS SEQ ID NO: 7223 T-003Hul
EVQLLESGGGLVQPGGSLRLSCAASG FTFDDI AMTWVRQAPG KG LE WVSSIYSYG PNTYYADSVKG RFTISTDSAKNTL
YLQMNSLRPEDTAVYYCAADSDLSTWVGPHDYWGQGTQVTVSS
SEQ ID NO: 7224 T-003Hul VTV (T-OOSHul)
EVQLLESGGGLVQPGGSLRLSCAASG FTFDDI AMTWVRQAPG KG LE WVSSIYSYG PNTYYADSVKG RFTISTDSAKNTL
YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVSS
SEQ ID NO: 7225 DVQ-T-005hul.VPAG
DVQLLESGGGLVQPGGSLRLSCAASGFTFDDIAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL
YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVPAG
SEQ ID NO: 7226 T-007
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAK
NTGYLEM NSLKVEDTAVYYCAATPSG KAYSYWGQGTQVTVSS
SEQ ID NO: 7227 T-007Hul
EVQLLESGGGLVQPGGSLRLSCAASG RTFSDYGMGWFRQAPGKDSEFVAAINWSNGRTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 7228 T-007Hul NAR (T-008HU1)
EVQLLESGGGLVQPGGSLRLSCAASG RTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 7229 T-008Hul-VPAG
EVQLLESGGGLVQPGGSLRLSCAASG RTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVPAG
SEQ ID NO: 7230 DVQ-T-008Hul-VPAG
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVPAG
SEQ ID NO: 7231 DVQ-T-008hul.A3.VPAG
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAAAPSGKAYSYWGQGTQVTVPAG
SEQ ID NO: 7232 DVQ-T-008hul.A4.VPAG
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATASGKAYSYWGQGTQVTVPAG
SEQ ID NO: 7233 DVQ-T-008hul.A5.VPAG
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQM NSLRVEDTAVYYCAATPAG KAYSYWGQGTQVTVPAG
SEQ ID NO: 7234 DVQ-T-008hul.A6.VPAG
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSAKAYSYWGQGTQVTVPAG SEQ ID NO: 7235 DVQ-T-008hul.A7.VPAG
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGAAYSYWGQGTQVTVPAG
SEQ ID NO: 7236 DVQ-T-008hul.A9.VPAG
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKAASYWGQGTQVTVPAG
SEQ ID NO: 7237 DVQ-T-008hul.A10.VPAG
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKAYAYWGQGTQVTVPAG
SEQ ID NO: 7238 DVQ-T-008hul.All.VPAG
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN TGYLQMNSLRVEDTAVYYCAATPSGKAYSAWGQGTQVTVPAG
SEQ ID NO: 7239 T-007Mu-2*01
EVQLVESGGGLVQPGESLKLSCEASGRTFSDYGMGWFRKTPEKDSEFVAAINWSNGRTNYPDTVERRFIISRDNAKNT
GYLQMSSLRVEDTALYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 7240 T-007Mu-4*01
EVQLVESGGGLVKPGGSLKLSCAASGRTFSDYGMGWFRQTPEKDSEFVAAINWSNGRTNYPDNVKGRFTISRDNAKN
NGYLQMSHLKVEDTAMYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 7241 T-007Mii-6*01
EVQLVESGGDLVKPGGSLKLSCAASGRTFSDYGMGWFRQTPEKDSEFVAAINWSNGRTNYPDSVKGRFTISRDNAKN
TGYLQMSSLKVEDTAMYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 7242 T-007Mii-9*01
EVMLVESGGGLVKPGGSLKLSCAASGRTFSDYGMGWFRQTPEKDSEFVAAINWSNGRTNYPDSVKGRFTISRDNAKN
TGYLQMSSLRVEDTALYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 7243 T-007Mu-12*01
EVKLVESGGGLVQPGGSLKLSCAASGRTFSDYGMGWFRQTPEKDSEFVAAINWSNGRTNYPDTVKGRFTISRDNAKN
TGYLQMSRLKVEDTAMYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 7244 T-007Mu-15*01
EVKLVESGGGLVQPGGSLKLSCAASGRTFSDYGMGWFRQAPRKDSEFVAAINWSNGRTNYADTVTGRFTISRDNAKN
TGYLEMSSLRVEDTAMYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 7245 T-007Mt!-16*01
EVKLVESEGGLVQPGSSLICLSCTASGRTFSDYGMGWFRQVPEKDSEFVAAINWSNGRTNYLDSVKSRFIISRDNAKNIG
YLQMSSLKVEDTATYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 7246 T-007Mu-17*01
EVQLVESGGGLVKPGGSLKLSCAASGRTFSDYGMGWFRQAPEKDSEFVAAINWSNGRTNYADTVKGRFTISRDNAKN TGFLQMTSLRVEDTAMYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 7247 T-007Mu-Consensus
EVQLVESGGGLVQPGGSLKLSCAASGRTFSDYGMGWFRQTPEKDSEFVAAINWSNGRTNYPDTVKGRFTISRDNAKN
TGYLQMSSLRVEDTAMYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 7248 T-007Mu-Graft-IGHV5~9*01
EVMLVESGGGLVKPGGSLKLSCAASGRTFSDYGMSWVRQTPEKRLEWVATINWSNGRTYYPDSVKGRFTISRDNAKN
TLYLQMSSLRSEDTALYYCAATPSGKAYSYWGQGTSVTVSS
SEQ ID NO: 7249 T-019
EVQLVESGGGLVQAGGSLRLSCAASGSIFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT
WYLQMSSLRPEDTAVYYCSVLGRDMVTYWGQGTQVTVSS
SEQ ID NO: 7250 T-019Hul
EVQLVESGGGLVQPGGSLRLSCAASGSIFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT
WYLQM NSLRP EDTAVYYCSVLG RDM VTYWGQGTQVTVSS
SEQ ID NO: 7251 DVQ-T-019hul.VPAG
DVQLVESGGGLVQPGGSLRLSCAASGSIFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT
WYLQM NSLRP EDTAVYYCSVLG RDM VTYWGQGTQVTVPAG
SEQ ID NO: 7252 DVQ-T-019hiil.LV,VPAG
DVQLVESGGGLVQPGGSLRLSCAASGSIFRFPPMGWYRQAPGKC^EQVAQLTSGGSTNYADSVKGRFTISRDNAKNT
WYLQM NSLRP EDTAVYYCSVLG RDLVTYWGQGTQVTVPAG
SEQ ID NO: 7253 DVQ-T-019hul,RV.VPAG
DVQLVESGGGLVQPGGSLRLSCAASGSIFRFPPMGWYRQAPGKC^EQVAQLTSGGSTNYADSVKGRFTISRDNAKNT
WYLQM NSLRP EDTAVYYCSVLG RDRVTYWGQGTQVTVPAG
SEQ ID NO: 7254 DVQ-T-019hul,IV.VPAG
DVQLVESGGGLVQPGGSLRLSCAASGSIFRFPPMGWYRQAPGKQREQVAQLTSGGSTNYADSVKGRFTISRDNAKNT
WYLQMNSLRPEDTAVYYCSVLGRDIVTYWGQGTQVTVPAG
SEQ ID NO: 72S8 anti-TNFR2 cluster D CDR2 consensus
INWSN(G/A)RT
SEQ ID NO: 7270 anti-TNFR2 CDR2 consensus
¥YQ(S/A)LSSPNYGQ(V/T)F
SEQ ID NO: 7271 anti-TNFR2 CDR3 consensus
AADSDLSTVV(V/T)GPHD¥
SEQ ID NO: 7272 anti-TNFR2 CDR3 consensus
AA(T/A)PSGKAYSY
SEQ ID NO: 7273 anti-TNFR2 CDR3 consensus ARV(G/R)G(T/A)PYEY(N/G)Y
SEQ ID NO: 7274 anti-TNFR2 CDR1 consensus GRTF(G/S)S(Y/L)(T/F)
SEQ ID NO: 7275 antl-TNFR2 CDR3 consensus SVLGRDM(M/V)TY
SEQ ID NO: 7276 anti-TNFR2 CDR3 consensus (A/V)A(A/S)PTGRAF(T/N)Y
SEQ ID NO: 7277 anti-TNFR2 CDR1 consensus GS(T/I)FRFPP
SEQ ID NO: 7281 T-009Hul CDR1 GRTFSDYG
SEQ ID NO: 7282 T-009Hul.NAR_.CDRl GRTFSDYG
SEQ ID NO: 7283 T-010Hul_CDRl GRTFSDYG
SEQ ID NO: 7284 T-010Hul.NAR_CDRl GRTFSDYG
SEQ ID NO: 7285 T-009HU1..CDR.2 INWSNGRT
SEQ ID NO: 7286 T-009Hul.NAR_CDR2 INWSNART
SEQ ID NO: 7287 T-010Hul..CDR2 INWSNGRT
SEQ ID NO: 7288 T-010Hul.NAR_.CDR2 INWSNART
SEQ ID NO: 7289 T-009Hul_CDR3 AATPTGKAYTY
SEQ ID NO: 7290 T-009Hul.NAR_.CDR3 AATPTGKAYTY
SEQ ID NO: 7291 T-010Hul_CDR3 AGTLSGKAYTY SEQ ID NO: 7292 T-010Hul.NAR_.CDR3
AGTLSGKAYTY
SEQ ID NO: 7293 T-009Hul_humanized VHH
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVSS
SEQ ID NO: 7294 T-009H ul.NAR_.hu ma nized VHH
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNARTTYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVSS
SEQ ID NO: 7295 T-OlOHulJwmanized VHH
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKN
TG YLQM NSLRVEDTAVYYCAGTLSG KAYTYWGQGTQVTVSS
SEQ ID NO: 7296 T-010Hul.NAR_.humanized VHH
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNARTSYADSAKGRFTISRDNAKN
TGYLQM NSLRVEDTAVYYCAGTLSG KAYTYWGQGTQVTVSS
SEQ ID NO: 7333 anti-TNFR2 CDR3 consensus
A(A/G;(T/Aj- P/L){S/T)GKAY(T/SjY
SEQ ID NO: 7342 C-007_.Group F, CDR1 consensus; amino acid sequence
GFTLDYYA
SEQ ID NO: 7343 C-007, CDR2 sequence, amino acid sequence
ISRDGDST
SEQ ID NO: 7344 C-007, CDR3 sequence, amino acid sequence
AAYVYPDYYCSEYVLLKYDY
SEQ ID NO: 7345 C-008, CDR1 sequence, amino acid sequence
GMPLVA
SEQ ID NO: 7346 C-008, CDR2 sequence, amino acid sequence
ISSGGNT
SEQ ID NO: 7347 C-008, CDR3 sequence, amino acid sequence
NIYRSQVPPTRYS
SEQ ID NO: 7348 C-009, CDR2 sequence, amino acid sequence
ISSTDGRT
SEQ ID NO: 7349 C-009CDR3 sequence, amino acid sequence
AAKRLGPMVHQYSLEVLTPLFLDEYDY
SEQ ID NO: 7350 C-010, CDR3 sequence, amino acid sequence AAKRLGPMVHRYSLEVLTPLFLDEYDY
SEQ ID NO: 7351 C-007, Full-length VHH; C-007, Group F, non-humanized VHH sequence, amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISRDGDSTNYGDSVKGRFTISRDNAKNTV YLQMNSLEPEDTAVYYCAAYVYPDYYCSEYVLLKYDYWGQGTQVTVSS
SEQ ID NO: 7352 C-008, Full-length VHH; C-008, Group G, non-humanized VHH sequence, amino add sequence EVQLVESGGGLVQAGGSLRLSCAASGMPLVAMGWYRQAPGKQRELVASiSSGGNTGYAEFVKGRFTISRDNAKKMV YLQM NSVKPEDTGVYYCN IYRSQVPPTRYSWGQGTQVTVSS
SEQ ID NO: 7353 C-009, Full-length VHH; C-009/C-010, Group H, non-humanized VHH sequence, amino add sequence
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVD
LQLNSLKPEDTALYYCAAKRLGPMVHQYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 7354 C-010, Full-length VHH; C-009/C-010, Group H, non-humanized VHH sequence, amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVD
LQLNSLKPEDTALYYCAAKRLGPMVHRYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 7355 C-007, Full-length VHH DNA, nucleotide sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACTTTGGATTATTACGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTCATG TATTAGTAGAGATGGTGATAGCACAAATTATGGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGC CAAGAACACGGTGTATCTGCAAATGAACAGCCTGGAACCTGAGGACACAGCCGTTTATTACTGTGCAGCCTACGTT TACCCTGATTACTACTGTTCAGAGTATGTCCTGTTAAAATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCT
CCTCA
SEQ ID NO: 7356 C-OOS, Full-length VHH DNA, nucleotide sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGGCTCTCCTGTGCAGCCTC TGGAATGCCCCTCGTTGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAAGTATCAG TAGTGGCGGTAATACAGGCTATGCAGAATTCGTGAAGGGCCGATTCACCATCTCGAGAGACAACGCCAAGAAGAT GGTGTATCTGCAAATGAACAGTGTGAAACCTGAGGACACAGGCGTCTATTATTGTAATATATATCGATCGCAAGTA
CCGCCTACCAGATACTCTTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 7357 C-009, Full-length VHH DNA, nucleotide sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACTTTGGATTATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTTATC
CATTAGTAGTACGGATGGCAGGACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATTTCCAGAGACAACCC CAAGAACACGGTCGATCTGCAATTGAACAGCCTGAAACCTGAGGACACAGCCCTTTATTACTGTGCAGCAAAACG ATTAGGTCCAATGGTTCATCAGTAITCTCTTGAAGTCC'TTACACCACTATTTCTAGATGAGTATGACTACTGGGGCC AGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 7358 C-010, Full-length VHH DNA, nucleotide sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACTTTGGATTATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTCTTATC
CATTAGTAGTACGGATGGCAGGACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATTTCCAGAGACAACCC
CAAGAACACGGTCGATCTGCAATTGAACAGCCTGAAACCTGAGGACACAGCCCTn'ATTACTGTGCAGCAAAACG
ATTAGGTC:CAATGGTTCATCGGTATTCTCTTGAAGTCCTTACACCACTATTTCTAGATGAGTATGACTACTGGGGCC
AGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 7359 C-007, Humanized VHH; C-007, Group F, humanized VHH sequence, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISRDGDSTNYADSVKGRFTISRDNAKNTV
YLQMNSLRPEDTAVYYCAAYVYPDYYCSEYVLLKYDYWGQGTQVTVSS
SEQ ID NO: 7360 C-008, Humanized VHH; C-008, Group G, humanized VHH sequence, amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGMPLVAMGWYRQAPGKQRELVASISSGGNTGYADSVKGRFTISRDNAKKTVY
LQMNSVRPEDTGVYYCNIYRSQVPPTRYSWGQGTQVTVSS
SEQ ID NO: 7361 C-009, Humanized VHH; C-009/C-010, Group H, humanized VHH sequence, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPIMVHQYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 7362 C-010, Humanized VHH; C-009/C-010, Group H, humanized VHH sequence, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPMVHRYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 7363 Group F, anti-CD25 CDR3 consensus, amino acid sequence
AA¥VYPDYYCS(D/E)YVLL(K/R)YDY
SEQ ID NO: 7364 Group F, anti-CD25 CDR2 consensus, amino acid sequence
IS(R/S)(D/S)G(D/G)ST
SEQ ID NO: 7365 Group G, anti-CD25 CDR3 consensus, amino acid sequence
NIYR(P/S)QVP(P/S/T)TRYS
SEQ ID NO: 7366 Group G, anti-CD25 CDR1 consensus, amino acid sequence
G(I/M)P(F/-)(A/-)L(P/V/Y)A, wherein can be absent
SEQ ID NO: 7367 C-010„Group H CDR3 consensus, amino acid sequence
AAKRLGPMVH(Q/R)YSLEVLTPLFLDEYDY
SEQ ID NO: 9411 C-009HulJ8, Group H, CDR3 sequence, amino acid sequence
AAKRLGPIVHQYSLEVLTPLFLDEYDY
SEQ ID NO: 9412 C-010Hul.l8, Group H, CDR3 sequence, amino acid sequence AAKRLGPIVHRYSLEVLTPLFLDEYDY SEQ ID NO: 9413 C-009Hul.A8, Group H, CDR3 sequence, amino acid sequence AAKRLGPAVHQYSLEVLTPLFLDEYDY
SEQ ID NO: 9414 C-010Hul.A8, Group H, CDR3 sequence, ammo acid sequence AAKRLGPAVHRYSLEVLTPLFLDEYDY
SEQ ID NO: 9415 C-009Hul.L8, Group H, CDR3 sequence, amino acid sequence AAKRLGPLVHQYSLEVLTPLFLDEYDY
SEQ ID NO: 9416 C-010Hul.L8, Group H, CDR3 sequence, amino acid sequence AAKRLGPLVHRYSLEVLTPLFLDEYDY
SEQ ID NO: 9417 C-009HulJ8, Group H, Humanized VHH sequence, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY LQLNSLRPEDTAVYYCAAKRLGPIVHQYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 9418 C-010Hul.l8, Group H, Humanized VHH sequence, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSiSSTDGRTYYADSVKGRFTiSRDNPKNTVY LQLNSLRPEDTAVYYCAAKRLGPIVHRYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 9419 C-009Hul.A8, Group H, Humanized VHH sequence, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY LQLNSLRPEDTAVYYCAAKRLGPAVHQYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 9420 C-010Hul.A8, Group H, Humanized VHH sequence, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY LQLNSLRPEDTAVYYCAAKRLGPAVHRYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 9421 C-009Hul,L8, Group H, Humanized VHH sequence, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY LQLNSLRPEDTAVYYCAAKRLGPLVHQYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 9422 C-010Hul.L8, Group H, Humanized VHH sequence, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 9423 C-009„C-010„Group H, CDR3 consensus, amino acid sequence
AAKRLGP(M/I/A/L)VH(Q/R)YSLEVLTPLFLDEYDY
SEQ ID NO: 9435 C-005 CDR2, amino acid sequence
IYSDSSGT
SEQ ID NO: 9436 C-005 CDR3, amino acid sequence
VKGRGSGSYYPFDDY
SEQ ID NO: 9437 C-005 Non-humanized VHH, amino acid sequence EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLKPEDTALYYCVKGRGSGSYYPFDDYWGQGTQVTVSS
SEQ ID NO: 9438 C-005 Non-humanized VHH, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGCGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACCTTCAGTAACTATGCCATGAGCTGGGCCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTGTCCGG
TATTTATAGTGATAGTAGTGGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC
AAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCACTGTATTACTGTGTAAAGGGTCGA GGGAGTGGTAGTTACTACCCCTTTGATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 9439 C-005 humanized VHH, amino acid sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVKGRGSGSYYPFDDYWGQGTQVTVSS
SEQ !D NO: 9440 C-005_Group D CDR3 consensus, amino acid sequence
( A/V) KG R(G/H/N )SGSYYP{W/F) D( D/E) Y
SEQ ID NO: 9441 C-005_Group D CDR2 consensus, amino acid sequence
IYSD(G/S)SGT
SEQ ID NO: 10214 C-OllHul , amino acid sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWTRQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCAKGRNSGSYYPFDDYWGQGTQVTVSS
SEQ ID NO: 10215 C-012Hul, amino add sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTiSRDNAKNT
VYLQMNSLRPEDTAVYYCAKGRNSGSYYPFDDYWGQGTQVTVSS
SEQ ID NO: 10216 C-013Hul, amino add sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTiSRDNAKNT
WLQMNSLRPEDTAVYYCSKGRHSGSYYPWDEYWGQGTQVTVSS
SEQ ID NO: 10217 C-014Hul, amino add sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCSKGARSGSYYPWDDVWGQGTQVTVSS
SEQ ID NO: 10218 C-015Hul, amino add sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCATGKHSGSYYPWDDYWGQGTQVTVSS
SEQ ID NO: 10219 C-005_Group D__CDR3 consensus - broadest, amino acid sequence
(A/V/S)(K/T)G(R/A/K)(G/H/N/R)SG(S/G)YYP(W/F/L)(D/EHD/E)(Y/V)
SEQ ID NO: 10220 C-016Hul, amino add sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCAKGRGSGSYYPLDDYWGQGTQVTVSS SEQ ID NO: 10221 C-017Hul, amino add sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCAKGRGSGGYYPFDDYWGQGTQVTVSS
SEQ ID NO: 10222 C-018Hul, amino add sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCAKGRGSGSYYPFEDYWGQGTQVTVSS
SEQ ID NO: 10223 C-019Hul, amino add sequence
EVQLVESGGGLVQPGGSLRLSCAASGRSFSTLIMAWYRQAPGKQRELVATIERGGTPTYADSVKGRFTISRDNAKNTVY
LQMNSLRPEDTAVYYCNALGYWGQGTQVTVSS
SEQ ID NO: 10224 C-020Hul, amino add sequence
EVQLVESGGGLVQPGGSLRLSCAASGRSFSTLiMAWYRQAPGKQRELVATiERGGTnYADSVKGRFTASRDNAKNTV
YLQM NSLRPEDTAVYYCN ALGYWGQGTQ.VTVSS
SEQ ID NO: 10225 C-021Hul? amino add sequence
EVQLVESGGGLVQPGGSLRLSCAASGRSFSTLiMAWYRQAPGKQRELVATiDRGGTTTYADSVKGRFTISRDNAKNTVY
LQMNSLRPEDTAVYYCNALLYWGQGTQVTVSS
SEQ ID NO: 10226 C-022Hiil? amino add sequence
EVQLVESGGGLVQPGGSLRLSCAASGRSFSTLIMGWYRQAPGKQRELVATIERGGTTTYADSVKGRFTISRDNAKNTVY
LQMNSLRPEDTAVYYCNALLYWGQGTQVTVSS
SEQ ID NO: 10227 C-023Hul, amino add sequence
EVQLVESGGGLVQPGGSLRLSCAASGRKFSTLiMAWYRQAPGKQRELVATVDRGGTTTYADSVKGRFTISRDNAKNTT
YLQMNSLRPEDTAVYYCNALRNWGQGTQVTVSS
SEQ ID NO: 10228 C-024Hul? amino add sequence
EVQLVESGGGLVQPGGSLRLSCAASGRSFSTLIMGWYRQAPGKQRELVATIERGGTTTYADSVKGRFTISRDNAKNTVY
LQMNSLRPEDTAVYYCNALRDWGQGTQVTVSS
SEQ ID NO: 10229 C-025Hul, amino add sequence
EVQLVESGGGLVQPGGSLRLSCAASGRRFSTLIMGWYRQAPGKQRELVATIERGGTPTYADSVKGRFTISRDNAKNTVY
LQMNSLRPEDTAVYYCKTLRYWGQGTQVTVSS
SEQ ID NO: 10230 C-026Hul, amino add sequence
EVQLVESGGGLVQPGGSLRLSCAASGRKFSTLIMGWYRQAPGKQRELVATIERDDTTTYADSVKGRFTISRDNAKNTVY
LQMNSLRPEDTAVYYCKTLRYWGQGTQVTVSS
SEQ ID NO: 10231 C-027HU1, amino add sequence
EVQLVESGGGLVQPGGSLRLSCAASGRKFSTLIMGWYRQAPGKQRELVATIERDDTTTYADSVKGRFTISRDNAKNTVY
LQMNSLRPEDTAVYYCTTLRYWGQGTQVTVSS
SEQ ID NO: 10232 C-028HU1., amino add sequence
EVQLVESGGGLVQPGGSLRLSCAASGRKFSTLIMAWYRQAPGKQRELVATIDRDGTTTYADSVKGRFTISRDNAKNTVY
LQMNSLRPEDTAVYYCKTLRYWGQGTQVTVSS SEQ ID NO: 10233 C-029Hul, amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAVSGRSFSTLIMAWYRQAPGKQRELVATiDRGGTTTYADSVKGRFTISRDNAKNTVY
LQMNSLRPEDTAVYYCKTLRYWGQGTQVTVSS
SEQ ID NO: 10234 C-030Hul, amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGRKFSTLIMAWYRQAPGKQRELVATIDRDGTTTYADSVKGRFTiSRDNAKNTVY
LQMNSLRPEDTAVYYCSTLRYWGQGTQVTVSS
SEQ ID NO: 10235 C-031Hul, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGRTFSSYGMGWFRQAPGKEREFVAAISQSGGRTRYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCAATNFLIATTISAYDYWGQGTQVTVSS
SEQ ID NO: 10236 C-032Hul, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGRTFASFGMGWFRQAPGKEREFVAAISRGGGRTRYADSVKGRFTISRDDAKNT
VYLQMNSLRPEDTAVYYCAASYFLLATTiSGYDYWGQGTQVTVSS
SEQ ID NO: 10237 C-033Hul, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGRTFSSYGMGWFRQAPGKEREFWAISGSGGRTRYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCAASDFLIATSiSAYDYWGQGTQVTVSS
SEQ ID NO: 10238 C-034Hul, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGRTFASFGMGWFRQAPGKEREFVAAISQGGGRTRYADSVKGRFTiSRDDAKNT
WLQMNSLRPEDTAVYYCAASYFLLATTISGYDYWGQGTQVTVSS
SEQ ID NO: 10239 C-035Hul? amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGRTFADFGMGWFRQAPGKEREFVAAISRSGGRTRYADSVKGRFTISRDDAKNT
VYLQMNSLRPEDTAVYYCAASYFLLAITiSGYDYWGQGTQVTVSS
SEQ ID NO: 10240 C-036Hul? amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGRTFSSYGMGWFRQAPGKEREFVAAISQSGGRTRYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCAATNFLIAATiSAYDYWGQGTQVTVSS
SEQ ID NO: 10241 C-037HU1,, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGRTFSSYGMGWFRQAPGKEREFVAAISQSGGRTRYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCAATKFLIATTISAYDYWGQGTQVTVSS
SEQ ID NO: 10242 C-038Hiil, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGRTFSSYGMGWFRQAPGKEREFVAAISQSGGRTRYADSVKGRFTISRDNVKNT
VYLQMNSLRPEDTAVYYCAATNFUATTISAHDYWGQGTQVTVSS
SEQ ID NO: 10243 C-039Hiil? amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGRTFSSYGMGWFRQAPGKEREFVAAiSQSGGRTRYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCAATNVLIA7TISAYDYWGQGTQVTVSS
SEQ ID NO: 10244 C-040Hul, amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGRTFSSYGMGWFRQAPGKEREFVAAiSQSGGRTRYADSVKGRFTiSRDNAKNT VYLQMNSLRPEDTAVYYCAATNFPIATTISAYDYWGQGTQVTVSS
SEQ ID NO: 10245 C-041Hul, amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGRTFSSYGMGWFRQAPGKEREFVAAISQSGGRTRYADSVKGRFTISRDNAKNT
VYLQM NSLRPEDTAVYYCAATN FU ATAiSAYDYWGQGTQVTVSS
SEQ ID NO: 10246 C-OllHul non-humanized counterpart, amino acid sequence
EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAMSWTRQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLKPEDTALYYCAKGRNSGSYYPFDDYWGQGTQVTVSS
SEQ ID NO: 10247 C-012Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGiYSDGSGTYYADSVKGRFTISRDNAKN
TVYLQMNSLKPEDTALYYCAKGRNSGSYYPFDDYWGQGTQVTVSS
SEQ ID NO: 10248 C-013Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSGIYSDGSGTYYADSVKGRFTISRDNAKN TVYLQMNSLKPEDTALYYCSKGRHSGSYYPWDEYWGQGTQVTVSS
SEQ ID NO: 10249 C-014Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDGSGTYYAD5VKGRFTISRDNAKN
TVYLQMSSLKPEDTALYYCSKGARSGSYYPWDDVWGQGTQVTVSS
SEQ ID NO: 10250 C-015Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSGIYSDGSGTYSADSVKGRFTiSRDNAKN
TVYLQMNSLKPDDTALYYCATGKHSGSYYPWDDYWGQGTQVTVSS
SEQ ID NO: 10251 C-016Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLKPEDTALYYCAKGRGSGSYYPLDDYWGQGTQVTVSS
SEQ ID NO: 10252 C-017Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSG7YYADSVKGRFTISRDNAKNT
VYLQMNSLKPEDTALYYCAKGRGSGGYYPFDDYWGQGTQVTVSS
SEQ ID NO: 10253 C-018Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLAQPGGSLRLSCAASGFTFSNYAIMSWARQAPGKGLEWVSGIYSDSSGIYYADSVKGRFTISRDNAKNT
VYLQMNSLKPEDTALYYCAKGRGSGSYYPFEDYWGQGTQVTVSS
SEQ ID NO: 10254 C-019Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRSFSTLiMAWYRQAPGKQRELVATiERGGTPTYADSVEGRFFISRDNAKNTVT
LQMNDLKPEDTATYYCNALGYWGQGTQVTVSS
SEQ ID NO: 10255 C-020Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLGLSCAASGRSFSTLIMAWYRQAPGKQRELVATIERGGTnYADSVKGRFFASRDNAKNTV
TLQM N NLKPDDTAIYYCN ALGYWGQGTQVTVSS SEQ ID NO: 10256 C-021Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRSFSTLIMAWYRQAPGKQRELVATIDRGGTnYADSVEGRFFISRDNAKNTVT
LQMNNLKPDDTALYYCNALLYWGQGTQVTVSS
SEQ ID NO: 10257 C~022Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRSFSTLIMGWYRQAPGKQRELVATIERGGTTTYADSVEGRFFiSRDNAKNTVT LQMNNLKPDDTAIYYCNALLYWGQGTQVTVSS
SEQ ID NO: 10258 C-023Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRKFSTLIMAWYRQAPGKQRELVATVDRGGTTTYSDSVKGRFFISRDNAKNTT
TLQMNNLKPDDTAIYYCNALRNWGQGTQVTVSS
SEQ ID NO: 10259 C-024Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRSFSTLIMGWYRQAPGKQRELVATIERGGTTTYADSVEGRFFISRDNAKNTVT
LQMNNLKPDDTAIYYCNALRDWGQGTQVTVSS
SEQ ID NO: 10260 C-025Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRRFSTLIMGWYRQAPGKQRELVATiERGGTPTYADSVKGRFIISRDNAKNTVT
LQMNNLKPDDTAIYYCKTLRYWGQGTQVTVSS
SEQ ID NO: 10261 C-026Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLS&IASGRKFSTLIMGWRQAPGKQRELVATIERDDTTTYADSVKGRFFISRDNAKNTVT
LQMNNLKPEDTAIYYCKTLRYWGQGTQVTVSS
SEQ ID NO: 10262 C-027Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRKFSTLIMGWYRQAPGKQRELVATIERDDTTTYADSVKGRFFISRDNAKNTVT
LQMNNLKPEDTAiYYCTTLRYWGQGTQVTVSS
SEQ ID NO: 10263 C-028Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRKFSTLIMAWYRQAEGKQRELVATiDRDGTTTYADSVKGRFTISRDNAKNTV
TLQM N NLKPEDTAVYYCKTLRYWGQGTQVTVSS
SEQ ID NO: 10264 C-029Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAVSGRSFSTLIMAWYRQAPGKQRELVATIDRGGTTTVTDSVKGRFFISRDNAKNTVT
LQMNNLKPEDTATYYCKTLRYWGQGTQVTVSS
SEQ ID NO: 10265 C-030Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRKFSTLIMAWYRQAEGKQRELVATIDRDGTTTYADSVKGRFTISRDNAKNTV
TLQM N NLKPEDTAVYYCSTLRYWGQGTQVTVSS
SEQ ID NO: 10266 C-031Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYGMGWFRQAPGEEREFVAAISQSGGRTRYSDSVKGRFTISRDNAKN
MVYLQMNSLKPEDTAVYYCAATNFLIATTISAYDYWGQGTQVTVSS
SEQ ID NO: 10267 C-032Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRTFASFGMGWFRQAiGKEREFVAAISRGGGRTRYSDSVKGRFTISRDDAKNM
VYLQMNSLKPEDTAVYYCAASYFLLATTISGYDYWGQGTQVTVSS SEQ ID NO: 10268 C-033Hul non-humanized counterpart, ammo add sequence
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYGMGWFRQAPGKEREFWAISGSGGRTRFSDSVKGRFTiSRDNAKN
MVYLQMNSLKPEDTAVYYCAASDFLIATSISAYDYWGQGTQVTVSS
SEQ ID NO: 10269 C-034Hul non-humanized counterpart, ammo acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRTFASFGMGWFRQPiGEEREFVAAISQGGGRTRYSDSVKGRFTiSRDDANN
MVYLQMNSLKPEDTAVYYCAASYFLLATTISGYDYWGQGTQ.VTVSS
SEQ ID NO: 10270 C-035Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRTFADFGMGWFRQAIGKEREFVAAISRSGGRTRYSDSVKGRFTISRDDAKNM
VYLQMNSLKPEDTAVYYCAASYFLLAITiSGYDYWGQGTQVTVSS
SEQ ID NO: 10271 C-036Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYGMGWFRQAPGEEREFVAAISQSGGRTRYSDSVKGRFTISRDNAKN
MVYLQMNSLKPEDTAVYYCAATNFLIAATiSAYDYWGQGTQVTVSS
SEQ ID NO: 10272 C-037Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYGMGWFRQAPGEEREFVAAISQSGGRTRYSDSVKGRFTISRDNAKN
MVYLQMNSLKPEDTAVYYCAATKFUATTISAYDYWGQGTQVTVSS
SEQ ID NO: 10273 C-038Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYGMGWFRQAPGEEREFVAAISQSGGRTRYSDSVKGRFTISRDNVKN
MVYLQMNSLKPEDTAVYYCAATNFLIATTISAHDYWGQGTQVTVSS
SEQ ID NO: 10274 C-039Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYGMGWFRQAPGEEREFVAAiSQSGGRTRYSDSVKGRFTISRDNAKN
MVYLQMNSLKPEDTAVYYCAATNVLIATTISAYDYWGQGTQVTVSS
SEQ ID NO: 10275 C-040Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYGMGWFRQAPGEEREFVAAISQSGGRTRYSDSVKGRFTISRDNAKN
MVYLQMNSLKPEDTAVYHCAATNFPIATTISAYDYWGQGTQVTVSS
SEQ ID NO: 10276 C-041Hul non-humanized counterpart, amino acid sequence
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYGMGWFRQAPGEEREFVAAISQSGGRTRYSDSVKGRFTISRDNAKN
MVYLQMNSLKPEDTAVYYCAATNFLIATAISAYDYWGQGTQVTVSS
SEQ ID NO: 10277 C-OllHul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGCGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACCTTCAGTAACTATGCCATGAGCTGGACCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTGTCCGG
TATTTATAGTGATGGTAGTGGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC
AAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCACTGTATTACTGTGCGAAAGGTCGT
AATAGTGGTAGTTACTACCCCTTTGATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10278 C-012Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGGTCTCTGAGATTGTCCTGTGCAGCCTC
TGGATTCACCTTCAGTAACTATGCCATGAGCTGGGCCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTGTCCGG TATTTATAGTGATGGTAGTGGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC
AAGAACACGGTGTATCTGCAAATGAACAGCCTAAAACCTGAGGACACGGCACTGTATTACTGTGCGAAAGGTCGT AATAGTGGTAGTTACTACCCCTTTGATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10279 C-013Hul nan-humanized counterpart, DMA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGCGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACCTTTAGTAACTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTCGAGTGGGTGTCCGG
TATTTATAGTGATGGTAGTGGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCACTGTATTACTGTTCAAAAGGGAGG CATAGTGGTAGTTACTACCCCTGGGATGAGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10280 C-014Hul nan-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGCGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACCTTCAGTAACTATGCCATGAGCTGGGCCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTGTCCGG TATTTATAGTGATGGTAGCGGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGCAAATGAGCAGCCTGAAACCTGAGGACACGGCACTGTATTACTGTTCAAAGGGGGCT
AGAAGTGGTAGTTACTACCCCTGGGATGACGTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10281 C-015Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGCGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACCTTCAGTAACTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTGTCCGG
TAnTATAGTGATGGTAGTGGCACATACTCTGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC
AAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGACGACACGGCACTGTATTACTGTGCAACAGGGAAA CATAGTGGTAG'TTACTACCCCTGGGATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10282 C-016Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGCGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACCTTCAGTAACTATGCCATGAGCTGGGCCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTGTCCGG
TATTTATAGTGATAGTAGTGGCACATACTATGCAGACTCCGTGAAGGGCCGATrCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCACTGTATTACTGTGCAAAGGGTCGA GGGAGTGGTAGTTACTACCCCCTTGATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10283 C-017Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGCGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACCTTCAGTAACTATGCCATGAGCTGGGCCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTGTCCGG
TATTTATAGTGATAGTAGTGGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCACTGTATTACTGTGCAAAGGGTCGA GGGAGTGGTGGTTACTACCCCTTTGATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10284 C-018Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCrFGGCGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGATTCACCTTCAGTAACTATGCCATGAGCTGGGCCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTGTCCGG
TATTTATAGTGATAGTAGTGGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCACTGTATTACTGTGCAAAGGGTCGA GGGAGTGGTAGTTACTACCCCTTTGAGGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10285 C-019Hul non-humanized counterpart, DNA sequence GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGACGAAGCn'CAGTACCCTTATTATGGCCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAC
TATTGAGAGGGGTGGTACGCCAACCTATGCAGACTCCGTGGAGGGCCGATTCTTCATCTCCAGAGACAACGCCAA
GAACACGGTGACTCTGCAAATGAACGACCTGAAACCTGAGGACACAGCCACCTATTACTGTAATGCCCTCGGGTA CTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10286 C-020Hul nan-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGGGACTCTCCTGTGCAGCCTC
TGGCAGAAGCTTCAGTACCCTTATTATGGCCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAC
TATTGAGAGGGGTGGTACGACAACCTATGCAGACTCCGTGAAGGGCCGATTCTTCGCCTCCAGAGACAACGCCAA
GAACACGGTGACTCTGCAAATGAACAACCTGAAACCTGATGACACAGCCATCTATTACTGTAATGCCCTCGGGTAC TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10287 C-021Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGCAGAAGCTTCAGTACCCTTATTATGGCCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAC
TATTGATAGGGGTGGTACGACAACCTATGCAGACTCCGTGGAGGGCCGATTCTTCATCTCCAGAGACAACGCCAA GAACACTGTGACTCTGCAAATGAACAACCTGAAACCTGATGACACAGCCCTCTATTACTGTAATGCCCTCCTGTACT GGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10288 C-022Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGCAGAAGCTTCAGTACCCTTATTATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAC
TATTGAGAGGGGTGGTACAACAACCTATGCAGACTCCGTGGAGGGTCGATTCTTCATCTCCAGAGACAACGCCAA GAACACGGTGACTCTGCAAATGAACAACCTGAAACCTGATGACACAGCCATCTATTACTGTAATGCCCTCCTGTAC TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10289 C-023Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGCAGAAAGTTCAGTACCCTGATTATGGCCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAC
TGTTGATAGGGGTGGTACGACAACCTATTCAGACTCCGTGAAGGGCCGATTCTTCATCTCCAGAGACAACGCCAA GAACACGACGACTCTGCAAATGAACAACCTGAAACCTGATGACACAGCCATCTATTACTGTAATGCCCTCCGGAAC TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10290 C-024Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGCAGAAGCTTCAGTACCCTTATTATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAC
TATTGAGAGGGGTGGTACAACAACCTATGCAGACTCCGTGGAGGGTCGATTCTTCATCTCCAGAGACAACGCCAA GAACACGGTGACTCTGCAAATGAACAACCTGAAACCTGATGACACAGCCATCTATTACTGTAATGCCCTCCGGGAC TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10291 C-025Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGCAGAAGGTTCAGTACCCTTATTATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAC
TATTGAGAGGGGTGGTACGCCAACCTATGCAGACTCCGTGAAGGGCCGATTTATCATCTCCAGAGACAACGCCAA GAACACGGTGACTCTGCAAATGAACAACCTGAAACCTGATGACACAGCCATCTATTACTGTAAGACCCTCCGGTAC TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA SEQ ID NO: 10292 C-026Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCAGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGCAGAAAGTTCAGTACCCTTATTATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAC TATTGAGAGGGATGATACGACAACCTATGCAGACTCCGTGAAGGGCCGATTCTTCATCTCCAGAGACAACGCCAA GAACACGGTGACTCTGCAAATGAACAACCTGAAACCTGAGGACACAGCCATCTATTACTGTAAGACCCTCCGGTAC TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10293 C-027Hul nan-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGCAGAAAGTTCAGTACCCTTATTATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAC TATTGAGAGGGATGATACGACAACCTATGCGGACTCCGTGAAGGGCCGATTCTTCATCTCCAGAGACAACGCCAA GAACACGGTGACTCTGCAAATGAACAACCTGAAACCTGAGGACACAGCCATCTATTACTGTACTACCCTCCGGTAC TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10294 C-028Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGCAGAAAATTCAGTACCCTTATTATGGCCTGGTACCGCCAGGCTGAAGGGAAGCAGCGCGAGTTGGTCGCGAC TATTGATAGGGATGGTACGACAACCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAA GAACACGGTGACTCTGCAAATGAACAACCTGAAACCTGAGGACACAGCCGTTTATTACTGTAAGACCCTCCGGTAC TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10295 C-029Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGTCTC
TGGACGAAGCTTCAGTACCCTGATTATGGCCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCGAC TATTGACAGGGGTGGTACGACAACCGTTACAGACTCCGTGAAGGGCCGATTCTTCATCTCCAGAGACAACGCCAA GAACACGGTGACTCTGCAAATGAACAACCTGAAACCTGAGGACACAGCCACGTATTACTGTAAGACCCTCCGGTA CTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10296 C-030Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTC
TGGCAGAAAATTCAGTACCCTTATTATGGCCTGGTACCGCCAGGCTGAAGGGAAGCAGCGCGAGTTGGTCGCGAC TATTGATAGGGATGGTACGACAACCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAA GAACACGGTGACTCTGCAAATGAACAACCTGAAACCTGAGGACACAGCCGTCTATTACTGTAGTACCCTCCGGTAC TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10297 C-031Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCGCTGAGACTCTCCTGTGCAGCCTC
TGGACGCACCTTCAGTAGCTATGGCATGGGCTGGTTCCGCCAGGCTCCAGGAGAGGAGCGTGAGTTTGTAGCAGC TATTAGTCAGAGTGGTGGGCGTACAAGGTACTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGC CAAGAACATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCCACAAA TTTCCTGATAGCGACTACTATATCTGCATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10298 C-032Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTC TGGACGCACCTTCGCTAGCTTTGGCATGGGCTGGTTCCGCCAGGCTATAGGGAAGGAGCGTGAATrTGTAGCAGC TATTAGTCGGGGCGGTGGACGTACAAGGTATTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGACGC CAAGAACATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCCTCATAT
TTCCTACTAGCGACTACTATATCTGGATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10299 C-033Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTC
TGGACGCACCTTCAGTAGCTATGGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGTAGC
TATTAGTGGGAGTGGTGGGCGTACACGC'mTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGC
CAAGAACATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCCTCGGA
TTTCTTGATAGCGACTAGTATATCCGCATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10300 C-034Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTC
TGGACGCACCn^CGCTAGCFrTGGCATGGGCTGGTTCCGCCAGCCGATAGGCGAGGAGCGTGAATTTGTTGCAGC
TATTAGTCAGGGTGGTGGGCGTACAAGGTATTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGACGC
CAACAACATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCCTCATAT
TTCCTGTTAGCGACTACTATATCTGGATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10301 C-035Hul nori-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTC
TGGACGCACCnCGCTGACTTTGGCATGGGCrGGTTCCGCCAGGCTATAGGGAAGGAGCGTGAATrTGTAGCAGC
TATTAGTCGGAGTGGTGGACGTACAAGGTATTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGACGC
CAAGAACATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTn"ATTACTGTGCAGCCTCATAT
TTCCTACTAGCGATTACTATATCTGGATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10302 C-03SHul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCGCTGAGACTCTCCTGTGCAGCCTC
TGGACGCACCTTCAGTAGCTATGGCATGGGCTGGTTCCGCCAGGCTCCAGGAGAGGAGCGTGAGTTTGTAGCAGC
TATTAGTCAGAGTGGTGGGCGTACAAGGTACTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGC
CAAGAACATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCCACAAA
TTTCCTGATAGCGGCTACTATATCTGCATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCrCCTCA
SEQ ID NO: 10303 C-037Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCGCTGAGACTCTCCTGCGCAGCCTC
TGGACGCACCTTCAGTAGCTATGGCATGGGCTGGTTCCGCCAGGCTCCAGGAGAGGAGCGTGAGTTTGTAGCAGC
TATTAGTCAGAGTGGTGGGCGTACAAGGTACTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGC
CAAGAACATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCCACAAA
GTTCCTGATAGCGACTACTATATCTGCATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10304 C-038Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCGCTGAGACTCTCCTGTGCAGCCTC
TGGACGCACCTTCAGTAGCTATGGCATGGGCTGGTTCCGCCAGGCTCCAGGAGAGGAGCGTGAGTTTGTAGCAGC
TATTAGTCAGAGTGGTGGGCGTACAAGGTACTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGT
CAAGAACATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCCACAAA
TTTCCTGATAGCGACTACTATATCTGCACATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10305 C-039Hul non-humanized counterpart, DNA sequence GAGGTGCAGCTGGTGGAGTCTGGGGGGGGATTGGTGCAGGCTGGGGGCTCGCTGAGACTCTCCTGTGCAGCCTC
TGGACGCACCTTCAGTAGCTATGGCATGGGCTGGTTCCGCCAGGCTCCAGGAGAGGAGCGTGAGTTTGTAGCAGC
TATTAGTCAGAGTGGTGGGCGTACAAGGTACTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGC
CAAGAACATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCCACAAA
TGTCCTGATAGCGACTACTATATCTGCATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10306 C-040Hul nan-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCGCTGAGACTCTCCTGTGCAGCCTC
TGGACGCACCTTCAGTAGCTATGGCATGGGCTGGTTCCGCCAGGCTCCAGGAGAGGAGCGTGAGTTTGTAGCAGC
TATTAGTCAGAGTGGTGGGCGTACAAGGTACTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGC
CAAGAACATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATCACTGTGCAGCCACAAA
TTTCCCGATAGCGACTACTATATCTGCATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO: 10307 C-041Hul non-humanized counterpart, DNA sequence
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCGCTGAGACTCTCCTGTGCAGCCTC
TGGACGCACCTTCAGTAGCTATGGCATGGGCTGGTTCCGCCAGGCTCCAGGAGAGGAGCGTGAGTTTGTAGCAGC
TATTAGTCAGAGTGGTGGGCGTACAAGGTACTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGC
CAAGAACATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCCACAAA
TTTCCTGATAGCGACTGCTATATCTGCATATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCrCCTCA
SEQ ID NO: 10308 C-006_Group E CDR.3 consensus, amino acid sequence
AA(S/T)(D/N/Y/K)(F/V)(L/P)(!/L)A(T/I/A)(T/S/A)IS(A/G)(Y/H)DY
SEQ ID NO: 10309 C-006__Group E CDR1 consensus, amino acid sequence
GRTF(A/S)(S/W/D)(F/N/Y)G
SEQ ID NO: 10310 C-006„Group E. CDR.2 consensus, amino acid sequence
IS(Q/R/G)(S/G)GGRT
SEQ ID NO: 10374 T-007hul CDR3 T/S, CDR3 sequence
AASPSGKAYSY
SEQ ID NO: 10375 T-007hul with CDR2 G/A, CDR3 T/S, Full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAASPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 10380 T-005, full-length VHH, camelid
EVQLVESGGGLVQPGGSLRLSCAASGFTFDDiAMTWVRQAPGKGLEWVSSIYSYGPNTYYADSVKGRFTISTDSAKNTL
YLQMNSLKPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVSS
SEQ ID NO: 10381 T-004, full-length VHH, camelid
EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTIFSYSSNTYYADSVKGRFTISTDSAKNTL
YLQMNSLKPEDTAVYYCAADSDLSTVWGPHDYWGQGTQVTVSS
SEQ ID NO: 10382 T-004Hul, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTIFSYSSNTYYADSVKGRFTISTDSAKNTL
YLQMNSLRPEDTAVYYCAADSDLSTVVVGPHDYWGQGTQVTVSS SEQ ID NO: 10383 T-004.VTV, full-length VHH, camelid
EVQLVESGGGLVQPGGSLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTIFSYSSNTYYADSVKGRFTISTDSAKNTL
YLQMNSLKPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVSS
SEQ ID NO: 10384 T-004Hul.VTV, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAMSWVRQAPGKGLEWVSTIFSYSSNTYYADSVKGRFTISTDSAKNTL
YLQMNSLRPEDTAVYYCAADSDLSTVTVGPHDYWGQGTQVTVSS
SEQ ID NO: 10385 T-009, full-length VHH, camelld
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNGRTTYADSVKGRFTISRDNAEN
TGYLEM NSLKVE DTALYYCAATPTG KAYTYWGQGTQVTVSS
SEQ ID NO: 10386 T-010, full-length VHH, camelld
EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYGMGWFRQAPGKEHEFVASINWSNGRTSYADSAKGRFTISRDNAKN
TGYLEM NSLKVE DTAVYYCAGTLSG KAYTYWGQGTQVTVSS
SEQ ID NO: 10387 anti-TNFR2 CDR3 consensus sequence
A(A/G)(T/A/S)(P/L)(S/T)GKAY(T/S)Y
SEQ ID NO: 10388 T-009HU1. QGR CDR2
INWSQGRT
SEQ ID NO: 10389 T-009Hul SGR CDR2
INWSSGRT
SEQ ID NO: 10390 T-007Hul.S3.EGR. CDR2
INWSEGRT
SEQ ID NO: 10391 T-007Hul.S3.NSR„CDR2
INWSNSRT
SEQ ID NO: 10392 T-007Hul.S3.NTR„CDR2
INWSNTRT
SEQ ID NO: 10393 T-007Hul.S3.SAR„CDR2
INWSSART
SEQ ID NO: 10394 T-009Hul„QGR, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSQGRTTYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVSS
SEQ ID NO: 10395 T-009Hul_ SGR, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKNT
GYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVSS
SEQ ID NO: 10396 T-009 Hul ..QGR..N/C, full-length VHH, humanized DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSQGRTTYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVPAG
SEQ ID NO: 10397 T-009Hul„NAR„N/C, full-length VHH, humanized
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSNARTTYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVPAG
SEQ ID NO: 10398 T-009Hul_ SGR_ N/C, full-length VHH, humanized
DVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPTGKAYTYWGQGTQVTVPAG
SEQ ID NO: 10399 T-007Hul.S3.QGR, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSQGRTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAASPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 10400 T-007Hul.S3.EGR, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSEGRTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAASPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 10401 T-007Hul.S3.SGR, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSSGRTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAASPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 10402 T-007Hul.S3.NSR, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNSRTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAASPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 10403 T-007Hul.S3.NTR, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNTRTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAASPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 10404 T-007Hul.S3.SAR, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSSARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAASPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 10405 T-007Hul.EQ.S3.QGR, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEQEFVAAINWSQGRTNYADSVKGRFTISRDNAK
NTG YLQM NSLRVEDTAVYYCAATPSG KAYSYWGQGTQVTVSS
SEQ ID NO: 10406 T-007Hul.EQ.S3.EGR, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASG RTFSDYG MG WFRQAPG KEQEFVAAI N WSEG RTNYADSVKG RFTISRDN AKN
TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 10407 T-007Hul.EQ.S3.SGR, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASG RTFSDYG MG WFRQAPG KEQEFVAAI NWSSG RTNYADSVKG RFTISRDN AKN TGYLQMNSLRVEDTAVYYCAATPSGKAYSYWGQGTQVTVSS SEQ ID NO: 10408 T-007Hul.EQ.S3,NSR, full-length V'HH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEQEFVAAINWSNSRTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAASPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 10409 antl-TNFR2 CDR2 consensus sequence
INWS(N/Q/E/S)(G/A)RT
SEQ ID NO: 10410 T-016hul.Al_CDR3
AASPTGRAFTY
SEQ ID NO: 10411 T-016hul.G2_CDR3
VGSPTGRAFTY
SEQ ID NO: 10412 T-016hul.A3_.CDR3
VAAPTGRAFTY
SEQ ID NO: 10413 T-016hul.A4_CDR3
VASATGRAFTY
SEQ ID NO: 10414 T-016hul.A5_.CDR3
VASPAGRAFTY
SEQ ID NO: 10415 T-016hul.A6_CDR3
VASPTARAFTY
SEQ ID NO: 10416 T-016hul.A7_.CDR3
VASPTGAAFTY
SEQ ID NO: 10417 T-016hul.A9_.CDR3
VASPTGRAATY
SEQ ID NO: 10418 T-016hul.A10_.CDR3
VASPTGRAFAY
SEQ ID NO: 10419 T-016hul.All_.CDR3
VASPTGRAFTA
SEQ ID NO: 10420 T-016Hul.A5.A7_CDR3
VASPAGAAFTY
SEQ ID NO: 10421 T-016Hul.A5.Al„CDR3
AASPAGRAFTY
SEQ ID NO: 10422 T-016Hul.A5.A3_CDR3
VAAPAGRAFTY
SEQ ID NO: 10423 T-016Hul.A5.A4„CDR3 VASAAGRAFTY
SEQ ID NO: 10424 T-016Hul.A5.A6„CDR3 VASPAARAFTY
SEQ ID NO: 10425 T~016Hul.A7.Al_CDR3 AASPTGAAFTY
SEQ ID NO: 10426 T-016Hul.A7.A3_CDR3 VAAPTGAAFTY
SEQ ID NO: 10427 T-016Hul.A7.A4„CDR3
VASATGAAFTY
SEQ ID NO: 10428 T-016Hul.A7.A6„CDR3
VASPTAAAFTY
SEQ ID NO: 10429 T-016Hul.Al.A4„CDR3
AASATGRAFTY
SEQ ID NO: 10430 T-016Hul.Al.A6„CDR3
AASPTARAFTY
SEQ ID NO: 10431 T-016Hul.A3.A4„CDR3
VAAATGRAFTY
SEQ ID NO: 10432 T-016Hul.A3.A6„CDR3
VAAPTARAFTY
SEQ ID NO: 10433 T-016Hul.A4.A6_CDR3
VASATARAFTY
SEQ ID NO: 10435 T-008Hul.Gl„CDR3
GATPSGKAYSY
SEQ ID NO: 10436 T-008Hul.G2_ CDR3
AGTPSGKAYSY
SEQ ID NO: 10437 T-008Hul.A4_CDR3
AATASGKAYSY
SEQ ID NO: 10438 T-008Hul.A5„CDR3
AATPAGKAYSY
SEQ ID NO: 10439 T-008Hul.A6„CDR3
AATPSAKAYSY SEQ ID NO: 10440 T-008Hul.A7_CDR3
AATPSGAAYSY
SEQ ID NO: 10441 T~008Hul.G8_CDR3 AATPSGKGYSY
SEQ ID NO: 10442 T-008Hul.A9„CDR3
AATPSGKAASY
SEQ ID NO: 10443 T-008Hul.A10_CDR3
AATPSGKAYAY
SEQ ID NO: 10444 T-008Hul.All_CDR3
AATPSGKAYSA
SEQ ID NO: 10445 T-009Hul . SGR.G1. CDR3 GATPTGKAYTY
SEQ ID NO: 10446 T-009Hul _SGR.G2_ CDR3 AGTPTGKAYTY
SEQ ID NO: 10447 T-009Hul . SGR.A3.. CDR3
AAAPTGKAYTY
SEQ ID NO: 10448 T-009Hul„SGR,A4„CDR3 AATATGKAYTY
SEQ ID NO: 10449 T-009Hul„SGR.A5„CDR3 AATPAGKAYTY
SEQ ID NO: 10450 T-009Hul„SGR.A6„CDR3 AATPTAKAYTY
SEQ ID NO: 10451 T-009Hul„SGR.A7„CDR3 AATPTGAAYTY
SEQ ID NO: 10452 T-009Hul„SGR.G8„CDR3 AATPTGKGYTY
SEQ ID NO: 10453 T-009Hul„SGR.A9„CDR3 AATPTGKAATY
SEQ ID NO: 10454 T-009Hul„SGR.A10„CDR3 AATPTGKAYAY
SEQ ID NO: 10455 T-009Hul„SGR.All„CDR3 AATPTGKAYTA SEQ ID NO: 10457 T-016hul.Al, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCAASPTGRAFTYWGQGTQVTVSS
SEQ ID NO: 10458 T-016hul.G2, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQM NSLRPEDTAVYYCVGSPTG RAFTYWGQGTQVTVSS
SEQ ID NO: 10459 T-016hul.A3, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVAAPTGRAFTYWGQGTQVTVSS
SEQ ID NO: 10460 T-016hul.A4, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASATGRAFTYWGQGTQ.VTVSS
SEQ ID NO: 10461 T-016hul.A5, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASPAGRAFTYWGQGTQVTVSS
SEQ ID NO: 10462 T-016hul.A6, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASPTARAFTYWGQGTQVTVSS
SEQ ID NO: 10463 T-016hul.A7, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASPTGAAFTYWGQGTQVTVSS
SEQ ID NO: 10464 T-016hul.A9, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASPTGRAATYWGQGTQVTVSS
SEQ ID NO: 10465 T-OlShul.AlO, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASPTGRAFAYWGQ.GTQVTVSS
SEQ ID NO: 10466 T-015hul.All, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASPTGRAFTAWGQGTQVTVSS
SEQ ID NO: 10467 T-016Hul.A5.A7, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASPAGAAFTYWGQGTQVTVSS
SEQ ID NO: 10468 T-016Hul.A5.Al, full-length VHH, humanized EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCAASPAGRAFTYWGQGTQVTVSS
SEQ ID NO: 10469 T-016Hul.A5.A3, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVAAPAGRAFTYWGQGTQVTVSS
SEQ ID NO: 10470 T-016Hul.A5.A4, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQM NSLRPEDTAVYYCVASAAG RAFTYWGQGTQVTVSS
SEQ ID NO: 10471 T-016Hul.A5.A6, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASPAARAFTYWGQGTQVTVSS
SEQ ID NO: 10472 T-016Hul.A7.Al, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCAASPTGAAFTYWGQGTQ.VTVSS
SEQ ID NO: 10473 T-016Hul.A7.A3, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVAAPTGAAFTYWGQGTQVTVSS
SEQ ID NO: 10474 T-016Hul.A7.A4, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASATGAAFTYWGQGTQVTVSS
SEQ ID NO: 10475 T-016Hul.A7.A6, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASPTAAAFTYWGQ.GTQVTVSS
SEQ ID NO: 10476 T-016Hul.Al.A3, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCAAAPTGRAFTYWGQGTQVTVSS
SEQ ID NO: 10477 T-016Hul.Al.A4, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCAASATGRAFTYWGQGTQVTVSS
SEQ ID NO: 10478 T-016Hul.Al.A6, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCAASPTARAFTYWGQGTQVTVSS
SEQ ID NO: 10479 T-016Hul.A3.A4, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVAAATGRAFTYWGQGTQVTVSS SEQ ID NO: 10480 T-016Hul.A3.A6, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVAAPTARAFTYWGQGTQVTVSS
SEQ ID NO: 10481 T-016Hul.A4.A6, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASATARAFTYWGQGTQVTVSS
SEQ ID NO: 10482 T-008Hul.Gl, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCGATPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 10483 T-008Hul.G2, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAGTPSGKAYSYWGQGTQVTVSS
SEQ ID NO: 10484 T-008Hul.A4, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATASGKAYSYWGQGTQVTVSS
SEQ ID NO: 10485 T-008Hul.A5, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TG YLQM NSLRVEDTAVYYCAATPAG KAYSYWGQGTQVTVSS
SEQ ID NO: 10486 T-008Hul.A6, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSAKAYSYWGQGTQVTVSS
SEQ ID NO: 10487 T-008Hul.A7, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGAAYSYWGQGTQVTVSS
SEQ ID NO: 10488 T-008Hul.G8, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKGYSYWGQGTQVTVSS
SEQ ID NO: 10489 T-008Hul.A9, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKAASYWGQGTQVTVSS
SEQ ID NO: 10490 T-008Hul.A10, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKAYAYWGQGTQVTVSS
SEQ ID NO: 10491 T-008Hul.All, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKDSEFVAAINWSNARTNYADSVKGRFTISRDNAKN
TGYLQMNSLRVEDTAVYYCAATPSGKAYSAWGQGTQVTVSS SEQ ID NO: 10492 T-009Hul_SGR.Gl, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKNT
GYLQMNSLRVEDTAVYYCGATPTGKAYTYWGQGTQ.VTVSS
SEQ ID NO: 10493 T-009Hul_SGR.G2, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKNT
GYLQMNSLRVEDTAVYYCAGTPTGKAYTYWGQGTQVTVSS
SEQ ID NO: 10494 T-009Hul_.SGR.A3, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKNT
GYLQMNSLRVEDTAVYYCAAAPTGKAYTYWGQGTQVTVSS
SEQ ID NO: 10495 T-009Hul„SGR,A4, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKNT
GYLQMNSLRVEDTAVYYCAATATGKAYTYWGQGTQVTVSS
SEQ ID NO: 10496 T-C09Hul . SGR.A5, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKNT
GYLQMNSLRVEDTAVYYCAATPAGKAYTYWGQGTQVTVSS
SEQ ID NO: 10497 T-C09Hul . SGR.A6, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKNT
GYLQMNSLRVEDTAVYYCAATPTAKAYTYWGQGTQVTVSS
SEQ ID NO: 10498 T-C09Hul.. SGR..A7, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKNT
GYLQMNSLRVEDTAVYYCAATPTGAAYTYWGQGTQVTVSS
SEQ ID NO: 10499 T-009Hul_SGR.G8, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKNT
GYLQMNSLRVEDTAVYYCAATPTGKGYTYWGQGTQVTVSS
SEQ ID NO: 10500 T-009Hul_ SGR.A9, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKNT
GYLQMNSLRVEDTAVYYCAATPTGKAATYWGQGTQVTVSS
SEQ ID NO: 10501 T-009Hul_.SGR.A10, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKNT
GYLQMNSLRVEDTAVYYCAATPTGKAYAYWGQGTQVTVSS
SEQ ID NO: 10502 T-009Hul„SGR. All, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFSDYGMGWFRQAPGKEREFVATINWSSGRTTYADSVKGRFTISRDNAKNT
GYLQMNSLRVEDTAVYYCAATPTGKAYTAWGQGTQVTVSS
SEQ ID NO: 10504 Group D_ CDR3 AKGRGSGSYYPFDDY SEQ ID NO: 10505 Group D_CDR3 VAGRGSGSYYPFDDY
SEQ ID NO: 10506 Group D.CDR3 VKARGSGSYYPFDDY
SEQ ID NO: 10507 Group D__CDR3 VKGAGSGSYYPFDDY
SEQ ID NO: 10508 Group D_CDR3 VKGRASGSYYPFDDY
SEQ ID NO: 10509 Group D...CDR3 VKGRGAGSYYPFDDY
SEQ ID NO: 10510 Group D_ CDR3 VKGRGSASYYPFDDY
SEQ ID NO: 10511 Group D.. CDR3 VKGRGSGAYYPFDDY
SEQ ID NO: 10512 Group D_CDR3 VKGRGSGSAYPFDDY
SEQ ID NO: 10513 Group D_ CDR3 VKGRGSGSYAPFDDY
SEQ ID NO: 10514 Group D.. CDR3 VKGRGSGSYYAFDDY
SEQ ID NO: 10515 Group D..CDR3 VKGRGSGSYYPADDY
SEQ ID NO: 10516 Group DJZDR3 VKGRGSGSYYPFADY
SEQ ID NO: 10517 Group D_CDR3 VKGRGSGSYYPFDAY
SEQ ID NO: 10518 Group D..CDR3 VKGRGSGSYYPFDDA
SEQ ID NO: 10519 Group H .CDR3 GAKRLGPLVHRYSLEVLTPLFLDEYDY
SEQ ID NO: 10520 Group H..CDR3 AGKRLGPLVHRYSLEVLTPLFLDEYDY
SEQ ID NO: 10521 Group H .CDR3
AAARLGPLVHRYSLEVLTPLFLDEYDY
SEQ ID NO: 10522 Group H_CDR3
AAKALGPLVHRYSLEVLTPLFLDEYDY
SEQ ID NO: 10523 Group HJ2DR3
AAKRAGPLVHRYSLEVLTPLFLDEYDY
SEQ ID NO: 10524 Group H .CDR3
AAKRLAPLVHRYSLEVLTPLFLDEYDY
SEQ ID NO: 10525 Group H_ CDR3
AAKRLGALVHRYSLEVLTPLFLDEYDY
SEQ ID NO: 10526 Group H.. CDR3
AAKRLGPLAHRYSLEVLTPLFLDEYDY
SEQ ID NO: 10527 Group HJZDR3
AAKRLGPLVARYSLEVLTPLFLDEYDY
SEQ ID NO: 10528 Group H_ CDR3
AAKRLGPLVHAYSLEVLTPLFLDEYDY
SEQ ID NO: 10529 Group H .CDR3
AAKRLGPLVHRASLEVLTPLFLDEYDY
SEQ ID NO: 10530 Group H_ CDR3
AAKRLGPLVHRYALEVLTPLFLDEYDY
SEQ ID NO: 10531 Group H_ CDR3
AAKRLGPLVHRYSAEVLTPLFLDEYDY
SEQ ID NO: 10532 Group HJZDR3
AAKRLGPLVHRYSLAVLTPLFLDEYDY
SEQ ID NO: 10533 Group HJZDR3
AAKRLGPLVHRYSLEALTPLFLDEYDY
SEQ ID NO: 10534 Group H_CDR3
AAKRLGPLVHRYSLEVATPLFLDEYDY
SEQ ID NO: 10535 Group H..CDR3
AAKRLGPLVHRYSLEVLAPLFLDEYDY SEQ ID NO: 10536 Group H .CDR3
AAKRLGPLVHRYSLEVLTALFLDEYDY
SEQ ID NO: 10537 Group H_CDR3
AAKRLGPLVHRYSLEVLTPAFLDEYDY
SEQ ID NO: 10538 Group H _CDR3
AAKRLGPLVHRYSLEVLTPLALDEYDY
SEQ ID NO: 10539 Group H_CDR3
AAKRLGPLVHRYSLEVLTPLFADEYDY
SEQ ID NO: 10540 Group H _CDR3
AAKRLGPLVHRYSLEVLTPLFLAEYDY
SEQ ID NO: 10541 Group H.. CDR3
AAKRLGPLVHRYSLEVLTPLFLDAYDY
SEQ ID NO: 10542 Group H .CDR3
AAKRLGPLVHRYSLEVLTPLFLDEADY
SEQ ID NO: 10543 Group H_ CDR3
AAKRLGPLVHRYSLEVLTPLFLDEYAY
SEQ ID NO: 10544 Group H_CDR3
AAKRLGPLVHRYSLEVLTPLFLDEYDA
SEQ ID NO: 10545 Group Dehumanized VHH amino add sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCAKGRGSGSYYPFDDYWGQGTQVTVSS
SEQ ID NO: 10546 Group D humanized VHH amino add sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVAGRGSGSYYPFDDYWGQGTQVTVSS
SEQ ID NO: 10547 Group D humanized VHH amino add sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVKARGSGSYYPFDDYWGQGTQVTVSS
SEQ ID NO: 10548 Group Dehumanized VHH amino add sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVKGAGSGSYYPFDDYWGQGTQVTVSS
SEQ ID NO: 10549 Group Dehumanized VHH amino add sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVKGRASGSYYPFDDYWGQGTQVTVSS SEQ ID NO: 10550 Group D_humanized VHH amino acid sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVKGRGAGSYYPFDDYWGQGTQVTVSS
SEQ ID NO: 10551 Group Dehumanized VHH amino acid sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVKGRGSASYYPFDDYWGQGTQVTVSS
SEQ ID NO: 10552 Group Dehumanized VHH amino acid sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVKGRGSGAYYPFDDYWGQGTQVTVSS
SEQ ID NO: 10553 Group Dehumanized VHH amino acid sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVKGRGSGSAYPFDDYWGQGTQVTVSS
SEQ ID NO: 10554 Group Dehumanized VHH amino acid sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVKGRGSGSYAPFDDYWGQGTQVTVSS
SEQ ID NO: 10555 Group Dehumanized VHH amino acid sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGiYSDSSGTYYADSVKGRFTlSRDNAKNT
VYLQMNSLRPEDTAVYYCVKGRGSGSYYAFDDYWGQGTQVTVSS
SEQ ID NO: 10556 Group Dehumanized VHH amino acid sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVKGRGSGSYYPADDYWGQGTQVTVSS
SEQ ID NO: 10557 Group Dehumanized VHH amino acid sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVKGRGSGSYYPFADYWGQGTQVTVSS
SEQ ID NO: 10558 Group Dehumanized VHH amino acid sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVKGRGSGSYYPFDAYWGQGTQVTVSS
SEQ ID NO: 10559 Group Dehumanized VHH amino acid sequence
EVQLLESGGGLAQPGGSLRLSCAASGFTFSNYAMSWARQAPGKGLEWVSGIYSDSSGTYYADSVKGRFTISRDNAKNT
VYLQMNSLRPEDTAVYYCVKGRGSGSYYPFDDAWGQGTQVTVSS
SEQ ID NO: 10560 Group Hehumanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCGAKRLGPLVHRYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10561 Group Hehumanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAGKRLGPLVHRYSLEVLTPLFLDEYDYWGQGTQVTVSS SEQ ID NO: 10562 Group H_humanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTiSRDNPKNTVY
LQLNSLRPEDTAVYYCAAARLGPLVHRYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10563 Group H__humanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTiSRDNPKNTVY
LQLNSLRPEDTAVYYCAAKALGPLVHRYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10564 Group H_ humanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTiSRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRAGPLVHRYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10565 Group H._ humanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLAPLVHRYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10566 Group HJiumanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSiSSTDGRTYYADSVKGRFTiSRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGALVHRYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10567 Group H_humanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLAHRYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10568 Group H_humanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVARYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10569 Group H_humanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHAYSLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10570 Group HJaumanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRASLEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10571 Group H_ humanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYALEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10572 Group H_ humanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSAEVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10573 Group H_humanized VHH amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLAVLTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10574 Group H__humanized VHH amino add sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEALTPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10575 Group H__humanized VHH amino add sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVATPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10576 Group H_ humanized VHH amino add sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTiSRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLAPLFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10577 Group HJiumanized VHH amino add sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTALFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10578 Group HJiumanized VHH amino add sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPAFLDEYDYWGQGTQVTVSS
SEQ ID NO: 10579 Group H_humanized VHH amino add sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPLALDEYDYWGQGTQVTVSS
SEQ ID NO: 10580 Group H_humanized VHH amino add sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSiSSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPLFADEYDYWGQGTQVTVSS
SEQ ID NO: 10581 Group HJiumanized VHH amino add sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPLFLAEYDYWGQGTQVTVSS
SEQ ID NO: 10582 Group H humanized VHH amino add sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPLFLDAYDYWGQGTQVTVSS
SEQ ID NO: 10583 Group H_ humanized VHH amino add sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPLFLDEADYWGQGTQVTVSS
SEQ ID NO: 10584 Group H_humanized VHH amino add sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPLFLDEYAYWGQGTQVTVSS SEQ ID NO: 10585 Group H_humanized VHH amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVLSISSTDGRTYYADSVKGRFTISRDNPKNTVY
LQLNSLRPEDTAVYYCAAKRLGPLVHRYSLEVLTPLFLDEYDAWGQGTQVTVSS anti-CD25 consensus sequence_CDR3 Group D
(A/V/S)(A/K/T)(A/G)(A/R/K)(A/G/H/N/R)(A/S)(A/G)(A/S/G)(A/Y)(A/Y)(A/P)(A/W/F/L)(A/D/E)(A/D/E)(A/Y/
V) anti-CD25 consensus sequence_CDR3 Group H
(A/G)(A/G)(A/K){A/R)(A/L)(A/G)(A/P)(M/I/A/L)(A/V)(A/H){A/R/Q)(A/Y)(A/S)(A/L)(A/E)(A/V)(A/L)(A/T)(A/P
)(A/L)(A/F)(A/L)(A/D)(A/E)(A/Y)(A/D)(A/Y)
SEQ ID NO: 10588 DVQ-T-008hul.A3.VPAG„nucleotide sequence
GACGTGCAATFGCTGGAGTCCGGGGGCGGnTGGTGCAGCCCGGGGGTAGCCTCAGATTGAGTTGCGCCGCATC
CGGGCGAACATTCAGTGACTACGGGATGGGGTGGTTTAGACAGGCACCTGGCAAGGACAGTGAGTTTGTGGCTG CGATTAATTGGTCCAACGCTAGGACCAACTACGCGGATTCAGTCAAGGGACGCTTCACCATCTCCAGAGACAATGC CAAGAACACAGGGTACCTGCAAATGAACTCCCTCAGGGTGGAGGACACCGCAGTGTACTACTGCGCTGCAGCCCC GTCAGGAAAGGCCTATTCCTACTGGGGGCAGGGGACTCAAGTCACGGTACCAGCAGGA
SEQ ID NO: 10589 DVQ-T-008hul.A4.VPAGmnucleotide sequence
GACGTCCAGTTGTTGGAGAGCGGAGGAGGCCTGGTCCAGCCGGGTGGCAGTCTTCGGCTGAGCTGTGCAGCCAG
TGGACGAACATTCAGTGACTACGGGATGGGGTGGTTTCGCCAGGCACCGGGCAAAGATTCAGAGTTCGTTGCTGC
GATCAATTGGTCCAACGCCAGGACAAATTACGCGGACTCCGTCAAGGGAAGATTCACTATATCCAGGGATAACGC TAAGAACACCGGGTACTTGCAGATGAATTCCCTGAGGGTCGAGGATACGGCTGTGTACTACTGTGCCGCAACGGC ATCCGGCAAGGCGTACAGCTATTGGGGGCAAGGCACCCAGGTGACGGTACCCGCCGGC
SEQ ID NO: 10590 DVQ-T-008hul.A5.VPAG„nucleotide sequence
GACGTCCAGCTTCTCGAATCAGGTGGCGGACTGGTCCAGCCAGGGGGCTCTTTGCGGTTGTCTTGTGCCGCCAGC
GGGAGAACCTTCTCAGACTACGGAATGGGGTGGTTTAGGCAGGCTCCTGGCAAAGACAGTGAGTTCGTGGCCGC
GATCAATTGGTCCAATGCAAGGACGAACTACGCGGATTCCGTCAAGGGCCGG'n'CACCATATCCCGGGATAATGC AAAGAATACGGGATACCTTCAGATGAATTCCTTGAGAGTGGAGGATACTGCCGTCTACTACTGCGCGGCCACCCC GGCAGGGAAAGCTTACTCCTATTGGGGGCAGGGGACACAGGTGACAGTACCTGCTGGT
SEQ ID NO: 10591 DVQ-T-008hul.A5.VPAG„nucleotide sequence
GACGTGCAGCTTCTTGAGTCCGGAGGGGGACTCGTCCAGCCGGGCGGATCCCTTAGGTTGAGCTGCGCCGCn'CA
GGAAGAACGTTCAGCGACTATGGGATGGGGTGGTTTAGACAGGCACCCGGCAAAGATTCTGAGTTCGTTGCCGC
GATTAACTGGTCCAATGCTAGAACAAACTACGCGGACTCTGTCAAGGGGCGATTCACAATTTCCAGAGACAACGC GAAGAACACTGGGTATCTCCAGATGAATTCCCTCAGAGTCGAAGATACGGCCGTATACTACTGCGCAGCCACGCC CAGTGCCAAGGCATATAGCTACTGGGGCCAAGGGACACAAGTAACAGTTCCAGCGGGC
SEQ ID NO: 10592 DVQ-T-008hul.A7.VPAG„nucieotide sequence
GATGTGCAGCTTCTCGAGTCCGGCGGGGGATTGGTACAGCCCGGAGGGAGCCTGCGATTGAGTTGCGCCGCGAG
TGGCAGGACTTTCAGTGACTATGGGATGGGGTGGTTTCGACAAGCCCCGGGTAAGGATTCAGAGTTTGTAGCCGC
AATTAACTGGAGCAATGCGCGGACCAACTACGCAGACAGTGTCAAGGGAAGATTCACTATTTCCAGAGATAACGC CAAGAACACCGGCTACCTCCAGATGAATTCCCTCAGAGTTGAGGATACGGCCGTGTACTACTGCGCAGCTACGCCC AGCGGGGCCGCCTACAGCTACTGGGGGCAAGGAACTCAGGTGACGGTACCAGCTGGA SEQ ID NO: 10593 DVQ"T-008hul.A9.VPAG„nucleotide sequence
GACGTTCAGTTGCTCGAGTCCGGAGGGGGTCTGGTACAGCCCGGGGGATCCCTCCGCCTGTCTTGCGCGGCTTCC
GGACGCACTTTCAGTGATTATGGGATGGGCTGGTTCAGGCAGGCCCCGGGGAAGGACAGCGAGTn'GTGGCTGC
CATTAACTGGTCAAACGCGCGGACCAACTATGCGGACAGCGTCAAGGGACGATTCACTATCTCCAGAGATAATGC CAAGAACACAGGGTACTTGCAAATGAACAGTCTGCGAGTCGAAGATACGGCCGTTTACTACTGTGCTGCTACGCC GAGTGGCAAGGCCGCTAGCTACTGGGGCCAGGGGACCCAGGTAACGGTACCGGCGGGA
SEQ ID NO: 10594 DVQ-P008hul.A10.VPAG„nucleodde sequence
GACGTGCAGCTTCTTGAGAGTGGCGGGGGATTGGTGCAGCCTGGGGGCTCTTTGAGACTCAGTTGCGCTGCGAG
CGGCCGCACCn'CTCAGACTATGGAATGGGGTGGTTTCGGCAGGCCCCAGGCAAGGATTCCGAGTTCGTGGCTGC
GATTAATTGGTCCAACGCCAGGACCAACTATGCGGATTCTGTCAAGGGCCGCTTTACCATTTCCAGAGATAATGCT AAGAATACCGGGTACCTTCAAATGAATTCCCTGAGGGTGGAAGACACGGCTGTGTATTACTGCGCCGCAACGCCC TCCGGGAAGGCTTATGCCTATTGGGGACAGGGGACACAGGTGACCGTGCCCGCTGGA
SEQ ID NO: 10595 DVQ-T-008hul.All.VPAG_nuc!eotide sequence
GATGTGCAGCTCCTCGAGTCTGGTGGGGGCCTTGTTCAGCCCGGGGGTTCACTCCGATTGAGTTGCGCAGCCAGC
GGAAGAACCTTCAGCGACTACGGCATGGGGTGGTTCCGACAGGCACCCGGCAAAGACTCCGAGTTCGTGGCCGC
GATAAATTGGTCCAACGCTCGCACCAACTACGCGGACAGTGTCAAGGGTAGGTTCACGATCTCTCGGGACAATGC TAAGAATACAGGATACCTGCAGATGAACTCCTTGCGAGTGGAGGATACCGCTGTGTATTACTGTGCCGCAACGCC GAGTGGGAAGGCATACTCAGCATGGGGCCAGGGGACACAAGTGACGGTACCAGCGGGC
SEQ ID NO: 10596 T-007Mu-2*01„nucleotide sequence
GAGGTGCAACTGGTGGAGAGTGGCGGAGGGCTCGTGCAGCCAGGGGAGAGTTTGAAGCTGAGTTGCGAGGCGT
CAGGGCGGACCTTCGGTTCCTACACAATGGGGTGGTTTCGGAAGACACCGGAAAAGGAGAGAGAGTTCTTGGCA
TCTATTAGGTGGACAGGGGGCTCTACATCCTACCCAGACACGGTAGAGAGACGCTTTATCATTTCCCGGGACGAT GCAAAGAAGACCGTGTACCTGCAGATGTCCTCCCTGAGGCCAGAGGACACGGCTCTCTACTATTGCGTAGCAAGT CCCACCGGCAGGGCTTrTACTTACTGGGGCCAAGGGACCCAGGTAACGGTCTCTTCA
SEQ ID NO: 10597 T-007Mu-4*01„nucleotide sequence
GAGGTGCAGCTTGTGGAGTCTGGGGGCGGACTGGTGAAGCCTGGGGGTAGTCTTAAACTCTCATGCGCTGCGTC
TGGTAGGACCmGGCTCCTACACCATGGGGTGGTTTCGGCAGACCCCGGAAAAGGAGAGAGAATrrCTGGCTTC
AATCCGATGGACAGGAGGGTCTACCAGCTACCCCGACAATGTGAAAGGCAGATTCACCATCTCCCGCGACGACGC AAAGAAGAACGTTTACCTCCAGATGTCCCACCTTAAGCCAGAAGACACTGCGATGTACTACTGCGTCGCCTCCCCT ACCGGCAGAGCATTTACGTATTGGGGCCAGGGCACCCAGGTGACAGTCTCATCA
SEQ ID NO: 10598 T-007Mu-6*01_nucleotide sequence
GAAGTGCAACTCGTGGAATCTGGGGGCGATCTTGTGAAGCCAGGGGGATCCCTGAAGCTTAGTTGCGCGGCTAG
TGGGAGGACTTTCGGGTCCTACACGATGGGGTGGTTCCGCCAGACACCAGACAAAGAAAGAGAGTnTTGGCCA
GCATACGGTGGACTGGAGGGTCAACAAGCTACCCCGATTCTGTTAAGGGAAGATTTACTATCTCCAGGGATGACG CCAAAAAGACAGTGTATFTGCAGATGAGCTCCCTGAAGCCCGAGGACACCGCAATGTACTACTGCGTGGCGAGCC CCACCGGGCGCGCCTTTACATACTGGGGCCAAGGGACACAGGTGACGGTCTCCTCC
SEQ ID NO: 10599 T-007Mu-9*01_nucleotlde sequence
GAGGTGATGCTGGTCGAATCTGGGGGTGGCCTTGTCAAGCCCGGAGGGTCCCTGAAGCTCAGCTGTGCCGCGAG
TGGCCGGACGTTCGGCAGTTACACGATGGGCTGGTTCAGGCAGACACCCGAAAAGGAAAGGGAGTTCCTGGCGT CAATTAGATGGACAGGTGGATCCACAAGTTACCCGGACTCCGTTAAAGGGAGGTTCACCATATCCAGGGATGACG CAAAGAAAACCGTTTACCTTCAGATGTCCTCTCTTCGACCAGAGGACACGGCTCTCTACTACTGCGTGGCCAGTCCT
ACAGGCAGAGCCTTCACTTACTGGGGGCAAGGGACACAGGTCACGGTAAGCTCA
SEQ ID NO: 10600 T4S07Mu-12*01_nudeotide sequence
GAGGTGAAGCTGGTGGAGTCCGGGGGCGGCCTGGTACAACCAGGCGGCAGCCTGAAACTTTCCTGTGCCGCATC
CGGCAGAACATTCGGGAGTTACACCATGGGGTGGTn'AGGCAGACACCGGAAAAGGAACGGGAATTCCTGGCGT
CCATAAGGTGGACAGGTGGGTCCACCAGTTACCCTGACACAGTAAAAGGAAGATTTACTATCAGTCGAGATGACG
CCAAGAAAACAGTCTACCTCCAAATGAGCCGCCTCAAGCCTGAAGACACCGCGATGTACTACTGTGTCGCGTCACC AACCGGGAGGGCCTTCACCTACTGGGGACAGGGGACGCAGGTAACGGTTTCCAGT
SEQ ID NO: 10601 T-007Mu-15*01_nudeotide sequence
GAAGTGAAACTGGTGGAAAGCGGGGGTGGACTTGTTCAGCCCGGAGGGTCCCTGAAGCTCAGCTGCGCAGCGTC
AGGAAGGACATTTGGATCCTATACCATGGGGTGGTTCAGGCAGGCTCCGCGGAAGGAAAGAGAATTCCTGGCAA
GTATTCGCTGGACCGGCGGGTCCACATCATACGCGGACACCGTGACCGGAAGATn'ACCATTAGCCGGGATGATG
CTAAGAAAACGGTCTACCTGGAGATGTCCAGCCTTCGCCCTGAAGATACGGCCATGTATTACTGTGTGGCATCCCC CACCGGAAGAGCATTCACTTACTGGGGGCAGGGGACACAGGTGACGGTAAGCTCT
SEQ ID NO: 10602 T-007Mu-16Wjnudeotide sequence
GAAGTGAAGCTTGTTGAAAGTGAGGGAGGTCTCGTGCAGCCCGGGAGCTCCn'GAAACTGTCCTGTACTGCGAGC
GGCCGCACG7TTGGGAGTTACACGATGGGCTGGTTCCGACAGGTCCCGGAGAAAGAGAGGGAGTTCCTTGCAAG
TATTCGATGGACCGGCGGGAGTACCTCATACCTGGACTCAGTAAAGTCCCGGTTCATCATCTCTAGAGACGATGCT
AAGAAGATTGTCTACCTCCAAATGAGCTCCCTTAAACCCGAGGACACCGCTACCTACTACTGCGTGGCGAGCCCAA
CAGGAAGGGCCTTTACCTACTGGGGTCAGGGGACTCAGGTAACGGTGAGTTCC
SEQ ID NO: 10603 T-007Mu47*01 _nucleotide sequence
GAGGTGCAGTTGGTGGAGAGCGGCGGCGGCCTCGTTAAGCCAGGGGGAAGTTTGAAGCTGAGCTGTGCCGCGT
CTGGTAGGACCTn'GGCTCCTATACGATGGGGTGG'TTCCGCCAAGCACCGGAAAAGGAGCGAGAA'TTCCTGGCTT
CCATTCGGTGGACCGGGGGATCTACAAGTTACGCAGACACCGTAAAAGGAAGATTCACAATCTCCCGGGACGACG
CGAAGAAGACCGTGTACCTCCAAATGACATCCTTGCGGCCAGAAGATACGGCCATGTACTACTGCGTCGCCTCCCC
TACCGGGCGCGCATTTACGTACTGGGGACAGGGGACCCAAGTGACGGTATCATCA
SEQ ID NO: 10604 T~007Mu-Consensus__niideotide sequence
GAAGTCCAACTGGTAGAGTCCGGCGGCGGGTTGGTCCAGCCGGGAGGATCTCTCAAGCTCAGCTGCGCAGCTTCC
GGACGCACGTTCGGGTCTTATACCATGGGTTGGTTCCGACAAACCCCGGAAAAGGAACGAGAGTTTCTTGCCAGC
ATCAGATGGACAGGCGGGTCTACAAGCTACCCTGACACAGTCAAGGGTAGGTTTACTATTTCCAGGGATGATGCC AAGAAGACGGTGTACTTGCAGATGTCATCCTTGAGACCCGAGGACACGGCTATGTATTACTGCGTGGCTTCCCCAA CCGGCCGGGCTTTCACCTACTGGGGGCAAGGGACTCAGGTCACCGTGTCAAGT
SEQ ID NO: 10605 T-007Mu-Graft-IGHV5-9*!01„nudeotide sequence
GAAGTGATGCTGGTAGAATCTGGGGGCGGCCTGGTCAAACCCGGGGGTTCCCTTAAATTGTCCTGCGCAGCGAGC
GGAAGGACATTTGGGAGTTATACTATGAGTTGGGTCAGGCAAACGCCGGAGAAGCGGTTGGAGTGGGTGGCCAC
CATAAGGTGGACAGGGGGCTCCACATACTACCCCGATAGCGTGAAGGGGCGCTTCACCATTTCCCGCGATAACGC
CAAGAATACCCTGTATCTGCAAATGTCTAGTCnCGATCCGAGGACACAGCGTTGTACTATTGCGTGGCTTCTCCGA CTGGAAGAGCTTTCACTTACTGGGGGCAGGGCACCAGTGTGACCGTGTCTAGT
SEQ ID NO: 10606 DVQ-T-019hul.VPAG__niicleotide sequence GATGTCCAGCTTGTTGAGAGTGGGGGTGGACTCGTGCAGCCAGGTGGTTCCCTGAGGCTGAGCTGCGCCGCGTCT
GGGTCCATATTCCGCTTTCCTCCGATGGGCTGGTACCGGCAGGCACCCGGGAAGCAGCGAGAGCAGGTGGCTCA
ACTGACAAGCGGCGGGAGCACAAACTACGCCGACTCAGTCAAAGGGCGGTTCACCATTAGTAGGGATAACGCGA
AAAACACGTGGTACCTTCAGATGAATAGCCTTAGGCCCGAAGACACAGCCGTCTACTATTGCTCCGTCCTGGGCAG
AGACATGGTCACGTATTGGGGGCAGGGGACTCAAGTGACGGTGCCAGCAGGA
SEQ ID NO: 10607 DVQ-T-019hul.LV.VPAG„nuc!eotide sequence
GACGTCCAGCTTGTCGAGAGTGGAGGCGGCTTGGTTCAGCCTGGGGGCTCACTTCGGCTGTCATGTGCGGCGTCT
GGGTCAATCTTCAGATTTCCGCCGATGGGGTGGTACCGACAGGCTCCGGGAAAACAAAGAGAACAGGTGGCGCA
ACTGACATCTGGCGGAAGCACCAACTACGCAGACTCAGTCAAGGGGCGGTTTACGATCAGTAGAGATAACGCCAA GAATACCTGGTACCTGCAGATGAACAGTCTGAGGCCTGAAGACACGGCCGTTTACTACTGCTCCGTGCTGGGGCG GGACCTGGTAACGTATTGGGGTCAGGGTACACAGGTGACAGTGCCAGCCGGT
SEQ ID NO: 10608 DVQ-T-019hul.RV,VPAG„nucleotide sequence
GACGTCCAGCTTGTGGAAAGCGGCGGCGGGCTTGTCCAGCCAGGGGGCTCACTCAGGCTn'CCTGCGCCGCGAGT
GGATCCATATTCCGCTTTCCACCGATGGGATGGTACAGGCAGGCCCCAGGGAAACAGCGAGAACAAGTGGCACA
GCTCACCTCCGGAGGGAGCACTAACTACGCTGACAGTGTTAAAGGGCGGTTTACCATn'CCCGCGACAACGCCAA
GAATACCTGGTACCTGCAGATGAACAGCCTGAGACCAGAGGACACGGCGGTTTATTACTG7TCTGTCTTGGGCAG
AGATCGAGTAACATACTGGGGGCAAGGGACGCAGGTGACGGTGCCAGCAGGT
SEQ ID NO: 10609 DVQ-T-O19hul.!V.VPAG_nucleotide sequence
GACGTTCAGCTCGTCGAATCAGGGGGAGGGCTCGTCCAGCCTGGGGGCAGTTTGCGCCTCAGTTGCGCCGCGAG
TGGATCAATTTTCAGGTTCCCTCCTATGGGGTGGTACCGGCAGGCTCCCGGGAAACAGAGGGAGCAGGTGGCTCA
ACTGACATCAGGTGGGTCTACAAACTATGCAGATTCTGTCAAGGGGAGG'nTACTATATCCAGAGATAATGCAAA GAACACTTGGTACCTCCAGATGAACTCCCTCCGCCCAGAGGACACGGCTGTATACTACTGTTCCGTGTTGGGCCGC GACATCGTGACGTATTGGGGTCAGGGGACCCAGGTGACAGTACCAGCGGGA
SEQ !D NO: 10610 T-009Hul_QGR__nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTGTTGCAACC
ATTAATTGGAGCCAGGGTCGTACCACCTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCAA
AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCAACCCCGAC CGGTAAAGCATATACCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10611 T~009Hul_.NAR_nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTGTTGCAACC
ATTAATTGGAGCAATGCACGTACCACCTATGCAGATAGCGTTAAAGGTCGTnTACCATrAGCCGTGATAATGCAA
AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTA'TTGTGCAGCAACCCCGAC CGGTAAAGCATATACCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10612 T~009Hul„SGR_nudeotlde sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTGTTGCAACC
ATTAATTGGAGCAGCGGTCGTACAACCTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCAA
AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTAITA'TTGTGCAGCAACCCCGAC CGGTAAAGCATATACCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC SEQ ID NO: 10613 T-009Hul„QGR_N/Cmnucleotide sequence
GATGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTAGCGATrATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTGTTGCAACC
ATTAATTGGAGCCAGGGTCGTACCACCTATGCAGATAGCGTTAAAGGTCGTTn’ACCATTAGCCGTGATAATGCAA
AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCAACCCCGAC CGGTAAAGCATATACCTATTGGGGTCAGGGTACCCAGGTTACCGTTCCGGCAGGT
SEQ ID NO: 10614 T-009Hul_NAR__N/C__nucleotide sequence
GATGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTGTTGCAACC
ATTAATTGGAGCAATGCACGTACCACCTATGCAGATAGCGnAAA.GGTCGTTTTACCATTAGCCGTGATAA.TGCAA
AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCAACCCCGAC CGGTAAAGCATATACCTATTGGGGTCAGGGTACCCAGGTTACCGTTCCGGCAGGT
SEQ ID NO: 10615 T-009Hul„SGR„N/C„nucleotide sequence
GATGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAA'nTG'n'GCAACC
ATTAATTGGAGCAGCGGTCGTACAACCTATGCAGATAGCGTTAAAGGTCGTnTACCATTAGCCGTGATAATGCAA
AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCAACCCCGAC CGGTAAAGCATATACCTATTGGGGTCAGGGTACCCAGGTTACCGTTCCGGCAGGT
SEQ ID NO: 10616 T-007Hul.S3.QGR_nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA
ATTAATTGGAGCCAGGGTCGTACCAATTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTA'TTATTGTGCAGCATCTCCGAG CGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ !D NO: 10617 T~007Hul.S3.EGR_nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTrAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA
ATTAATTGGAGCGAAGGTCGTACCAATTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCAA
AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTAITA'TTGTGCAGCATCTCCGAG CGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10618 T-007Hul.S3.SGR_nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA
ATTAATTGGAGCAGCGGTCGTACAAATTATGCAGATAGCGTTAAAGGTCGmTACCATTAGCCGTGATAATGCAA
AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTAITA'TTGTGCAGCATCTCCGAG CGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10619 T-007Hul.S3.NSR_nucleotide sequence
GAAGTTCAGCTGCJGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTrAGCGATTATGGTATGGGTTGGTn'CGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA
ATTAATTGGAGCAATAGCCGTACCAATTATGCAGATAGCGTTAAAGGTCGTnTACCATTAGCCGTGATAATGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCATCTCCGAG
CGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10620 T-007Hul.S3.NTR_nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTnCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA ATTAATTGGAGCAATACCCGTACCAATTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCAA AAAATACI:GGTTATCT£3CAGATGAATA{3CCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCATCTC:CGAG CGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10621 T-007Hul.S3.SAR_nucleotlde sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA ATTAATTGGAGCAGCGCACGTACCAATTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTA'TTATTGTGCAGCATCTCCGAG CGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10622 T-007Hul.EQ.QGR„nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGG7AAAGAACAGGMTTTGTTGCAGC AATTAATTGGAGCCAGGGTCGTACCAATTATGCAGATAGCGTTAAAGGTCGTnTACCATTAGCCGTGATAATGCA AAAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATrATTGTGCAGCAACCCCGA GCGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10623 T-007Hul.EQ,EGR._nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACAGGAATTTGTTGCAGC AATTAATTGGAGCGAAGGTCGTACCAATTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCA AAAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCAACCCCGA GCGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10624 T~007Hul.EQ.SGR_nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTn'CGTCAGGCACCGGGTAAAGAACAGGAATTTGTTGCAGC AATTAATTGGAGCAGCGGTCGTACAAATTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCA AAAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCAACCCCGA GCGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10625 T-007Hul.EQ.S3.NSR„nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTrAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACAGGAATTTGTTGCAGC AATTAATTGGAGCAATAGCCGTACCAATTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCA AAAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCATCTCCGA GCGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10626 T~016hul.Al_nucleotide sequence GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTGGTAGCTATACCATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTCTGGCAAGC
ATTCGTTGGACCGGTGGTAGCACCAGCTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATGATGCAA AAAAAACCGTTTATCTGCAGATGAATAGCCTGCGTCCGGAAGATACCGCAGTTTATTATTGTGCAGCATCTCCGAC CGGTCGTGCCTTTACCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10627 T~016hul.G2_nucleQtide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTGGTAGCTATACCATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTCTGGCAAGC
ATTCGTTGGACCGGTGGTAGCACCAGCTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATGATGCAA
AAAAAACCGTTTATCTGCAGATGAATAGCCTGCGTCCGGAAGATACCGCAGTTTATTATTGTGTTGGTAGCCCGAC CGGTCGTGCCTTTACCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10628 T-016hul.A3„nudeotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTGGTAGCTATACCATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTCTGGCAAGC
ATTCGTTGGACCGGTGGTAGCACCAGCTATGCAGATAGCGTTAAAGGTCGTnTACCATTAGCCGTGATGATGCAA
AAAAAACCGTTTATCTGCAGATGAATAGCCTGCGTCCGGAAGATACCGCAGTTTATrATTGTGTTGCAGCACCGAC CGGTCGTGCCnTACCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10629 T~016hul.A4_nudeotlde sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTGGTAGCTATACCATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATnCTGGCAAGC
ATTCGTTGGACCGGTGGTAGCACCAGCTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATGATGCAA
AAAAAACCGTTTATCTGCAGATGAATAGCCTGCGTCCGGAAGATACCGCAGTn'ATTA'TTGTGTTGCAAGCGCAAC CGGTCGTGCCTTTACCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10630 T-016hul.A5__nudeotlde sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTrGGTAGCTATACCATGGGTTGGTFTCGTCAGGCACCGGGTAAAGAACGTGAATTTCTGGCAAGC
ATTCGTTGGACCGGTGGTAGCACCAGCTATGCAGATAGCGTTAAAGGTCGTnTACCATTAGCCGTGATGATGCAA
AAAAAACCGTTTATCTGCAGATGAATAGCCTGCGTCCGGAAGATACCGCAGTTTATTATTGTGTTGCAAGCCCGGC AGGTCGTGCCTTTACCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10631 T~016hul.A6_nudeotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTnGGTAGCTATACCATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTCTGGCAAGC
ATTCGTTGGACCGGTGGTAGCACCAGCTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATGATGCAA
AAAAAACCGTTTATCTGCAGATGAATAGCCTGCGTCCGGAAGATACCGCAGTTTATTATTGTGTTGCAAGCCCGAC CGCACGTGCCTTTACCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10632 T-016hul.A7„nudeotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTGGTAGCTATACCATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTCTGGCAAGC
ATTCGTTGGACCGGTGGTAGCACCAGCTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATGATGCAA
AAAAAACCGTTTATCTGCAGATGAATAGCCTGCGTCCGGAAGATACCGCAGTTTATTAITGTGTTGCAAGCCCGAC CGGTGCAGCCnTACCTA'TTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC SEQ ID NO: 10633 T-016hul.A9__nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTGGTAGCTATACCATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTCTGGCAAGC
ATTCGTTGGACCGGTGGTAGCACCAGCTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATGATGCAA
AAAAAACCGTTTATCTGCAGATGAATAGCCTGCGTCCGGAAGATACCGCAGTTTATTATTGTGTTGCAAGCCCGAC CGGTCGTGCAGCAACCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10634 T-016hul.A10_nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTGGTAGCTATACCATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAA.TTTCTGGCAAGC
ATTCGTTGGACCGGTGGTAGCACCAGCTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATGATGCAA
AAAAAACCGTTTATCTGCAGATGAATAGCCTGCGTCCGGAAGATACCGCAGTTTATTATTGTGTTGCAAGCCCGAC CGGTCGTGCATTTGCATATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10635 T-016hul.All„nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG
CGGTCGTACCTTTGGTAGCTATACCATGGGTTGGTrTCGTCAGGCACCGGGTAAAGAACGTGAATTTCTGGCAAGC
ATTCGTTGGACCGGTGGTAGCACCAGCTATGCAGATAGCGTTAAAGGTCGTnTACCATTAGCCGTGATGATGCAA
AAAAAACCGTTTATCTGCAGATGAATAGCCTGCGTCCGGAAGATACCGCAGTTTA7TATTGTGTTGCAAGCCCGAC CGGTCGTGCCTTTACCGCATGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10636 T-016Hul.A5.A7_nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTTTGAGGCTGAGCTGCGCCGCCAG
CGGCAGGACCTTCGGCAGCTACACCATGGGCTGGTTCAGACAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCCA
GCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACGAC
GCCAAGAAGACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGGCCAG CCCCGCCGGAGCTGCCTTCACCTACTGGGGCCAGGGCACACAGGTGACCGTGAGCAGC
SEQ SD NO: 10637 T~016Hul.A5.Al__nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTTTGAGGCTGAGCTGCGCCGCCAG
CGGCAGGACCTTCGGCAGCTACACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCCA
GCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGGGACGAC
GCCAAGAAGACCGTGTACCTGCAGATGAACAGCCTGAGACCCGAGGACACCGCCGTGTACTACTGCGCCGCCTCT CCCGCCGGAAGGGCCTTCACCTACTGGGGCCAGGGCACACAGGTGACCGTGAGCAGC
SEQ ID NO: 10638 T-016Hul.A5,A3mnucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGACTGAGCTGCGCCGCCA
GCGGCAGAACCTTCGGCAGCTACACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCC
AGCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACGA
CGCCAAGAAGACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGGCCG CCCCTGCTGGAAGGGCCTTCACCTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10639 T~016Hul.A5.A4_nucleot!de sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCAGGACCTTCGGCAGCTACACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGAGAGTTCCTGGCC
AGCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACGA CGCCAAGAAGACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGGCCA
GCGCCGCCGGAAGGGCCTTCACCTACTGGGGCCAGGGCACACAGGTGACCGTGAGCAGC
SEQ ID NO: 10640 T"016Hul.A5.A6_nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTTTGAGGCTGAGCTGCGCCGCCAG
CGGCAGGACCTTCGGCAGCTACACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCCA
GCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGGGACGAC GCCAAGAAGACCGTGTACCTGCAGATGAACAGCCTGAGACCCGAGGACACCGCCGTGTACTACTGCGTGGCCAGC CCCGCCGCTAGGGCCTTCACCTACTGGGGCCAGGGCACACAGGTGACCGTGAGCAGC
SEQ ID NO: 10641 T-016Hul.A7.Al__nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTTTGAGGCTGAGCTGCGCCGCCAG
CGGCAGAACCTTCGGCAGCTACACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCCA
GCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACGAC
GCCAAGAAGACCGTGTACCTGCAGATGAACAGCCTGAGACCCGAGGACACCGCCGTGTACTACTGCGCCGCCTCT CCCACCGGCGCTGCCTTCACCTACTGGGGCCAGGGCACACAGGTGACCGTGAGCAGC
SEQ ID NO: 10642 T-016Hul.A7.A3„nucieotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTTTGAGGCTGAGCTGCGCCGCCAG
CGGCAGGACCTTCGGCAGCTACACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCCA
GCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGGGACGAC
GCCAAGAAGACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGGCCGC CCCCACCGGAGCTGCCTTCACCTACTGGGGCCAGGGCACACAGGTGACCGTGAGCAGC
SEQ ID NO: 10643 T-016Hul.A7.A4„nuc!eotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGACTGAGCTGCGCCGCCA
GCGGCAGGACCTTCGGCAGCTACACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGAGAGTTCCTGGCC
AGCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACGA
CGCCAAGAAGACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGGCCA GCGCCACCGGCGCTGCCTTCACCTACTGGGGCCAGGGCACACAGGTGACCGTGAGCAGC
SEQ ID NO: 10644 T~016Hul.A7.A6__nucleQtide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCAGGACCTTCGGCAGCTACACCATGGGCTGGTTCAGACAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCC
AGCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGGGACGA CGCCAAGAAGACCGTGTATCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGGCCA GCCCCACCGCCGCTGCCTTCACCTACTGGGGCCAGGGCACACAGGTGACCGTGAGCAGC
SEQ ID NO: 10645 T-016Hul.Al.A3__nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCAGAACCTTCGGCAGCTACACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCC
AGCATCAGATGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACGA CGCCAAGAAGACCGTGTATCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCGC CCCCACAGGCAGAGCCnCACCTACTGGGGCCAGGGCACACAGGTGACCGTGAGCAGC
SEQ ID NO: 10646 T~016Hul.Al.A4__nucleot!de sequence GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTTTGAGGCTGAGCTGCGCCGCCAG CGGCAGAACCTTCGGCAGCTACACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGAGAGTTCCTGGCCA GCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACGAC GCCAAGAAGACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCTCT GCCACCGGAAGGGCCTTCACCTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10647 T~016Hul.Al.A6__nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA GCGGCAGGACCTTCGGCAGCTACACCATGGGCTGGTTCAGACAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCC AGCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACGA CGCCAAGAAGACCGTGTATCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAG CCCCACAGCTAGGGCCTTCACCTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10648 T-016Hul.A3.A4„nuc!eotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTTTGAGGCTGAGCTGCGCCGCCAG CGGCAGGACCTTCGGCAGCTACACCATGGGCTGGTTCAGACAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCCA GCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGATTOACCATCAGCAGGGACGAC GCCAAGAAGACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGGCCGC CGCCACAGGAAGGGCCTTCACCTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10649 T-016Hul.A3.A6__nucleotlde sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTTTGAGGCTGAGCTGCGCCGCCAG CGGCAGGACCTTCGGCAGCTACACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCCA GCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACGAC GCCAAGAAGACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGGCCGC CCCCACCGCCAGAGCCTTCACCTACTGGGGCCAGGGCACACAGGTGACCGTGAGCAGC
SEQ ID NO: 106S0 T-016Hul.A4.A6__nucleotlde sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGACTGAGCTGCGCCGCCA GCGGCAGGACCTTCGGCAGCTACACCATGGGCTGGTTCAGACAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCC AGCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATTAGCAGAGACGA CGCCAAGAAGACCGTGTACCTGCAGATGAACTCTCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGGCCAG CGCCACCGCCAGAGCCTTCACCTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10651 T4J08Hul.Gl_nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA ATTAATTGGAGCAATGCACGTACCAATTATGCAGATAGCGTTAAAGGTCGTnTACCATrAGCCGTGATAATGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTA'TTGTGGTGCAACCCCGAG CGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10652 T-008Hul.G2__nucteotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTn'AGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA ATTAATTGGAGCAATGCACGTACCAATTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTA7TATTGTGCAGGTACCCCGAG CGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC SEQ ID NO: 10653 T-008Hul.A3_nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTTAGCGATrATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA ATTAATTGGAGCAATGCACGTACCAATTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCAGCACCGAG CGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10654 T-008Hul.A4_nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTrAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA ATTAATTGGAGCAATGCACGTACCAATTATGCAGATAGCGnAAA.GGTCGTTTTACCATTAGCCGTGATAA.TGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCAACCGCAAG CGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10655 T-008Hul.A5„nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGATAGCGAA'nTG'n'GCAGCA ATTAATTGGAGCAATGCACGTACCAATTATGCAGATAGCGTTAAAGGTCGTnTACCATTAGCCGTGATAATGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCAACCCCGGC AGGTAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10656 T-008Hul.A6_nudeotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA ATTAATTGGAGCAATGCACGTACCAATTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTA'TTATTGTGCAGCAACCCCGAG CGCAAAAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ !D NO: 10657 T~008Hul.A7_nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTrAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA ATTAATTGGAGCAATGCACGTACCAATTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTrTAITA'TTGTGCAGCAACCCCGAG CGGTGCAGCATATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10658 T-008Hul.G8mnucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA ATTAATTGGAGCAATGCACGTACCAATTATGCAGATAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAATGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTAITA'TTGTGCAGCAACCCCGAG CGGTAAAGGTTATAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10659 T~008Hul.A9mniicleotide sequence
GAAGTTCAGCTGCJGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTrAGCGATTATGGTATGGGTTGGTn'CGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA ATTAATTGGAGCAATGCACGTACCAATTATGCAGATAGCGTTAAAGGTCGTnTACCATTAGCCGTGATAATGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCAACCCCGAG
CGGTAAAGCAGCAAGCTATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10660 T"008Hul.A10__nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTTAGCGATTATGGTATGGGTTGGTTTCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA ATTAATTGGAGCAATGCACGTACCAATTATGCAGATAGCGTTAAAGGTCG'TTTTACCATTAGCCGTGATAATGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCAACCCCGAG CGGTAAAGCATATGCATATTGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10661 T-008Hul.All__nucleotide sequence
GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAG CGGTCGTACCTTTAGCGATTATGGTATGGGTTGG'TTTCGTCAGGCACCGGGTAAAGATAGCGAATTTGTTGCAGCA ATTAATTGGAGCAATGCACGTACCAATTATGCAGATAGCGTTAAAGGTCGrnTACCATTAGCCGTGATAATGCAA AAAATACCGGTTATCTGCAGATGAATAGCCTGCGTGTTGAAGATACCGCAGTTTATTATTGTGCAGCAACCCCGAG CGGTAAAGCATATAGCGCATGGGGTCAGGGTACCCAGGTTACCGTTAGCAGC
SEQ ID NO: 10662 C-005Hul.Al„nucletoide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCTTGAGACTGAGCTGCGCCGCCA GCGGCTTCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGGGACAA CGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCAAGG GCAGGGGCAGCGGCAGCTACTACCCCTTCGACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10663 C-005Hul.A2_nucletoide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCn’GAGACTGAGCTGCGCCGCCA GCGGCTFCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CGCCAAGAACACCGTGTATCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGGCCGG CAGGGGCAGCGGAAGCTACTACCCCTTCGACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10664 C-005Hul.A3__nucletoide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA GCGGCTTCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGAAGGC CAGAGGCAGCGGCAGCTACTACCCCTTCGACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10665 C-005Hul.A4_nucletoide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGC7TCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGACCCGAGGACACCGCCGTGTACTACTGCGTGAAGG GCGCCGGCAGCGGCAGCTACTACCCCTTCGACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10666 C-005Hul.A5_nucletoide sequence GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC
GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA
CGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGAAGG
GGAGAGCCAGCGGCAGCTACTACCCCTTCGACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10667 G005Hul.A6__nucletoide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC
GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA
CGCCAAGAACACCGTGTATCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGAAGG
GGAGAGGCGCCGGCAGCTACTACCCCTTCGACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10668 C-005HuLA7„nuc!etoide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCTTGAGACTGAGCTGCGCCGCCA
GCGGCTTCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC
GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA
CGCCAAGAACACCGTGTATCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGAAGG
GCCGGGGCAGCGCCAGCTACTACCCCTTCGACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10669 C-005Hul.A8_nucletoide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC
GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA
CGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGAAGG
GCCGGGGCAGCGGCGCCTACTACCCCTTCGACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10670 C-005Hul.A9__nucletoide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC
GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA
CGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGAAGG GCCGGGGCAGCGGCAGCGCCTACCCCTTCGACGACTACTGGGGCCAAGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10671 C-005Hul.A10_nt!cletoide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC
GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA
CGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGAAGG GCCGGGGCAGCGGCAGCTACGCCCCCTTCGACGACTACTGGGGCCAAGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10672 C-005Hul.All_nucletoide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC
GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGAGACAA CGCCAAGAACACCGTGTATCTGCAGATGAACTCTCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGAAGGG AAGGGGCAGCGGCAGCTACTACGCCTTCGACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC SEQ ID NO: 10673 C-005Hul.A12__nucletosde sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCTTGAGACTGAGCTGCGCCGCCA
GCGGCTTCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CGCCAAGAACACCGTGTATCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGAAGG GCCGGGGCAGCGGCAGCTACTACCCCGCCGACGACTACTGGGGCCAAGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10674 C-005Hul.A13_nucletoide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGAAGG GCCGGGGCAGCGGCAGCTACTACCCCTTCGCCGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10675 C-005Hul.A14„nuc!etoide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCTTGAGACTGAGCTGCGCCGCCA
GCGGCTTCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGAAGG GGAGAGGCAGCGGCAGCTACTACCCCTTCGACGCCTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10676 C-005Hul.A15_nucletoide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGCCCAGCCTGGTGGAAGCn'GAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCTTCAGCAACTACGCCATGAGCTGGGCCAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGC GGCATCTACAGCGACAGCAGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGAAGG GCCGGGGCAGCGGCAGCTACTACCCCTTCGACGACGCCTGGGGCCAAGGCACCCAGGTGACCGTGAGCAGC
SEQ !D NO: 10677 C-010Hul.L8.Gl_nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG AGCATCAGCAGCACCGACGGCAGAACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CCCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGGCGCCAA GAGGCTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCTCTGTTCCTGGACGAGTACGACTACTG
GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10678 C-010Hul.L8.G2_nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTTTGAGGCTGAGCTGCGCCGCCAG
CGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGAGAGGGCGTGCTGA
GCATCAGCAGCACCGACGGCAGAACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGAGACAAC CCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGACCCGAGGACACCGCCGTGTACTACTGCGCCGGCAAG AGGCTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCTCTGTTCCTGGACGAGTACGACTACTGG GGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10679 C-010Hul.L8.A3_nucleotide sequence GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTTTGAGGCTGAGCTGCGCCGCCAG
CGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTGA GCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAAC CCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCGCC AGACTGGGCCCTCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCTCTGTTCCTGGACGAGTACGACTACTGG
GGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10680 C-010Hul.L8.A4__nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGAGACAA CCCCAAGAACACCGTGTATCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GGCCCTGGGCCCTCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCTCTGTTCCTGGACGAGTACGACTACTG
GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10681 C-010Hul.L8.A5„nuc!eotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CCCCAAGAACACCGTGTATCTGCAGCTGAACAGCCTGAGACCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGGGCCGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCTCTGTTCCTGGACGAGTACGACTACTG
GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10682 C-010Hul.L8.A6_nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGAGACAA CCCCAAGAACACCGTGTATCTGCAGCTGAACAGCCTGAGACCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGACTGGCCCCCTTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCTCTGTTCCTGGACGAGTACGACTACTG
GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10683 C-010Hul.L8.A7_nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTTTGAGGCTGAGCTGCGCCGCCAG
CGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTGA
GCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAAC CCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAAG AGGCTGGGCGCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCCTTGTTCCTGGACGAGTACGACTACTGG GGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10684 C-010Hul.A8„nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG
AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGAGACAA CCCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGGCTGGGCCCCGCCGTGCATAGGTACAGCCTGGAAGTGCTGACCCCTCTGTTCCTGGACGAGTACGACTACTG GGGCCAGGGCACCCAGGTGACCGTGAGCAGC SEQ ID NO: 10685 C-010Hul.L8.A9„nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGACAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG
AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGGGACAA CCCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGGCTGGGCCCCCTGGCCCATAGGTACAGCCTGGAAGTGCTGACCCCCTTGTTCCTGGACGAGTACGACTACTG GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10686 C-010Hul.L8.A10__nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG AGCATCAGCAGCACCGACGGCAGAACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGGGACAA CCCCAAGAACACCGTGTACCTGCAGCTGAACTCTCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGGCTGGGCCCCCTGGTGGCTAGGTACAGCCTGGAAGTGCTGACCCCCTTGTTCCTGGACGAGTACGACTACTG
GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10687 C-010Hul.L8.All__nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTn'GAGGCTGAGCTGCGCCGCCAG
CGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTGA
GCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAAC CCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAAG AGGCTGGGCCCCCTGGTGCACGCCTACAGCCTGGAAGTGCTGACCCCTCTGTTCCTGGACGAGTACGACTACTGG GGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10688 C-010Hul.L8.A12„nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGACTGAGCTGCGCCGCCA
GCGGCTFCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA
CCCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA
GAGGCTGGGCCCCCTGGTGCACAGGGCCAGCCTGGAAGTGCTGACCCCCTTGTTCCTGGACGAGTACGACTACTG GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10689 C-010Hul.L8.A13__nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGAGACAA CCCCAAGAACACCGTGTATCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGGCTGGGCCCCCTGGTGCACAGGTACGCCCTGGAAGTGCTGACCCCTCTGTTCCTGGACGAGTACGACTACTG
GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10690 C-010Hul.L8.A14__nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CCCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGACCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGGCTGGGCCCCCTGGTGCACAGGTACAGCGCCGAGGTGCTGACCCCTCTGTTCCTGGACGAGTACGACTACTG
GGGCCAGGGCACCCAGGTGACCGTGAGCAGC SEQ ID NO: 10691 C-010Hul.L8.A15„nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG
AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CCCCAAGAACACCGTGTATCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGGCTGGGCCCCCTGGTGCACAGATACAGCCTGGCCGTGCTGACCCCTCTGTTCCTGGACGAGTACGACTACTG GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10692 C-010Hul.L8.A16_nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGACTGAGCTGCGCCGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG
AGCATCAGCAGCACCGACGGCAGAACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CCCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGACCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGGCTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGCCCTGACCCCTCTGTTCCTGGACGAGTACGACTACTG GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10693 C-010Hul.L8.A17„nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGACTGAGCTGCGCCGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG
AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CCCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGACTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGGCCACCCCTCTGTTCCTGGACGAGTACGACTACTG GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10694 C-010Hul.L8.A18__nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG
AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CCCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGACTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGGCCCCTCTGTTCCTGGACGAGTACGACTACTG GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10695 C-010Hul.L8.A19_nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTTTGAGGCTGAGCTGCGCCGCCAG
CGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGAGAGGGCGTGCTGA
GCATCAGCAGCACCGACGGCAGAACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAAC CCCAAGAACACCGTGTATCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAAG AGGCTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCGCCCTGTTCCTGGACGAGTACGACTACTGG GGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10696 C-010Hiil.L8.A20__nucteotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA CCCCAAGAACACCGTGTACCTGCAGCTGAACTCTCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGGCTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCCGCCTTCCTGGACGAGTACGACTACTG
GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10697 C-010Hul.L8.A21„nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG
AGCATCAGCAGCACCGACGGCAGAACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA
CCCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGGCTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCTCTGGCCCTGGACGAGTACGACTACTG GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10698 C-010Hul.L8.A22_nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG
AGCATCAGCAGCACCGACGGCAGAACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA
CCCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA
GAGGCTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCTCTGTTCGCCGACGAGTACGACTACTG GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10699 C-010Hul.L8.A23__nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGATCTTTGAGGCTGAGCTGCGCCGCCAG
CGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGACAGGCCCCCGGCAAGGAGAGAGAGGGCGTGCTGA
GCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGAGACAAC
CCCAAGAACACCGTGTACCTGCAGCTGAACTCTCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAAG AGGCTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCTCTGTTCCTGGCCGAGTACGACTACTGG GGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10700 C-010Hul.L8.A24__nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG
AGCATCAGCAGCACCGACGGCAGAACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA
CCCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGACCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA
GAGGCTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCTCTGTTCCTGGACGCCTACGACTACTG GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10701 C-010Hul.L8.A2S_nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG
AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAA
CCCCAAGAACACCGTGTATCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA GAGGCTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCTCTGTTCCTGGACGAGGCCGACTACTG GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10702 C-010Hul.L8.A26__nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGACTGAGCTGCGCCGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAAGGCGTGCTG
AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAA CCCCAAGAACACCGTGTACCTGCAGCTGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA
GAGGCTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCCTTGTTCCTGGACGAGTACGCCTACTG
GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10703 C-010Hul.L8.A27_nucleotide sequence
GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGTGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCTTCACCCTGGACTACTACGCCATCGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGAGAGGGCGTGCTG
AGCATCAGCAGCACCGACGGCAGGACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGAGACAA
CCCCAAGAACACCGTGTATCTGCAGCTGAACAGCCTGAGACCCGAGGACACCGCCGTGTACTACTGCGCCGCCAA
GAGGCTGGGCCCCCTGGTGCACAGGTACAGCCTGGAAGTGCTGACCCCTCTGTTCCTGGACGAGTACGACGCCTG
GGGCCAGGGCACCCAGGTGACCGTGAGCAGC
SEQ ID NO: 10704 T-022 __nucleotide sequence
GACGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGAGGAAGCTTGAGGCTGAGCTGCGCTGCCA
GCGGCAGAACCTTCGGCAGCTACACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCC
AGCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACGA
CGCCAAGAAGACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGGCCA
GCCCCACCGGAAGGGCCTTCACCTACTGGGGCCAGGGCACCCAGGTGACCGTGCCCGCCGGAGGCGGATCTGAC
AAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGGCTGCTGGAGGACCTTCTGTGTTCCTGTTCCCTCCCAAGCC
CAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCG
AGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTA
CAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGT
GCAAGGTGAGCAACAAGGCCCTGGCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGA
GCCCCAGGTGTACACCCTGCCCCCTAGCAGGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGA
AGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACC
CCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAG
GGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAAAAATCTCTTTCTCTGTCTC
CTGGAAAATGA
SEQ ID NO: 10706 T-023 ^nucleotide sequence
GACGTCCAGCTGCTCGAGTCAGGAGGCGGGCrTGTGCAACCTGGCGGCAGCCTGAGACTn'CTTGTGCCGCAAGT
GGAAGGACCTTTGGTAGCTATACCATGGGTTGGTTTCGGCAGGCTCCCGGCAAAGAACGGGAGTTCCTGGCn'CT
ATAAGGTGGACAGGAGGCAGCACCAGCTATGCCGACTCCGTTAAGGGGAGG'nTACTATTAGTCGGGATGATGC
AAAGAAGACAGTTTATCTGCAAATGAACAGCCTTCGCCCAGAAGATACTGCTGTGTACTATTGTGTGGCCTCACCC
GCGGGCGCCGCATTCACCTACTGGGGGCAGGGAACCCAGGTCACAGTACCAGCCGGAGGCGGCTCCGATAAAAC
TCATACGTGTCCCCCATGCCCTGCCCCGGAAGCCGCTGGGGGACCTTCAGTTTTCCTGTTTCCACCCAAGCCAAAA
GACACACTCATGATCTCCAGAACCCCAGAAGTAACATGCGTCGTGGTGGACGTGTCTCATGAGGACCCAGAGGTC
AAATTCAATTGGTACGTCGATGGCGTCGAGGTACACAACGCTAAAACTAAGCCCCGCGAAGAGCAATACAACTCT
ACGTACCGAGTGGTGTCTGTCCTTACAGTGCTGCACCAGGACTGGCTGAACGGGAAAGAGTACAAGTGCAAAGT
GTCAAACAAGGCACTCGCGGCCCCTATCGAAAAGACCATTTCCAAGGCAAAAGGCCAGCCTCGAGAGCCCCAGGT
TTATACACTGCCGCCCTCCAGAGAAGAAATGACTAAGAATCAGGTTAGTCTGACGTGCCTCGTGAAAGGGTTCTAC
CCCTCTGACATCGCCGTAGAGTGGGAGTCCAATGGGCAACCGGAGAATAATTATAAGACTACCCCTCCTGTTCTCG
ATrCAGATGGTAGTTTCTTTCTGTACAGCAAGCTTACCGTGGACAAATCTCGCTGGCAGCAGGGAAATGTGTTTAG
CTGTTCCGTGATGCATGAGGCTTTGCACAACCACTATACTCAGAAGAGCTTGTCCTTGAGTCCTGGTAAGTGA SEQ ID NO: 10708 T-024_niideotide sequence
GATGTCCAGCTGTTGGAAAGTGGCGGAGGGCTGGTGCAGCCAGGCGGCTCTCTTAGACTCTCATGCGCAGCAAGT GGACGCACGTTCGGTTCTTACACCATGGGCTGGTTCCGGCAGGCTCCCGGTAAAGAGAGGGAGTTCCTGGCATCC ATCCGCTGGACTGGCGGATCCACTTCTTATGCCGATTCCGTCAAGGGGCGGTTTACAATCTCACGAGATGACGCTA AAAAAACCGTTTATCTTCAAATGAATTCCCTGAGGCCTGAGGACACGGCGGTGTATTACTGCGCCGCATCCCCCGC CGGTCGAGCCTTTACTTACTGGGGACAAGGGACACAGGTGACAGTACCCGCCGGCGGCGGGAGCGACAAGACCC ATACTTGCCCACCCTGTCCCGCTCCCGAAGCCGCTGGTGGGCCATCTGTATTCTTGTn'CCACCGAAGCCAAAAGAC ACTCTCATGATCAGCAGGACACCTGAAGTTACCTGTGTGGTTGTTGACGTGAGCCACGAAGATCCTGAGGTGAAG TTTAATTGGTACGTGGATGGCGTGGAAGTGCACAACGCCAAAACCAAACCTCGGGAGGAGCAGTATAACTCAACA TATAGAGTAGTGTCTGTTCTTACGGTGTTGCATCAGGATTGGCTGAATGGGAAAGAGTACAAGTGTAAAGTAAGC AACAAAGCTCTGGCCGCCCCGATTGAGAAGACCATTTCCAAGGCTAAGGGACAGCCTAGGGAGCCTCAGGTCTAC
ACCCTGCCCCCTAGTCGCGAGGAGATGACAAAGAACCAAGTCAGCTTGACCTGCCTCGTGAAGGGGTTCTATCCG TCTGACATAGCGGTCGAATGGGAATCTAATGGCCAACCCGAAAATAATTACAAGACTACTCCACCAGTGCTCGACT CCGATGGAAGTTTCTTTCTGTACAGCAAGTTGACCGTGGACAAATCAAGATGGCAGCAGGGAAACGTCTTTAGCT GTAGCGTCATGCACGAGGCACTGCACAACCATTATACACAGAAGTCACTCTCCCTGAGTCCAGGCAAGTGA
SEQ ID NO: 10710 T-025_jwdeotide sequence
GACGTTCAACTGCTGGAAAGTGGCGGAGGTCTGGTCCAACCCGGGGGCAGCCTGCGCCTCAGCTGTGCAGCTAG TGGCAGAACCTTCGGCTCATACACAATGGGTTGGTTCCGCCAAGCCCCCGGGAAGGAACGGGAATTTCTCGCCTC CATCAGATGGACAGGAGGTAGTACCTCTTACGCCGACTCTGTCAAGGGGCGGTTCACTATAAGCAGGGACGACGC AAAGAAGACTGTGTACCTGCAGATGAATTCACTTCGCCCTGAGGATACAGCAGTCTACFACTGCGTGGCCGCCCCT GCCGGGAGGGCTTTCACATATTGGGGTCAGGGGACTCAGGTGACCGTACCAGCCGGAGGAGGATCAGATAAGAC TCACACGTGCCCTCCATGCCCAGCTCCGGAAGCTGCAGGCGGACCTAGCGTTTTTCTTTTTCCACCGAAGCCCAAA GACACCCTCATGATTAGCAGAACTCCGGAAGTGACCTGTGTAGTCGTGGATGTCTCCCACGAGGATCCCGAGGTG AAATTCAATTGGTACGTCGATGGAGTTGAAGTTCATAACGCCAAGACGAAGCCTCGGGAGGAGCAATACAATTCC ACATACAGGGTGGTGTCAGTGCTCACGGTGTTGCATCAGGACTGGCTGAACGGGAAAGAATATAAATGCAAGGT ATCCAATAAGGCGCTCGCTGCTCCCATTGAGAAGACCATCTCCAAAGCGAAAGGCCAGCCTCGAGAGCCCCAGGT
TTATACTCTCCCTCCCTCACGAGAAGAGATGAC^AAAAACCAGGTCAGCTTGACCTGTCTCGTGAAAGGCTTTTATC CATCCGACATCGCCGTTGAGTGGGAGAGCAACGGCCAGCCAGAGAATAATTATAAGACCACCCCTCCCGTGTT'GG ATAGTGACGGCTCTTrmTCTGTATTCTAAACTGACAGTGGATAAGTCTAGGTGGCAGCAGGGCAACGTGTTCTC TTGTTCTGTAATGCATGAGGCACTGCACAACCACTATACTCAGAAGTCCCTTAGCCTGAGTCCAGGGAAATGA
SEQ ID NO: 10712 T"026_nucleotide sequence
GACGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCTGGAGGATCTCTGAGGCTGAGCTGCGCCGCCAG CGGCAGGACCTTCGGCAGCTACACCATGGGCTGGTTCAGGCAGGCCCCCGGCAAGGAGAGGGAGTTCCTGGCCA GCATCAGGTGGACCGGCGGCAGCACCAGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACGAC GCCAAGAAGACCGTGTACCTGCAGATGAACAGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGCGTGGCCAG CGCCGCTGGAAGGGCCTTCACCTACTGGGGCCAGGGCACCCAGGTGACCGTGCCCGCCGGAGGCGGATCTGACA AGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGGCTGCTGGAGGACCTTCTGTGTTCCTGTTCCCTCCCAAGCCC AAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGA GGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTAC AACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTG CAAGGTGAGCAACAAGGCCCTGGCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGC
CCCAGGTGTACACCCTGCCCCCTAGCAGGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAG
GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCT
CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGG
CAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAAAAATCTTTGTCTTTGTCTCCTG GAAAATGA
SEQ !D NO: 10714 T-027__nucleotide sequence
GACGTGCAGCTCCTGGAGTCTGGGGGCGGGCTCGTTCAGCCGGGCGGGTCATTGCGACTCAGTTGTGCTGCGTCT
GGCGGCACATTTGGCTCATACACTATGGGATGGTTTCGGCAGGCTCCTGGCAAGGAAAGAGAGTTCCTGGCTAGC
ATCCGCTGGACTGGGGGTAGCACCTCATATGCCGATTCCGTGAAGGGCAGGTTTACCATTAGCAGAGACGACGCA
AAAAAGACAGTATATCTTCAAATGAACTCTCTGCGCCCAGAGGATACAGCAGTGTACTATTGTGTGGCTAGCCCTG
CAGCCAGGGCCTTTACCTACTGGGGGCAAGGAACACAGGTGACGGTGCCCGCCGGCGGGGGTAGTGATAAAACA
CACACCTGCCCTCCTTGTCCTGCACCGGAAGCTGCTGGCGGTCCATCTGTATTCCTGTrTCCCCCTAAACCCAAAGA
TACCCTGATGATCTCCCGGACACCCGAGGTAACCTGTGTGGTCGTCGACGTCAGCCACGAGGACCCTGAGGTCAA
GTTCAATTGGTACGTGGATGGAGTGGAAGTTCACAACGCCAAGACTAAGCCACGGGAGGAACAGTACAATAGCA
CCTATAGGGTCGTGAGCGTGCTGACTGTGTTGCATCAAGATTGGCTTAACGGAAAGGAGTATAAGTGCAAGGTTT
CCAACAAAGCACTGGCGGCCCCCATTGAAAAGACGATCTCCAAAGCCAAAGGACAGCCCAGAGAGCCTCAGGTGT
ACACACTCCCACCATCACGAGAGGAAATGACCAAGAACCAGGTTTCCCTTACTTGCCTGGTGAAGGGGTTTTATCC
CAGTGATATAGCCGTTGAATGGGAGAGCAACGGCCAACCAGAGAATAATTATAAGACCACTCCGCCCGTTCn'GA
CTCCGACGGAAGTTTCTTCCTn'ACTCTAAGCTGACTGTCGACAAAAGTAGGTGGCAGCAGGGTAATGTCTTCTCCT
GCTCAGTAATGCATGAAGCCCTCCACAATCATTACACGCAGAAATCTTTGTCCCTGTCTCCAGGAAAATGA
SEQ ID NO: 10716 T-028__nucleotide sequence
GACGTCCAGCTGCTGGAATCTGGCGGTGGCCTTGTCCAGCCTGGAGGGTCCrrGAGACTCAGTTGCGCTGCCAGT
GGGCGAACGTTTGGGTCATACACCATGGGATGGTTTAGGCAGGCCCCGGGTAAAGAAAGAGAGTTCCTTGCTAG
TATCCGCTGGACCGGGGGTAGCACAAGCTACGCTGACTCCGTAAAGGGCCGCTTCACCATATCTCGCGATGATGC
CAAGAAGACAGTGTACTTGCAAATGAACTCACTGCGACCCGAGGACACGGCCGTGTATTACTGTGTTGCAAGTGC
AACTGGCGCGGCCTTCACTTACTGGGGCCAGGGCACCCAGGTGACAGTGCCAGCCGGAGGCGGCAGCGATAAAA
CTCACACCTGTCCACCGTGCCCTGCTCCTGAGGCGGCAGGTGGACCCAGTGTGTTCCTGTTCCCACCCAAGCCAAA
AGACACTCTGATGATCTCTAGGACGCCTGAGGTAACCTGCGTCGTCGTTGACGTTAGCCATGAGGACCCTGAGGTT
AAGTTTAATTGGTATGTCGATGGCGTGGAGGTGCATAACGCCAAAACAAAGCCTCGGGAAGAACAGTACAACTCT
ACCTATAGGGTTGTGAGCGTGTTGACTGTGCTGCACCAGGATTGGTTGAATGGGAAGGAGTATAAGTGCAAGGT
ATCTAATAAAGCTCTCGCCGCCCCCATTGAAAAAACCATTTCCAAGGCTAAAGGACAGCCTAGAGAGCCCCAAGTA
TATACACTTCCACCCTCACGGGAAGAGATGACCAAGAATCAGGTGTCCCTGACTTGTCTCGTCAAGGGGTTTTACC
CCAGCGACATCGCAGTGGAGTGGGAATCCAACGGGCAACCGGAAAACAATTATAAAACAACTCCCCCAGTGCTCG
ATTCCGACGGCTCCTTCTrTCTTTATTCTAAGCTGACCGTTGATAAATCACGGTGGCAGCAGGGAAATGTGTTTAGC
TGTTCAGTCATGCACGAGGCACTCCATAACCACTACACACAAAAATCTCTGTCCCTGAGCCCAGGAAAGTGA
SEQ ID NO: 10718 T-029_jwdeotide sequence
GACGTTCAACTCCTTGAGTCCGGAGGGGGCCTGGTTCAGCCAGGAGGCTCCCTTCGCCTCTCTrGTGCTGCTAGTG
GTCGGACCTTCGGATCCTACACCATGGGGTGGTTCAGACAAGCACCTGGTAAGGAGAGGGAGTTTCTGGCCTCCA
TCCGGTGGACAGGCGGATCAACCTCCTATGCGGACAGCGTCAAGGGGAGGTTCACAATTAGCCGCGACGATGCT AAAAAGACTGTCTACCTCCAGATGAACTCACTGAGGCCCGAAGACACCGCCGTATATTATTGCGCTGCTTCCGCCA
CCGGCCGGGCCTTTACTTACTGGGGACAGGGGACCCAGGTGACAGTGCCCGCCGGGGGTGGAAGTGATAAGACT
CACACATGTCCGCCCTGTCCTGCACCGGAGGCAGCCGGGGGCCCATCTGTGTTCCTGTTTCCCCCAAAACCGAAAG
ACACTCTTATGATTTCCAGAACACCTGAGGTTACTTGCGTGGTCGTTGATGTGTCCCATGAGGATCCTGAAGTCAA
ATTTAATTGGTATGTGGATGGCGTGGAGGTGCACAATGCAAAGACTAAGCCCCGAGAGGAGCAATACAATTCAAC
ATACAGGGTGGTGTCAGTACTGACCGTACTGCATCAGGATTGGTTGAATGGAAAAGAGTACAAGTGCAAGGTGA
GCAATAAGGCACTTGCTGCCCCAATAGAAAAGACCATCTCTAAGGCGAAAGGCCAGCCTAGAGAACCCCAGGTCT
ATACGCTGCCCCCTAGTCGAGAGGAAATGACAAAAAACCAAGTCTCTTTGACCTGTCTCGTCAAGGGCTTTTATCC
TAGCGACATCGCCGTGGAATGGGAAAGCAACGGCCAGCCAGAAAATAACTACAAGACGACACCCCCAGTGCTCG
ATAGTGACGGTTCTTTTTTCCTGTACTCAAAGCTTACGGTTGACAAAAGCCGCTGGCAGCAGGGGAACGTGTTCAG
CTGCTCTGTCATGCACGAGGCCCTGCATAACCACTATACTCAGAAATCTCTGAGTTTGAGCCCAGGCAAATGA
SEQ ID NO: 10720 monomer V-body in the fusion protein T-022_amino acid sequence
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASPTGRAFTYWGQGTQVTVPAG
SEQ ID NO: 10721 monomer V-body in the fusion protein T-023 _amino acid sequence
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASPAGAAFTYWGQGTQVTVPAG
SEQ ID NO: 10722 monomer V-body in the fusion protein T-024_amino acid sequence
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCAASPAGRAFTYWGQGTQVTVPAG
SEQ ID NO: 10723 monomer V-body in the fusion protein T-025_amino acid sequence
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVAAPAGRAFTYWGQGTQVTVPAG
SEQ ID NO: 10724 monomer V-body in the fusion protein T-026 amino acid sequence
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQM NSLRPEDTAVYYCVASAAG RAFTYWGQGTQVTVPAG
SEQ ID NO: 10725 monomer V-body in the fusion protein T-027_amino acid sequence
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASiRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQM NSLRPEDTAVYYCVASPAA RAFTYWGQGTQVTVPAG
SEQ ID NO: 10726 monomer V-body in the fusion protein T-02g__amino acid sequence
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASATGAAFTYWGQGTQVTVPAG
SEQ ID NO: 10727 monomer V-body in the fusion protein T-029__amino acid sequence
DVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCAASATGRAFTYWGQGTQVTVPAG SEQ ID NO: 10790 anti-CD25 CDR3 consensus sequence
(A/V/S)(K/T)G(R/A/K)(G/H/N/R)SG(S/G)YYP(W/F/L)D(D/E)(Y/V) anti-CD25 CDR3 consensus sequence
(A/V/S){A/K/T){A/G)(A/R/K)(A/G/H/N/R){A/S)(A/G)(A/S/G)(A/Y)(A/Y)(A/P)(A/W/F/L)(A/D)(A/D/E){A/Y/V)
SEQ ID NO: 10811 T-016Hul.S5.A4_CDR3
VASASGRAFTY
SEQ ID NO: 10812 T-016Hul.S5.A4, full-length VHH, humanized
EVQLLESGGGLVQPGGSLRLSCAASGRTFGSYTMGWFRQAPGKEREFLASIRWTGGSTSYADSVKGRFTISRDDAKKT
VYLQMNSLRPEDTAVYYCVASASGRAFTYWGQGTQVTVSS
SEQ ID NO: 10813 Anti-TNFR2 CDR3 consensus
AADSDL(S/R)TV(V/T)VGPHDY
Anti-TNFR2 CDR3 consensus
(T/A/V)(A/G)(s/A)(A/P)(A/T)(A/G)(A/R)A(A/F)(T/N/A)(A/Y)

Claims

□aims
1. A multispecific antigen-binding protein comprising: a) one or more antigen-binding domains that specifically bind to tumor necrosis factor receptor 2 (TNFR2); and b) one or more antigen-binding domains that specifically bind to cluster of differentiation 25 (CD25).
2. The multispecific antigen-binding protein of claim 1, wherein the multispecific antigen-binding protein is bivalent, trivalent, tetravalent, pentavalent, hexavalent, septivalent, or octavalent for both antigens.
3. The multispecific antigen-binding protein of claim 1 or claim 2, wherein the multispecific antigenbinding protein comprises at least two, at least four, or at least six TNFR2 antigen-binding domains, and at least two CD25 antigen-binding domains.
4. The multispecific antigen-binding protein of any of claims 1-3, wherein the one or more anti-TNFR2 antigen-binding domains comprise a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from: a) (Y/F)YQ(S/A)LS(T/S)(P/A)N(Y/F)GQ(V/T)F (SEQ ID NO: 60}; b) AADSDL(S/R)TV(V/T)(V/T)GPHDY (SEQ. ID NO: 61); c) AKDAG(S/G)WG(T/R)GPFG(Y/F)(E/D)YDY (SEQ ID NO: 62); d) A(A/G)(T/A/S)(P/L)(S/T)GKAY(T/S)Y (SEQ ID NO: 10387); e) ATPGPY(T/S/M)YCAPYGSSWSRGYDY (SEQ ID NO: 64); f) ARV(R/G)G(T/S/A)PY(E/D)Y(N/G)Y (SEQ ID NO: 65); g) (T/A/V)A(S/A)PTGRAF(T/N/A)Y (SEQ ID NO: 66); h) AGSAFDF (SEQ ID NO: 42); i) S(V/M)(V/L)GRDM(M/V)TY (SEQ ID NO: 67); j) AVGDFEGELVLKGDY (SEQ ID NO: 6635); and k) AAD(L/V)G(F/V/Y)LY(A/T/V)DYV(P/R)LH(M/T)HHFGS (SEQ ID NO: 7086); wherein one or more non-alanine residues in the CDR3 sequence are optionally replaced with an alanine, and/or one or more alanine residues In the CDR3 sequence are optionally replaced with a glycine.
5. The multispecific antigen-binding protein of ciaim 4, wherein the CDR3 of the one or more anti- TNFR2 antigen-binding domains comprises an amino acid sequence a). {T/A/V)(A/G)(S/A)(A/P){A/T/S)(A/G)(A/R)A{A/F)(T/N/A)(A/Y}; or b). (A/G)(A/G)(T/A/S)(A/P/L)(A/S/T)(A/G)(A/K)(A/G)(A/Y)(A/T/S)(A/Y).
6. The multispecific antigen-binding protein of claim 4, wherein the CDR3 of the one or more anti- TNFR2 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 3, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 63, 6635, 6639, 6643, 7093, 7099, 7289-7292, 7333, 10374, 10410-10433, 10435-10455, and 10811.
7. The multispecific antigen-binding protein of any of claims 1-6, wherein the one or more anti-TNFR2 antigen-binding domains further comprise a CDR1 comprising an amino acid sequence selected from: a) GS!(V/F)(R/S)(T/A)(N/D)(S/G/A); b) GFT(F/L)DD(I/Y)A (SEQ ID NO: 69); c) GFTFS(S/R/G)YA (SEQ ID NO: 70); d) GRTFSDYG (SEQ ID NO: 16); e) G(L/F)TLDYYA (SEQ ID NO: 71); f) GF(T/N)FSMYS (SEQ ID NO: 72); g) GRTF(G/R/S)(N/S)(Y/L)(T/F) (SEQ ID NO: 73); h) GASLSRNA (SEQ ID NO: 40); i) GS(I/T)FRFPP (SEQ ID NO: 74); and k). G(F/V)(S/T)LD(D/Y)(H/Y)T (SEQ ID NO: 7088).
8. The multispecific antigen-binding protein of claim 7, wherein the CDR1 of the one or more anti- TNFR2 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 1, 5, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 6633, 6637, 6641, 7089, and 7281-7284.
9. The multispecific antigen-binding protein of any one of claims 1-8, wherein the one or more anti- TNFR2 antigen-binding domains further comprise a CDR2 comprising an amino acid sequence selected from: a) IRSDGF(T/I) (SEQ ID NO: 75); b) l(Y/F)SY(S/G)(S/P) NT (SEQ ID NO: 76); c) i(Y/S)(S/D)DGS(E/D)T (SEQ ID NO: 77); d) INWS(N/Q/E/S)(G/A)RT (SEQ ID NO: 10409); e) l(S/N)(V/T)(S/G)DGST (SEQ ID NO: 78); f) IDT(R/G)GST (SEQ iD NO: 79); g) IR(W/R/Y)(T/P)G(G/L)(S/I)T (SEQ ID NO: 80); h) IYDDGET (SEQ ID NO: 41); i) LTSGGST (SEQ ID NO: 45);and j) l(N/S)SNDG(S/T)(T/V) (SEQ ID NO: 7087).
10. The multispecific antigen-binding protein of claim 9, wherein the CDR2 of the one or more anti- TNFR2 antigen-binding domains comprises an amino acid sequence selected from SEQ ID Nos: 2, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 6634, 6638, 6642, 7096, 7268, 7285-7288, and 10388-10393.
11. The multispecific antigen-binding protein of any one of claims 4, 7, and 9, wherein the one or more anti-TNFR2 antigen-binding domains comprise i) a CDR1 comprising an amino acid sequence of GSI(V/F)(R/S)(T/A)(N/D)(S/G/A), a CDR2 comprising an amino acid sequence of SEQ ID NO: 75, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 60; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 69, a CDR2 comprising an amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 61; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 70, a CDR2 comprising an amino acid sequence of SEQ ID NO: 77, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 62; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10409, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10387; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 71, a CDR2 comprising an amino acid sequence of SEQ ID NO: 78, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 64; vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 72, a CDR2 comprising an amino acid sequence of SEQ ID NO: 79, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 65; vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 73, a CDR2 comprising an amino acid sequence of SEQ ID NO: 80, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 66; x) a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 42; ix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 74, a CDR2 comprising an amino acid sequence of SEQ ID NO: 45, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 67; x) a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6635; xi). a CDR1 comprising an amino acid sequence of SEQ ID NO: 7088, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7087, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7086; xii). a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10409, and a CDR3 comprising an amino acid sequence of (A/G)(A/G)(T/A/S)(A/P/L)(A/S/T)(A/G)(A/K)(A/G)(A/Y)(A/T/S)(A/Y); or xiii) , a CDR1 comprising an amino acid sequence of SEQ ID NO: 73, a CDR2 comprising an amino acid sequence of SEQ ID NO: 80, and a CDR3 comprising an amino acid sequence of (T/A/V)(A/G)(S/A){A/P)(A/T/S)(A/G)(A/R)A(A/F)(T/N/A){A/Y).
12, The multispecific antigen-binding protein of claim 11, wherein the one or more anti-TNFR2 antigenbinding domains comprise a) a CDR1 comprising an amino acid sequence of SEQ ID NO: 69, a CDR2 comprising an amino acid sequence of SEQ ID NO: 76, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 61; b) a CDR1 comprising an amino acid sequence of SEQ. ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7263, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10387; c) a CDR1 comprising an amino acid sequence of SEQ ID NO: 73, a CDR2 comprising an amino acid sequence of SEQ ID NO: 80, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 66; d) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7263, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 63; or e) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7263, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7333,
13. The multispecific antigen-binding protein of any one of claims 4, 7, 9, and 11, wherein the one or more anti-TNFR2 antigen-binding domains comprise i) a CDR1 comprising an amino acid sequence of GSI (V/F)(R/S)(A/T)(N/D)(G/A), a CDR2 comprising an amino acid sequence of IRSDGFT (SEQ ID NO: 2), and a CDR3 comprising an amino acid sequence of YYQ(S/A)LSSPNYGQ(V/T)F (SEQ ID NO: 7270); ii) a CDR1 comprising an amino acid sequence of GFT(F/L)DD(I/Y)A (SEQ ID NO: 69), a CDR2 comprising an amino acid sequence of i(Y/F)SY(S/G)(S/P) NT (SEQ ID NO: 76), and a CDR3 comprising an amino acid sequence of AADSDLSTW(V/T)GPHDY (SEQ ID NO: 7271); iii) a CDR1 comprising an amino acid sequence of GFTFSRYA (SEQ ID NO: 12), a CDR2 comprising an amino acid sequence of ISDDGSDT (SEQ ID NO: 13), and a CDR3 comprising an amino acid sequence of AKDAGSWGTGPFGYEYDY (SEQ ID NO: 14); iv) a CDR1 comprising an amino acid sequence of GRTFSDYG (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of INWSN(G/A)RT (SEQ ID NO: 7268), and a CDR3 comprising an amino acid sequence of A(A/G)(T/A/S)(P/L)(S/T)GKAY(T/S)Y (SEQ ID NO: 10387); v) a CDR1 comprising an amino acid sequence of GLTLDYYA (SEQ ID NO: 20), a CDR2 comprising an amino acid sequence of ISTSDGST (SEQ ID NO: 21), and a CDR3 comprising an amino acid sequence of ATPGPYTYCAPYGSSWSRGYDY (SEQ ID NO: 22); vi) a CDR1 comprising an amino acid sequence of GF(T/N)FSMYS (SEQ ID NO: 72), a CDR2 comprising an amino acid sequence of IDT(R/G)GST (SEQ ID NO: 79), and a CDR3 comprising an amino acid sequence of ARV(G/R)G(T/A)P¥EY(N/G)Y (SEQ ID NO: 7273); vii) a CDR1 comprising an amino acid sequence of GRTF(G/S)S(Y/L)(T/F) (SEQ ID NO: 7274), a CDR2 comprising an amino acid sequence of IR(W/R/Y)(T/P)G(G/L)(S/I)T (SEQ ID NO: 80), and a CDR3 comprising an amino acid sequence of (A/V)A(A/S)PTGRAF(T/N)Y (SEQ ID NO: 7276); x) a CDR1 comprising an amino acid sequence of GASLSRNA (SEQ ID NO: 40), a CDR2 comprising an amino acid sequence of IYDDGET (SEQ ID NO: 41), and a CDR3 comprising an amino acid sequence of AGSAFDF (SEQ ID NO: 42); ix) a CDR1 comprising an amino acid sequence of GS(T/I)FRFPP (SEQ ID NO: 7277), a CDR2 comprising an amino acid sequence of LTSGGST (SEQ ID NO: 45), and a CDR3 comprising an amino acid sequence of SVLGRDM(M/V)TY (SEQ ID NO: 7275); x) a CORI comprising an amino acid sequence of GFTLDDYA (SEQ ID NO: 6633), a CDR2 comprising an amino acid sequence of IFSYSSNT (SEQ ID NO: 6634), and a CDR3 comprising an amino acid sequence of AVGDFEGELVLKGDY (SEQ ID NO: 6635); or xi) a CDR1 comprising an amino acid sequence of GFTLDYYT (SEQ ID NO: 6637), a CDR2 comprising an amino acid sequence of ISSNDGSV (SEQ ID NO: 6638), and a CDR3 comprising an amino acid sequence of AADLGYLYVDYVRLHTHHFGS (SEQ ID NO: 6639).
14. The multispecific antigen-binding protein of claim 11 or claim 12, wherein the antigen-binding protein comprise comprises a) a CDR1 comprising an amino acid sequence of GFT(F/L)DD(I/Y)A (SEQ ID NO: 69), a CDR2 comprising an amino acid sequence of l(Y/F)SY(S/G)(S/P) NT (SEQ ID NO: 76), and a CDR3 comprising an amino acid sequence of AADSDLSTW(V/F)GPHD¥ (SEQ ID NO: 7271); b) a CDR1 comprising an amino acid sequence of GRTFSDYG (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of INWSN(G/A)RT (SEQ ID NO: 7268), and a CDR3 comprising an amino acid sequence of A(A/G)(T/A/S)(P/L)(S/T)GKAY(T/S)Y (SEQ ID NO: 10387); or c) a CDR1 comprising an amino acid sequence of GRTF(G/S)S(Y/L)(T/F) (SEQ ID NO: 7274), a CDR2 comprising an amino acid sequence of IR(W/R/Y)(T/P)G(G/L)(S/I)T (SEQ ID NO: 80), and a CDR3 comprising an amino acid sequence of (A/V)A(A/S)PTGRAF(T/N)Y (SEQ ID NO: 7276).
15. The multispecific antigen-binding protein of any one of claims 1-14, wherein the one or more anti-
TNFR2 antigen-binding domains comprise: i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 3; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 5, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NQ: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 12, a CDR2 comprising an amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 14; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 20, a CDR2 comprising an amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 22; vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 24, a CDR2 comprising an amino acid sequence of SEQ ID NO: 25, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 26; viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 28, a CDR2 comprising an amino acid sequence of SEQ ID NO: 29, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 30; ix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 33, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34; x) a CDR1 comprising an amino acid sequence of SEQ ID NO: 36, a CDR2 comprising an amino acid sequence of SEQ ID NO: 37, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 38; xi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 40, a CDR2 comprising an amino acid sequence of SEQ ID NO: 41, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 42; xii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 44, a CDR2 comprising an amino acid sequence of SEQ ID NO: 45, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 46; xiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ iD NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6635; xiv). a CDR1 comprising an amino acid sequence of SEQ ID NO: 6637, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6638, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6639; xv). a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6643; xvi) . a CDR1 comprising an amino acid sequence of SEQ ID NO: 7089, a CDR2 comprising an amino acid sequence of SEQ iD NO: 45, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 46; xvii). a CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093; xviii). a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; xix). a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7099; xx). a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; xxi), a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; xxi i) . a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7291; xxiii). a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 7291; xxiv). a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxv). a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10; xxvi). a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 7093; xxviija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; xxviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; xxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10388, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10391, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxiiija CDR1 comprising an amino acid sequence of SEQ ID NO: IS, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10392, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10393, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; xxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino add sequence of SEQ ID NO: 10338, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; xxxvija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10390, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; xxxviija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; xxxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10410; xxxixja CDR1 comprising an amino acid sequence of SEQ ID NQ: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10411; xl)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10412; xli)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10413; xlii)a CDR1 comprising an amino acid sequence of SEQ ID NQ: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10414; xliiija CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10415; xliv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10416; xlv)a CDR1 comprising an amino acid sequence of SEQ ID NQ: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10417; xlvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10418; xlviija CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10419; xlviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10420; xlix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10421; l)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10422; li)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10423; lii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10424; liiija CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10425; liv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10426; lv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10427; lvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10428; lvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 34; lviii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10429; lix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10430; lx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10431; lxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10432; lxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10433;
Ixiii) CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10811; lxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10435; lxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10436; lxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10437; lxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10438; ixviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10439;
Ixvixja CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10440; ixx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10441; lxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10442;
Ixxiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10443;
Ixxilija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10444; lxxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10445; lxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NQ: 10446; lxxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10447; lxxvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10448;
Ixxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10449; lxxvix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ ID NO: lxxx)a CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10451; lxxxi)a CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10452; ixxxiija CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10453;
Ixxxiiija CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10454; or
Ixxxivja CDR1 comprising an amino acid sequence of SEQ iD NO: 16, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10389, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10455.
16. The multispecific antigen-binding protein of any one of claims 1-15, wherein the one or more anti-
TNFR2 antigen-binding domains comprise a) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; b) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 18; c) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7099; d) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; e) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7289; f) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino add sequence of SEQ ID NO: 7285, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7291; g) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7291; h) a CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10; i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 8, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093; j) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6643; k) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10414; l) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10421; m) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10422; n) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10423; o) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10429; p) a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6642, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10811; q) a CDR1 comprising an amino acid sequence of SEQ ID NO: 16, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7096, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10374; r) a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10; or s) a CDR1 comprising an amino acid sequence of SEQ ID NO: 6633, a CDR2 comprising an amino acid sequence of SEQ ID NO: 6634, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7093,
17. The multispecific antigen-binding protein of any one of claims 1-3, wherein the one or more anti- TNFR2 antigen-binding domains comprise a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1128-1686, 6745-6806, and 7102-7125; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 1637-2245, 6807-6863, and 7126-7149; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID NOs: 2246-2804, 6869- 6930, and 7150-7173.
18. The multispecific antigen-binding protein of any one of claims 1-17, wherein the one or more anti- TNFR2 antigen-binding domains comprise at least one anti-TNFR2 single-domain antibody.
19. The multispecific antigen-binding protein of claim 18, wherein the anti~TNFR2 single-domain antibody is an anti-TNFR2 VHH, an anti-TNFR2 VNAR, or an anti-TNFR2 VH domain.
20. The multispecific antigen-binding protein of claim 19, wherein the anti-TNFR2 VHH is a camelid anti- TNFR2 VHH.
21. The multispecific antigen-binding protein of claim 20, wherein the anti-TNFR2 VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 93-640, 2805-3363, 6636, 6640, 6644, 7090, 6651-6697, 6931-6992, 7174-7197, 10380, 10381, 10383, 10385, and 10386, or a sequence having at least 75% identity thereto.
22. The multispecific antigen-binding protein of claim 20 or 21, wherein the anti-TNFR2 VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43 47, 6636, 6640, 6644, 7090, 10380, 10381, 10383, 10385, and 10386, or a sequence having at least 75% identity thereto,
23. The multispecific antigen-binding protein of claim 19, wherein the anti-TNFR2 VHH is a humanized anti-TNFR2 VHH.
24. The multispecific antigen-binding protein of claim 23, wherein the humanized anti-TNFR2 VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 6648, 6649, 6650, 7092, 7095, 7098, 7101,7293-7296, 641-1127, 6698-6744, 10375, 10382, 10384, 10394-10408, 10457-10502, 10720-10727, and 10812, or a sequence having at least 75% identity thereto.
25. The multispecific antigen-binding protein of claim 24, wherein the humanized anti-TNFR2 VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 81-92, 6648, 6649, 6650, 7092, 7095, 7098, 7101, 7293-7296, 10375, 10382, 10384, 10394-10408, 10457-10502, 10720-
10727, and 10812, or a sequence having at least 75% identity thereto. , The multispecific antibody of any of ciaims 1-3, wherein the one or more antigen-binding domains that specifically bind to TNFR2 comprise TNF or a variant thereof. , The multispecific antigen-binding protein of any one of claims 1-26, wherein the one or more anti- TNFR2 antigen-binding domains have an agonist effect upon binding to TNFR2. , The mu Itispecif ic antigen-binding protein of claim 27, wherein the one or more anti-TNFR2 antigenbinding domains have an agonist effect upon binding to TNFR2 with an ECso of about 1-100 nM. , The multispecific antigen-binding protein of any one of claims 1-28, wherein the one or more anti- TNFR2 antigen-binding domains bind to human TNFR2. , The multispecific antigen-binding protein of claim 29, wherein the one or more anti-TNFR2 antigenbinding domains bind to human TNFR2 with a Ko of less than about 3xl0'6 M. , The multispecific antigen-binding protein of claim 30, wherein the one or more anti-TNFR2 antigenbinding domains bind to human TNFR2 with a fo of about IxiO"10 to 5x10 s M. , The multispecific antigen-binding protein of any one of claims 1-28, wherein the one or more anti- TNFR2 antigen-binding domains bind to cyno TNFR2. , The multispecific antigen-binding protein of ciaim 32, wherein the one or more anti-TNFR2 antigenbinding domains bind to cyno TNFR2 with a KD of less than about 3xl0"6 M. , The multispecific antigen-binding protein of claim 33, wherein the at least one or more anti-TNFR2 antigen-binding domains bind to cyno TNFR2 with a Ka of about IxlO"9 to 2xl0”7 M.
35. The multispecific antigen-binding protein of any one of claims 1-34, wherein the one or more anti- TNFR2 antigen-binding domains bind to the same epitope(s) on TNFR2 as antibody clone MR2-1.
36. The multispecific antigen-binding protein of any one of claims 1-35, wherein the one or more anti- TNFR2 antigen-binding domains do not bind to the same epitope(s) on TNFR2 as antibody clone MR2-1.
37. The multispecific antigen-binding protein of any one of claims 1-36, wherein the one or more anti- TNFR2 antigen-binding domains increase expression of one or more proteins selected from a protein in the NF-kB pathway, FOXP3, HELIOS, EZH2, HLA-DR, ICAM-1, OX-40, ICOS, and CCR8.
33. The multispecific antigen-binding protein of any one of claims 1-37, wherein the one or more anti- TNFR2 antigen-binding domains comprise one or more modifications that reduce binding of said TNFR2 antigen-binding domain by pre-existing antibodies found in human blood or serum.
39. The multispecific antigen-binding protein of any one of claims 19-38, wherein the anti-TNFR2 singledomain antibody comprises one or more modifications at the amino-terminus and/or the carboxyterminus.
40. The multispecific antigen-binding protein of claim 39, wherein the anti-TNFR2 single-domain antibody comprises the amino acid sequence VP AG (SEQ, ID NO: 7267) or VAGG (SEQ ID NO: 7266) at the carboxy-terminus starting from position 111 according to Chothia.
41. The multispecific antigen-binding protein of claim 39 or 40, wherein the anti-TNFR2 single-domain antibody comprises a substitution of amino acid residue Glu with Asp (EID) at the first position of the amino-terminus,
42. The multispecific antigen-binding protein of any one of claims 1-41, wherein the one or more anti- TNFR2 antigen-binding domains bind to the same epitope on TNFR2.
43. The multispecific antigen-binding protein of any one of claims 1-41, wherein the one or more anti- TNFR2 antigen-binding domains bind to different epitopes on TNFR2.
44. The multispecific antigen-binding protein of any one of claims 1-43, wherein the one or more anti- CD25 antigen-binding domain comprises a complementarity determining region 3 (CDR3) comprising an amino add sequence selected from: a) NAL(G/L/P/Q/W)Y (SEQ ID NO: 4131); b) NALR(D/H/N/F) (SEQ ID NO: 4134); c) (K/S/T)TLRY (SEQ ID NO: 4136); d) (A/V/S)(K/T)G(R/A/K)(G/H/N/R)SG(S/G)YYP(W/F/L)(D/E)(D/E)(Y/V) (SEQ ID NO: 10219); e) AA(S/T)(D/N/Y/K)(F/V)(L/P)(I/L)A(T/I/A)(T/S/A)IS(A/G)(Y/H)DY (SEQ ID NO: 10308); f) AAYVYPDYYCS(D/E)YVLL(K/R)YDY (SEQ ID NO: 7363); g) NIYR(P/S)QVP(P/S/T)TRYS (SEQ ID NO: 7365); and h) AAKRLGP(M/I/A/L)VH(Q/R)YSLEVLTPLFLDEYDY (SEQ ID NO: 9423), wherein one or more non-alanine residues in the CDR3 sequence are optionally replaced with an alanine, and/or one or more alanine residues in the CDR3 sequence are optionally replaced with a glycine.
45. The multispecific antigen-binding protein of claim 44, wherein the CDR3 comprises an amino acid sequence a).(A/V/S)(A/K/T)(A/G)(A/R/K)(A/G/H/N/R)(A/S)(A/G)(A/S/G)(A/Y)(A/Y)(A/P)(A/W/F/L)(A/D/E)(A/D/E )(A/Y/V); or b). (A/G)(A/G)(A/K)(A/R)(A/L)(A/G)(A/P)(M/I/A/L)(A/V)(A/H)(A/R/Q)(A/Y)(A/S)(A/L)(A/E)(A/V) (A/L)(A/T)(A/P)(A/L)(A/F)(A/L)(A/D)(A/E)(A/Y)(A/D)(A/Y).
45. The multispecific antigen-binding protein of claim 44, wherein the CDR3 of the one or more anti- CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4103, 4107, 4111, 4115, 4119, 4139, 4141, 5337, 5339, 5371, 5375, 5398, 5401, 5431, 5515, 5519, 5521, 5528, 5532, 5542, 5544, 5545, 5547, 5548, 7344, 7347, 7349, 7350, 7367, 9411-9416, 9436, 9440, 9887, 9966, 9975, 9978, 9979, 9980, and 10504-10544.
47. The multispecific antigen-binding protein of any one of claims 1-46, wherein the one or more anti-
CD25 antigen-binding domains further comprises a CDR1 comprising an amino acid sequence selected from: a) GR(K/R/S)FSTU (SEQ ID NO: 4137); b) GFTFS(N/S)YA (SEQ ID NO: 4140); c) GRTF(A/S)(S/W/D)(F/N/Y)G (SEQ ID NO: 10309); d) GFTLDYYA (SEQ ID NO: 7342); and e) G(I/M)P(F/-)(A/-)L(P/V/Y)A (SEQ ID NO: 7366).
48, The mu Itispecif ic antigen-binding protein of claim 47, wherein the CDR1 of the one or more anti- CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4101,
4105, 4109, 4113, 4117, 4132, 4142, 4905, 4909, 4918, 7342, and 7345.
49. The multispecific antigen-binding protein of any one of claims 1-48, wherein the one or more anti- CD25 antigen-binding domains further comprises a CDR2 comprising an amino acid sequence selected from: a) (l/V)(D/E)R(D/G)(D/G)T(A/P/T); b) IYSD(G/S)SGT (SEQ ID NO: 9441); c) IS(Q/R/G)(S/G)GGRT (SEQ ID NO: 10310); d) IS(R/S)(D/S)G(D/G)ST (SEQ ID NO: 7364); e) ISSGGNT (SEQ ID NO: 7346); and f) ISSTDGRT (SEQ ID NO: 7348).
50, The multispecific antigen-binding protein of claim 49, wherein the CDR2 of the one or more anti- CD25 antigen-binding domains comprises an amino acid sequence selected from SEQ ID NOs: 4102,
4106, 4110, 4114, 4118, (i/V)(D/E)R(D/G)GT(A/P/T), 4135, l(D/E)R(D/G)(D/G)T(P/T), 5042, 5046, 5059, 5067, 5092, 5214, 5215, 5216, 5217, 7343, 7346, 7348, and 9435,
51. The multispecific antigen-binding protein of any one of claims 44, 45, 47, and 49, wherein the one or more anti-CD25 antigen-binding domains comprise i) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; ii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4136; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)GT(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; v) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4135, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4137, a CDR2 comprising an amino acid sequence of l(D/E)R(D/G)(D/G)T(P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4136; viij a CDR1 comprising an amino acid sequence of SEQ ID NO: 4132, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; viii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)(D/G)T(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4134; lx) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4132, a CDR2 comprising an amino acid sequence of (l/V)(D/E)R(D/G)GT(A/P/T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4131; x) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino add sequence of SEQ ID NO: 4135, and a CDR3 comprising an amino add sequence of SEQ ID NO: 4134; xi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9441, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10219; xii) a CDRI comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9441, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9440; xiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4139; xiv) a CDR1 comprising an amino acid sequence of SEQ iD NO: 10309, a CDR2 comprising an amino acid sequence of SEQ iD NO: 10310, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10308; xv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4142, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4141; xvi) a CDRI comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7364, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7363; xvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7366, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7346, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7365; xviii) a CDRi comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9423; xix) a CDRI comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ !D NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7367; xx) a CDRI comprising an amino acid sequence of SEQ ID NO: 4140, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9441, and a CDR3 comprising an amino acid sequence of (A/V/S){A/K/T){A/G){A/R/K)(A/G/H/N/R){A/S)(A/G)(A/S/G)(A/Y)(A/Y)(A/P)(A/W/F/L)(A/D/EKA/D/E){A/Y/ V); or xxi) a CDRI comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of (A/G)(A/G)(A/K){A/R)(A/L)(A/G)(A/P)(M/I/A/L)(A/V)(A/H){A/R/Q)(A/Y)(A/S)(A/L)(A/E)(A/V)(A/L)(A/T)(A/P )(A/L)(A/F)(A/L){A/D)(A/E)(A/Y)(A/D)(A/Y).
52. The multispecific antigen-binding protein of any one of claims 1-51, wherein the one or more anti- CD25 antigen-binding domains comprise: i) a CDRI comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4102, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4103; ii) a CDR1 comprising an amino add sequence of SEQ ID NO: 4105, a CDR2 comprising an amino add sequence of SEQ ID NO: 410S, and a CDR3 comprising an amino add sequence of SEQ ID NO: 4107; iii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4109, a CDR2 comprising an amino add sequence of SEQ ID NO: 4110, and a CDR3 comprising an amino add sequence of SEQ ID NO: 4111; iv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4115; v) a CDR1 comprising an amino add sequence of SEQ ID NO: 4117, a CDR2 comprising an amino add sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4119; vi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7343, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7344; vii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7345, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7346, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 7347; viii) a CDR1 comprising an amino add sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7349; ix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 7350; x) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 9411; xi) a CDR1 comprising an amino add sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 9412; xii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9413; xiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9414; xiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9415; xv) a CDR1 comprising an amino add sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 9416; xvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9975; xvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 5431; xviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 9887 xix) a CDR1 comprising an amino acid sequence of SEQ iD NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4114, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 9966; xx) a CDR1 comprising an amino acid sequence of SEQ ID NQ: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 9436; xxi) a CDR1 comprising an amino acid sequence of SEQ iD NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 9978; xxii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 9979; xxi si) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9980; xxiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ iD NO: 4110, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5339; xxv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ iD NO: 5046, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5339; xxvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ iD NO: 5059, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 5337; xxvii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ iD NO: 5046, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 5337; xxvlii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino add sequence of SEQ ID NO: 5067, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5371; xxix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino add sequence of SEQ ID NO: 5046, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5375; xxx) a CDR1 comprising an amino add sequence of SEQ ID NO: 4109, a CDR2 comprising an amino add sequence of SEQ iD NO: 4110, and a CDR3 comprising an amino add sequence of SEQ ID NO: 4111; xxxi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino add sequence of SEQ ID NO: 5092, and a CDR3 comprising an amino add sequence of SEQ ID NO: 4111; xxxii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino acid sequence of SEQ iD NO: 5092, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5398; xxxiii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5042, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4111; xxxiv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4105, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5059, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4111; xxxv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4101, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5042, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5401; xxxvi) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5515; xxxvii) a CDR1 comprising an amino acid sequence of SEQ, ID NO: 4909, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5214, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5519; xxxviii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5216, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5521; xxxix) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4909, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5217, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5519; xl) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4918, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5215, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5528; xii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5532; xlii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino acid sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 5542; xliii) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino add sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5544; xliv) a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino add sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5545; xiv) a CDR1 comprising an amino add sequence of SEQ ID NO: 4905, a CDR2 comprising an amino add sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5547; xlvij a CDR1 comprising an amino acid sequence of SEQ ID NO: 4905, a CDR2 comprising an amino add sequence of SEQ ID NO: 4118, and a CDR3 comprising an amino add sequence of SEQ ID NO: 5548; xlvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10504; xlviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10505; xlix)a CDR1 comprising an amino acid sequence of SEQ iD NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ !D NO: 10506; l)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10507; li)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10508; lii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10509; liii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10510; liv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10511; lv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10512; lvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10513; lvii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ iD NO: 9435, and a CDR3 comprising an amino acid sequence of SEQ iD NO: 10514; lviil)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10515; lix)a CDR1 comprising an amino add sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10516; lx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 4113, a CDR2 comprising an amino add sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10517; lxi)a CDR1 comprising an amino add sequence of SEQ ID NO: 4113, a CDR2 comprising an amino acid sequence of SEQ ID NO: 9435, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10518; Ixi i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10519;
Ixiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10520; lxiv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10521; lxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10522; lxvi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10523;
Ixvi i)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10524;
Ixviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10525; lxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9414; lxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10526; lxxi)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10527; lxxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10528;
Ixxiiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10529;
Ixxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10530; lxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10531;
Ixxvija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino add sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino add sequence of SEQ ID NO: 10532; Ixxviija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10533;
Ixxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10534; lxxix)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10535; lxxx)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10536;
Ixxxija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10537; lxxxii)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ !D NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10533;
Ixxxiiija CDR1 comprising an amino acid sequence of SEQ iD NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10539;
Ixxxivja CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10540; lxxxv)a CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10541;
Ixxxvija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ !D NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10542;
Ixxxviija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ iD NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10543; or
Ixxxviiija CDR1 comprising an amino acid sequence of SEQ ID NO: 7342, a CDR2 comprising an amino acid sequence of SEQ ID NO: 7348, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 10544,
53. The multispecific antigen-binding protein of any one of claims 1-3, wherein the one or more anti- CD25 antigen-binding domains comprise a CDR1 comprising an amino acid sequence selected from any one of SEQ ID NOs: 4726-5030, 7931-8226, and 9660-9770; a CDR2 comprising an amino acid sequence selected from any one of SEQ ID NOs: 5031-5335, 8227-8522, and 9771-9880; and/or a CDR3 comprising an amino acid sequence selected from any one of SEQ ID Nos: 5336-5640, 8523- 8818, and 9881-9991.
54. The multispecific antigen-binding protein of any one of claims 1-53, wherein the one or more anti- CD25 antigen-binding domains comprise at least one anti-CD25 single-domain antibody.
55. The multispecific antigen-binding protein of claim 54, wherein the anti-CD25 single-domain antibody is an anti-CD25 VHH, an anti-CD25 VNAR, or an anti-CD25 VH domain.
56. The multispecific antigen-binding protein of claim 55, wherein the anti-CD25 VHH is a camelid anti- CD25 VHH.
57. The multispecific antigen-binding protein of claim 56, wherein the anti-CD25 VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, 4143- 4442, 5641-5945, 7351-7354, 7368-7659, 8819-9114, 9437, 9442-9551, 9992-10102, and 10246- 10276, or a sequence having at least 75% identity thereto.
53. The multispecific antigen-binding protein of claim 56 or 57, wherein the anti-CD25 VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4104, 4108, 4112, 4116, 4120, 7351- 7354, 9437, and 10246-10276, or a sequence having at least 75% identity thereto.
59. The multispecific antigen-binding protein of claim 55, wherein the anti-CD25 VHH is a humanized antl-CD25 VHH.
60. The multispecific antigen-binding protein of claim 59, wherein the humanized anti-CD25 VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4126-4130, 4443-4725, 7359-7362, 7660-7930, 9417-9422, 9439, 9552-9659, 10214-10245, and 10545-10585, or a sequence having at least 75% identity thereto.
61. The multispecific antigen-binding protein of claim 59, wherein the humanized anti-CD25 VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 4126-4130, 7359-7362, 9417-9422, 9439, 10214-10245, and 1054540585, or a sequence having at least 75% identity thereto.
62. The multispecific antibody of any of claims 1-43, wherein the one or more antigen-binding domains that specifically bind to CD25 comprise IL-2 or a variant thereof.
63. The multispecific antigen-binding protein of any one of claims 1-62, wherein the one or more anti- CD25 antigen-binding domains bind to human CD25.
64. The mu Itispecific antigen-binding protein of claim 63, wherein the one or more anti-CD25 antigenbinding domains bind to human CD25 with a KB of less than about 3.5 xlO”7 M.
65. The multispecific antigen-binding protein of ciaim 64, wherein the one or more anti-CD25 antigenbinding domains bind to human CD25 with a KD of about lxlO“10 to about 5xl0~8 M.
66. The multispecific antigen-binding protein of any one of claims 1-65, wherein the one or more anti- CD25 antigen-binding domains bind to cyno CD25.
67. The multispecific antigen-binding protein of ciaim 66, wherein the one or more anti-CD25 antigenbinding domains bind to cyno CD25 with a KB of less than about IxlO"6 M.
68. The multispecific antigen-binding protein of claim 67, wherein the one or more anti-CD25 antigenbinding domains bind to cyno CD25 with a KD of about 1x10 s to about 4xl0"7 M.
69. The multispecific antigen-binding protein of any one of claims 1-61 and 63-68, wherein the one or more anti-CD25 antigen-binding domains bind to the same epitope(s) on CD25 as IL-2.
70. The multispecific antigen-binding protein of any one of claims 1-61 and 63-69, wherein the one or more anti-CD25 antigen-binding domains compete for binding to CD25 with IL-2 .
71. The multispecific antigen-binding protein of claim 69 or 70, wherein the one or more anti-CD25 antigen-binding domains have an antagonistic effect upon binding to CD25.
72. The muitispecific antigen-binding protein of any one of ciaims 1-61 and 63-71, wherein the one or more anti~CD25 antigen-binding domains do not bind to the same epitope(s) on CD25 as I L-2.
73. The multispecific antigen-binding protein of any one of claims 1-61, 63-68 and 72, wherein the one or more anti-CD25 antigen-binding domains do not compete for binding to CD25 with IL-2.
74. The multispecific antigen-binding protein of any one of claims 1-73, wherein the one or more a nt i- CD25 antigen-binding domains comprise one or more modifications that reduce binding of said antigen-binding domains by pre-existing antibodies found in human blood or serum.
75. The multispecific antigen-binding protein of any one of claims 54-74, wherein the anti-CD25 singledomain antibody comprises one or more modifications at the amino-terminus and/or the carboxyterminus.
76. The multispecific antigen-binding protein of claim 75, wherein the anti-CD25 single-domain antibody comprises the amino acid sequence VPAG (SEQ. ID NO: 7267) or VAGG (SEQ ID NO: 7266) at the carboxy-terminus starting from position 111 according to Chothia.
77. The multispecific antigen-binding protein of claim 75 or 76, wherein the anti-CD25 single-domain antibody comprises a substitution of amino acid residue Glu with Asp (EID) at the first position of the amino-terminus.
78. The multispecific antigen-binding protein of any one of claims 1-77, wherein the one or more anti- CD25 antigen-binding domains bind to the same epitope on CD25.
79. The multispecific antigen-binding protein of any one of claims 1-77, wherein the one or more a nt i-
CD25 antigen-binding domains bind to different epitopes on CD25.
80. The multispecific antigen-binding protein of any one of claims 1-79, further comprising at least one antigen-binding domain that binds to another antigen.
81. The multispecific antigen-binding protein of claim 80, wherein the other antigen comprises serum albumin.
82. The multispecific antigen-binding protein of any one of claims 1-81, which further comprises an immunoglobulin Fc region.
83. The multispecific antigen-binding protein of claim 80, wherein the immunoglobulin Fc region is an Fc region of a human immunoglobulin.
84. The multispecific antigen-binding protein of claim 83, wherein the immunoglobulin Fc region is an Fc region of human IgGl, lgG2, lgG3 or lgG4, or a variant thereof.
85. The multispecific antigen-binding protein of claim 84, wherein the immunoglobulin Fc region is an Fc region of human IgGl, or a variant thereof.
86. The multispecific antigen-binding protein of ciaim 85, wherein the Fc region of human IgGl comprises one or more mutations selected from Leu234Ala (L.234A), Leu234Gly (L234G), Leu234Ser (L234S), Leu234Thr (L234T), Leu234Ala (L234A), Leu235Ala (L235A), Leu235Giu (L235E), Leu235Ser (L235S), Leu235Thr (L235T), Leu235Val (L235V), Leu235Gin (L235Q), Giy236Arg (G236R), Met252Tyr (M252Y), Ser254Thr (S254T), Thr256Giu (T256E), Asp265Asn (D265N), Asp265Aia (D265A), Asp270Asn (D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A), Pro239Gly (P329G), Asn325Giu (N325E) and/or Ala327Ser (A327S) according to EU numbering.
87. The multispecific antigen-binding protein of claim 86, wherein the Fc region of human IgGl comprises a set of mutations selected from
1JL234A and L235A;
2)L234A, L.235A, and P329A;
3JD265A, N297A and P329A; 4JL234A, L235A, and G237A;
5)L234G, L235S, and G236R;
6) L234S, L235T, and G236R;
7)L234S, L235V, and G236R;
8)L234T, L235Q, and G236R;
9)L234T, L235T, and G236R;
10JL234A, L235A, and P329G; and
11JM252Y, S254T, and T256E. , The mu Itispecif ic antigen-binding protein of any one of claims 85-87, the immunoglobulin Fc region is an Fc region of human SgGl comprising L234A, L235A, and P329A. , The multispecific antigen-binding protein of claim 84, wherein the immunoglobulin Fc region is an Fc region of human lgG4, or a variant thereof. , The multispecific antigen-binding protein of claim 89, wherein the Fc region of human lgG4 comprises one or more mutations selected from Ser228Pro (S228P), Leu235Glu (L235E), Leu235Ala (L235A), Phe234Ala (F234A), and/or Pro329Gly (P329G) according to EU numbering. , The multispecific antigen-binding protein of claim 90, wherein the Fc region of human lgG4 comprises a set of mutations selected from
1).S228P and L235E;
2J.S228P and L235A;
3J.S228P, F234A, and L235E;
4J.S228P, F234A, and L235A; and
5) P329G, S228P, and L235E. , The multispecific antigen-binding protein of any of claims 1-91, wherein the multispecific antigenbinding protein comprises at least one linker, optionally selected from the group consisting of SEQ ID NOs: 3969-4027, and 6261-6362.
93. The multispecific antigen-binding protein of claim 92, wherein the at least one linker is a rigid linker.
94. The multispecific antigen-binding protein of ciaim 93, wherein the at least one rigid linker is selected from the group consisting of PAPAPAPAPAPAPAPAP (SEQ ID NO: 4009), GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 4012), GGGGSPAPAPAPAPGGGGS (SEQ ID NO: 6361), GGGGSPAPAPAPAPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 6362), and A(EAAAK)nA (SEQ ID NO: 4027), where n is any integer, e.g., 1, 2, 3, 4, 5, 6, or 7, 8, 9 or 10.
95. The multispecific antigen-binding protein of claim 92, wherein the at least one linker is a flexible linker.
96. The multispecific antigen-binding protein of claim 95, wherein the at least one flexible linker is selected from the group consisting of GnS (SEQ ID NO: 4013), SGn (SEQ ID NO: 4014), where n is any integer, e.g,, 1, 2, 3, 4, 5, 6, or 7, 8, 9 or 10, and (GGGGS)n (SEQ ID NO: 4015), where n is any integer, e.g,, 1, 2, 3, 4, 5, 6, or 7, 8, 9 or 10.
97. The multispecific antigen-binding protein of any of claims 1-96, wherein the multispecific antigenbinding protein comprises the amino acid sequence of any one of SEQ ID NOs: 6401-6520 and 10792-10810, or a sequence having at least 70% identity thereto.
98. A conjugate comprising the multispecific antigen-binding protein of any one of claims 1-97, wherein the multispecific antigen-binding protein is conjugated to a second moiety.
99. The conjugate of claim 98, wherein the second moiety is selected from a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytokine, a cytotoxic agent, a chemotherapeutic agent, and a diagnostic agent, or a combination thereof.
100.A polynucleotide molecule encoding the muitispecific antigen-binding protein of any one of claims 1-97.
101.The polynucleotide molecule of claim 100, which comprises the nucleotide sequence of any one of SEQ ID NOs: 6521-6632 encoding a multispecific antigen-binding protein, or a sequence having at least 70% identity thereto.
102.A recombinant vector comprising the polynucleotide molecule of any one of claims 100-101.
103.A host cell comprising the polynucleotide molecule of any one of claims 100-101, or the recombinant vector of claim 102.
104.A kit comprising the multispecific antigen-binding protein of any one of claims 1-97, the conjugate of any one of claims 98-99, the polynucleotide molecule of any one of claims 100-101, the recombinant vector of claim 102, or the host cell of claim 103, and optionally, instructions and/or packaging for the same.
105.A pharmaceutical composition comprising the multispecific antigen-binding protein of any one of claims 1-97, the conjugate of any one of claims 98-99, the polynucleotide molecule of any one of claims 100-101, or the recombinant vector of claim 94, and a pharmaceutically acceptable carrier and/or excipient,
106. A method for preparing a multispecific antigen-binding protein that specifically binds TNFR2 and CD25, comprising the steps of:
(a) culturing the host cell of claim 103 in a culture medium under conditions suitable for expression of the multispecific antigen-binding protein, and
(b) isolating the multispecific antigen-binding protein from the host cell and/or the culture medium.
107.A method for promoting proliferation, activating and/or enhancing suppressive function, and/or stabilizing immunosuppressive phenotype of a population of regulatory T cells (Treg) comprising contacting the population of Treg with the multispecific antigen-binding protein of any one of claims 1-97, or the conjugate of any one of claim 98-99. lOS.The method of claim 107, wherein said contacting occurs in vitro.
109.The method of claim 107, wherein said contacting occurs in vivo.
110.The method of claim 109, wherein the method further comprises administering the multispecific antigen-binding protein or the conjugate into a subject in need thereof.
111.A method of treating or preventing a disease or disorder in a subject in need thereof, said method comprising administering to the subject an effective amount of the multispecific antigen-binding protein of any one of claims 1-97, or the conjugate of any one of claim 98-99.
112.The method of claim 111, wherein the disease or disorder is an immunological disease, inflammatory disease, cancer, cardiovascular disease, or an infertility and pregnancy-associated disease.
113.The method of claim 112, wherein the immunological disease is selected from an autoimmune disease, a neurological condition, an allergy, asthma, macular degeneration, muscular atrophy, a disease related to miscarriage, atherosclerosis, bone loss, a musculoskeletal disease, obesity, a graft- versus-host disease, and an allograft rejection.
114. The method of claim 113, wherein the autoimmune disease is selected from lupus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpastures disease, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, hypothyroidism, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, juvenile arthritis, lichen planus, lichen sclerosis, lgG4-related disease, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disease, pemphigus vulgaris or related blistering skin disease, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatics, polymyositis and dermatomyositis, premature ovarian failure, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, primary ovarian insufficiency, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthritis, stiff-man syndrome, type I diabetes, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis (Granulomatosis with polyangiitis) or other immune vasculitis.
IlS.The method of claim 114, wherein the lupus is systemic lupus erythematosus (SLE), cutaneous lupus, lupus nephritis, neonatal lupus, or drug-induced lupus.
IlS.The method of claim 115, wherein the cutaneous lupus is acute cutaneous lupus, chronic cutaneous lupus erythematosus, discoid lupus erythematosus (DIE), or subacute cutaneous lupus erythematosus,
IlT.The method of claim 113, wherein the neurological condition is selected from a brain tumor, a brain metastasis, a spinal cord injury, schizophrenia, epilepsy, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Huntington's disease, Parkinson's disease, and stroke.
118.The method of claim 113, wherein the allergy is selected from food allergy, seasonal allergy, pet allergy, hives, hay fever, allergic conjunctivitis, poison ivy allergy oak allergy, mold allergy, drug allergy, dust allergy, cosmetic allergy, and chemical allergy.
119.The method of claim 113, wherein the allograft rejection is selected from skin graft rejection, bone graft rejection, vascular tissue graft rejection, ligament graft rejection, and organ graft rejection.
120.The method of claim 119, wherein the ligament graft rejection is selected from cricothyroid ligament graft rejection, caudal cruciate ligament graft rejection, periodontal ligament graft rejection, suspensory ligament of the lens graft rejection, palmar radiocarpal ligament graft rejection, dorsal radiocarpal ligament graft rejection, ulnar collateral ligament graft rejection, radial collateral ligament graft rejection, suspensory ligament of the breast graft rejection, anterior sacroiliac ligament graft rejection, posterior sacroiliac ligament graft rejection, sacrotuberous ligament graft rejection, sacrospinous ligament graft rejection, inferior pubic ligament graft rejection, superior pubic ligament graft rejection, anterior cruciate ligament graft rejection, lateral collateral ligament graft rejection, posterior cruciate ligament graft rejection, medial collateral ligament graft rejection, cranial cruciate ligament graft rejection, and patellar ligament graft rejection.
IZl.The method of claim 119, wherein the organ graft rejection is selected from heart graft rejection, lung graft rejection, kidney graft rejection, liver graft rejection, pancreas graft rejection, intestine graft rejection, and thymus graft rejection.
122.The method of claim 113, wherein the graft-versus-host disease arises from a bone marrow transplant or one or more blood cells selected from B-cells, T-cells, basophils, common myeloid progenitor cells, common lymphoid progenitor cells, dendritic cells, eosinophils, hematopoietic stem cells, neutrophils, natural killer cells, megakaryocytes, monocytes, or macrophages.
123.The method of claim 112, wherein the inflammatory disease is acute or chronic inflammation.
124, The method of claim 112, wherein the inflammatory disease is selected from osteoarthritis, atopic dermatitis, endometriosis, polycystic ovarian syndrome, inflammatory bowel disease, fibrotic lung disease, and cardiac inflammation.
125,The method of claim 112, wherein the cancer is selected from adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dube syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal carcinoma, HIV/AIDS-related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B~cell prolymphocytic leukemia and hairy ceil leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic T-cell lymphocytic leukemia, eosinophilic leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, hodgkin lymphoma, non- hodgkin lymphoma, lynch syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or M¥H)-associated polyposis, myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian, fallopian tube, and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, soft tissue sarcomas, skin cancer (non-melanoma), small bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Waldenstrom macroglobulinemia (lymphoplasmacytic lymphoma), Werner syndrome, Wilms tumor, or xeroderma pigmentosum. .The method of claim 112, wherein the cardiovascular disease is selected from atherosclerosis, heart failure, left heart failure with reduced ejection fraction, left heart failure with preserved ejection fraction, right ventricular failure, congestive heart failure, restrictive cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, idiopathic cardiomyopathy, and hypertension.
127.The method of claim 112, wherein the infertility and pregnancy-associated diseases is selected from recurrent pregnancy loss, pre-eclampsia, preterm labor, fetal growth restriction, and intrauterine growth restriction.
128. A method of regenerating a tissue or organ comprising one or more TNFR2+ and/or CD25+ cells, said method comprising contacting the tissue or organ with an effective amount of the multispecific antigen-binding protein of any one of claims 1-97, or the conjugate of any one of claim 98-99.
129.The method of claim 128, wherein said tissue or organ is selected from pancreas, salivary gland, pituitary gland, kidney, heart, lung, hematopoietic system, cranial nerves, heart, aorta, olfactory gland, ear, nerve, eye, thymus, tongue, bone, liver, small intestine, large intestine, gastrointestinal, lung, brain, skin, peripheral nervous system, central nervous system, spinal cord, breast, embryonic structures, embryo, and testes tissue.
130.The method of claim 128 or 129, wherein said contacting occurs in vitro.
131.The method of claim 128 or 129, wherein said contacting occurs in vivo.
132.The method of claim 131, wherein the method further comprises administering the multispecific antigen-binding protein, or the conjugate into a subject in need thereof.
133.A method for inducing tolerance to a foreign agent and/or preventing or reducing immune response to a foreign agent in a subject in need thereof, said method comprising administering to the subject an effective amount of the multispecific antigen-binding protein of claim 1-97, or the conjugate of any one of claims 98-99.
134.The method of claim 133, wherein the foreign agent is a therapeutic protein or peptide, a viral, bacterial, or fungal vector, a biochemical vector, a lipid, carbohydrate, a nucleic acid, a sperm, an oocyte, or an embryo.
135.The method of claim 134, wherein the viral vector is a DNA or RNA vector.
136.The method of any one of claims 111-127 and 131-135, wherein the subject is a mammal.
137,The method of claim 136, wherein the mammal is human.
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