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WO2024251884A1 - Protéines d'activation de cellules nk comprenant un anticorps anti-cd20 et ant-nkp46, liées à il-2 dans le traitement de lnh-b r/r - Google Patents

Protéines d'activation de cellules nk comprenant un anticorps anti-cd20 et ant-nkp46, liées à il-2 dans le traitement de lnh-b r/r Download PDF

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Publication number
WO2024251884A1
WO2024251884A1 PCT/EP2024/065622 EP2024065622W WO2024251884A1 WO 2024251884 A1 WO2024251884 A1 WO 2024251884A1 EP 2024065622 W EP2024065622 W EP 2024065622W WO 2024251884 A1 WO2024251884 A1 WO 2024251884A1
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amino acid
domain
acid sequence
cells
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PCT/EP2024/065622
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Olivier DEMARIA
Marianna MULLER
Carine Paturel
Romain REMARK
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Innate Pharma
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Publication of WO2024251884A1 publication Critical patent/WO2024251884A1/fr

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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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • 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
    • C07K2317/30—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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/66—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a swap of domains, e.g. CH3-CH2, VH-CL or VL-CH1
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the disclosure relates to multispecific binding proteins comprising a first and a second antigen binding domains (ABDs), a cytokine moiety, wherein the first ABD binds specifically to human CD20 and the second ABD bind specifically to human NKp46, for use in treating refractory and/or relapsed CD20 positive B-cell non Hodgkin lymphoma.
  • ABDs antigen binding domains
  • NKp46 bind specifically to human NKp46
  • BACKGROUND Rituximab has become widely used in the treatment of B cell lymphomas and is effective in many patients.
  • Ritixumab is believed to have multiple modes of action, including inter alia, the ability to mediate ADCC toward malignant CD20-expressing cells.
  • NK cells mediate ADCC and are believed to have an important role in the anti-tumor immunity of rituximab.
  • NK cells are a subpopulation of lymphocytes that are involved in non- conventional immunity. NK cells provide an efficient immunosurveillance mechanism by which undesired cells such as tumor or virally-infected cells can be eliminated.
  • NK cells Characteristics and biological properties of NK cells include the expression of surface antigens including CD16, CD56 and/or CD57, the absence of the ⁇ / ⁇ or ⁇ / ⁇ TCR complex on the cell surface, the ability to bind to and kill cells in a MHC-unrestrictive manner and in particular cells that fail to express "self" MHC/HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate and shape the immune response.
  • the addition of rituximab into combination therapies for DLBCL have greatly improved patient outcomes.
  • T cell engagers that bind, via an antigen binding domain, to CD20 on the malignant cells and, via another antigen binding domain, to CD3 on effector T cells have shown great promise in treating B cell lymphomas.
  • TCEs therapy and their ability to mediate tumor cell killing by effector T cells, had promise for treatment of tumors who are R/R after rituximab treatment.
  • NK cell activity is diminished such that rituximab would lack or have reduced efficacy. Diminished NK cell activity following rituximab treatment might be attributable to different causes, including for example hypoactivity of NK cells caused by the prior therapy, increased expression of inhibitory receptors on NK cells or decreased expression of activating receptors (e.g. CD16A) on NK cells.
  • CRS cytokine release syndrome
  • CAR-T chimeric antigen receptor T
  • CAR-T cells are autologous genetically modified T cells formed by combining the antigen-binding site of an antibody with the intracellular domain of a T-cell activation receptor.
  • axicabtagene ciloleucel axi-cel, Yescarta ®
  • tisagenlecleucel tisa-cel, Kymriah ®
  • lisocabtagene maraleucel liso-cel, Breyanzi ®
  • CRS cytokine release syndrome
  • ICANS immune effector cell-associated neurotoxicity syndrome
  • the invention is based on the finding that CD20-expressing B-NHL cells from patients having relapsed or refractory NHL can be targeted in a highly effective manner with an NK cell engager (NKCE) embodied as a multispecific protein that specifically binds NKp46 on NK cells and CD20 on NHL cells, moreover despite the patients having had prior treatment with immunotherapy that mediates effector cell cytotoxicity toward the B-NHL cells.
  • NKCE NK cell engager
  • the NKCE showed highly effective anti-tumor activity despite that the NKCE targets the same tumor antigen (CD20) which had been targeted by the prior anti-CD20 agent (anti-CD20 agent used in a prior line of therapy). Additionally, the CD20-targeting NKCE was effective even where the prior anti-CD20 therapy was used in combination with chemotherapy.
  • the multispecific binding proteins that bind to NKp46 and to CD20 were highly effective in an ex vivo study of rituximab-experienced R/R B- NHL patients, and surprisingly even more effective than a gold standard CD20-targeting T cell engager (TCE) despite the fact that the TCE does not need to rely on NK cell activity.
  • the CD20-targeting NKCE caused more complete target cell elimination compared to the TCE, and the NKCE may thus be able to trigger elimination of resistant B-NHL cells that are not eliminated through use of agents such as TCEs that solely mediate effector T cell cytotoxicity.
  • CD20-targeting NKCE remains highly effective to eliminate tumor cells that are not eliminated by other NK or T cell based therapies provides an advantageous treatment option for patients who have had prior treatment with such therapies that direct NK or T cell mediated cytotoxicity towards B-NHL cells.
  • the R/R B-NHL patients with prior immunotherapy showed low numbers of NK cells in lymphoid tissues, while administration of the NKCE in non-human primates showed that it has a strong ability to induce CD20+ B-cell depletion and NK cell expansion within lymphoid tissues.
  • the NKCE may therefore have a particular ability to treat the R/R B-NHL patients due to its high efficacy in inducing NK cell cytotoxicity toward B-NHL cells while increasing NK cells in lymphoid tissues.
  • a hematological cancer e.g. a B-NHL, particularly a R/R B-NHL
  • that comprise administering to an individual in need thereof e.g.
  • the multispecific protein comprises a binding domain that binds human CD20, a binding domain that binds human NKp46, a binding domain that binds to the human IL-2R without binding to the CD25 subunit thereof (e.g., a cytokine, cytokine variant or fragment thereof), and a binding domain that binds CD16A (e.g. an Fc domain).
  • the treatment is a second (or third or further) line of treatment for R/R B-NHL.
  • the B-NHL is a DLBCL.
  • the individual is in leukemic phase.
  • methods and compositions for treating a hematological cancer e.g. a B-NHL, particularly a R/R B-NHL or R/R/ DLBCL, that comprise administering to an individual in need thereof (e.g.
  • an individual having an R/R NHL) a multispecific protein that binds to NKp46, to CD20 and to the human IL-2R without binding to the CD25 subunit thereof, and optionally further to CD16A, wherein the individual was previously treated with a composition comprising an immunotherapy comprising an antigen-binding domain that specifically binds an antigen expressed by B-NHL cells (e.g. CD19, CD20, CD79b, CD30).
  • the immunotherapy may be specified as directing (or capable of directing) immune effector cells to eliminate B-NHL cells and/or cells expressing the antigen (e.g. CD19, CD20, CD79b, CD30).
  • the immunotherapy agent may be for example an antibody or antibody fragment that binds B-NHL cells (e.g. an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD79b antibody, an anti-CD30 antibody) and mediates ADCC, a T cell engager that comprises antibody or antibody fragment that binds B-NHL cells (e.g. an anti-CD20 antibody fragment, an anti-CD19 antibody fragment, an anti-CD79b antibody fragment, an anti-CD30 antibody fragment), or a cell (e.g. T cell) that expresses a chimeric antigen receptor comprising an antibody fragment that binds B-NHL cells (e.g. an anti-CD19, anti-CD20, anti-CD79b or anti- CD30 antibody fragment).
  • a T cell engager that comprises antibody or antibody fragment that binds B-NHL cells (e.g. an anti-CD20 antibody fragment, an anti-CD19 antibody fragment, an anti-CD79b antibody fragment, an anti-CD30 antibody fragment)
  • a cell
  • the individual is in leukemic phase.
  • the multispecific protein comprises a binding domain that binds human CD20, a binding domain that binds human NKp46, a binding domain that binds human CD122 (e.g., a cytokine, cytokine variant or fragment thereof), and a binding domain that binds CD16A (e.g. an Fc domain)
  • a B-NHL (or an individual having a B-NHL) can be specified as R/R following treatment a prior therapy, e.g. with the immunotherapy.
  • the prior therapy is a first line therapy for B-NHL.
  • the prior therapy comprises a therapeutic agent (e.g.
  • the prior therapy comprises rituximab (e.g. a treatment regimen comprising rituximab and/or additional therapeutic agents, optionally where the additional agent(s) is one or more chemotherapy agents).
  • the prior therapy or immunotherapy is an anti-CD20 agent, for example an agent that comprises an antibody or antibody fragment that binds CD20 (e.g.
  • the prior therapy comprises a protein (e.g. bispecific antibody) that comprise an antigen binding domain (e.g. antibody or antibody fragment) that specifically binds to CD20 and an antigen binding domain (e.g. antibody or antibody fragment) that specifically binds to CD3.
  • the prior therapy comprises an immune effector cell (e.g. T cell, CAR-T cell) that is genetically modified to express a chimeric antigen receptor comprising an antigen binding domain (e.g. antibody fragment) that specifically binds to CD19 or CD20.
  • the prior therapy is selected from the group consisting of rituximab, ofatumumab, veltuzumab, ocrelizumab, epcoritamab, odronextamab, glofitamab, mosunetuzumab and plamotamab.
  • the prior therapy is selected from the group consisting of axicabtagene ciloleucel, tisagenlecleucel, and lisocabtagene maraleucel.
  • a method of eliminating CD20+ B cells in lymphoid tissues e.g.
  • lymph nodes, spleen in an individual having a R/R B-NHL or R/R DLBCL, optionally wherein the individual has received prior treatment with an immunotherapy (e.g., an immunotherapy agent comprising an antigen-binding domain that specifically binds an antigen (e.g. CD19, CD20) expressed by B-NHL cells), the method comprising administering to the individual a multispecific protein that binds to human CD20, human NKp46, human CD122, and optionally to CD16A.
  • an immunotherapy e.g., an immunotherapy agent comprising an antigen-binding domain that specifically binds an antigen (e.g. CD19, CD20) expressed by B-NHL cells
  • the method comprising administering to the individual a multispecific protein that binds to human CD20, human NKp46, human CD122, and optionally to CD16A.
  • a method of increasing the number, anti-tumor and/or cytotoxic activity of NK cells in lymph tissues e
  • lymph nodes in an individual having a R/R B-NHL, optionally wherein the individual has received prior treatment with an immunotherapy (e.g., an immunotherapy agent comprising an antigen-binding domain that specifically binds an antigen (e.g. CD19, CD20) expressed by B-NHL cells), the method comprising administering to the individual having a R/R B-NHL a multispecific protein that binds to human CD20, human NKp46, human CD122, and optionally to CD16A.
  • an immunotherapy e.g., an immunotherapy agent comprising an antigen-binding domain that specifically binds an antigen (e.g. CD19, CD20) expressed by B-NHL cells
  • an immunotherapy e.g., an immunotherapy agent comprising an antigen-binding domain that specifically binds an antigen (e.g. CD19, CD20) expressed by B-NHL cells
  • the method comprising administering to the individual having a R/R B-NHL a multispecific protein
  • Exemplary embodiments include a method of treating an individual having a R/R B- NHL or R/R DLBCL, wherein the individual has received prior treatment with an agent selected from the group consisting of rituximab, ofatumumab, veltuzumab, ocrelizumab, epcoritamab, odronextamab, glofitamab, mosunetuzumab and plamotamab, the method comprising administering to the individual a multispecific protein that binds to human CD20, human NKp46, human CD122, and CD16A.
  • Exemplary embodiments include a method of treating an individual having a R/R B-NHL or R/R DLBCL, wherein the individual has received prior treatment with a CAR-T cell therapy, the method comprising administering to the individual a multispecific protein that binds to human CD20, human NKp46, human CD122, and optionally CD16A.
  • the multispecific protein that binds to human CD20, human NKp46, human CD122, and CD16A is a protein comprising or consisting of: (I) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 1, and (II) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70.
  • the B-NHL is Diffuse Large B Cell Lymphoma (DLBCL).
  • the B-NHL is High-grade B-cell lymphoma (HGBCL).
  • the B- NHL is Primary mediastinal large B-cell lymphoma (PMBCL).
  • the B-NHL is Follicular Lymphoma (FL).
  • the B-NHL is Mantle cell lymphoma (MCL).
  • MZL marginal zone lymphoma (MZL) (nodal, extranodal or splenic).
  • the patient or B-NHL can be specified as being R/R, e.g.
  • the multispecific protein that specifically binds to NKp46 and to CD20 is a multispecific protein that binds to NKp46 and a cytokine receptor (e.g.
  • the multispecific protein is capable of increasing NK cell cytotoxicity toward a target cell that expresses CD20 (e.g., a B-NHL cell).
  • the multispecific protein can in any embodiment be characterized as being capable of increasing NK cell cytotoxicity toward a target cell that expresses CD20 (e.g., a B-NHL cell) via both activation of NKp46 signaling and activation of CD16A signaling in the NK cells.
  • the multispecific protein is capable of increasing NK cell activation and/or proliferation via activation of IL2 receptor signaling in NK cells.
  • the multispecific protein can be specified to have monovalent binding to NKp46 (e.g. the multispecific protein comprises only one ABD that binds NKp46), monovalent (or optionally bivalent) binding to CD20 (one or two ABDs that bind CD20), monovalent binding to CD16A (e.g. the multispecific protein comprises only one Fc domain dimer or one ABD that binds CD16A), and monovalent binding to cytokine receptor (e.g., the multispecific protein comprises only one ABD that binds a cytokine receptor).
  • the NKp46 binding domain (exemplified as a VH/VL pair comprised in a Fab or scFv), the CD16- binding Fc domain and the cytokine were placed adjacent to one another in series within the protein, each separated from the adjacent element (i.e. NKp46 ABD, Fc domain or cytokine) solely by a short flexible peptide linker.
  • NKp46 ABD adjacent element
  • Fc domain or cytokine cytokine
  • the multispecific protein can be characterized as capable of (or having binding domains arranged to be capable of) co-engaging NKp46, CD16A and CD122 on the same cell surface plane, e.g, on an NK cell.
  • the multispecific protein comprises a first and a second antigen binding domain (ABDs) that comprises an immunoglobulin heavy variable domain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR1, CDR2, and CDR3); and wherein: (i) the first antigen binding domain (ABD) specifically binds to human CD20 and comprises: - a VH1 comprising a CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 29 (HCDR1), SEQ ID NO: 32 (HCDR2), SEQ ID NO: 35 (HCDR3), and - a VL1 comprising a CDR1, CDR2 and CDR3 corresponding
  • the multispecific protein comprises a variant IL-2 polypeptide that bind CD122, said variant IL-2 comprising the amino acid sequence of SEQ ID NO: 65 or an amino acid sequence having at least 70%, 80% or 90% of sequence identity thereto.
  • the multispecific protein comprises a first (I) polypeptide having the amino acid sequence of SEQ ID NO: 1, and a second (II) polypeptide having the amino acid sequence of SEQ ID NO: 70.
  • the multispecific protein comprises a first (I) polypeptide chain having the amino acid sequence of SEQ ID NO: 1, a second (II) polypeptide chain having the amino acid sequence of SEQ ID NO: 9, and a third (III) polypeptide chain having the amino acid sequence of SEQ ID NO: 17.
  • the multispecific protein comprises a first (I) polypeptide chain having the amino acid sequence of SEQ ID NO: 1, a second (II) polypeptide chain having the amino acid sequence of SEQ ID NO: 73, and a third (III) polypeptide chain having the amino acid sequence of SEQ ID NO: 74.
  • the multispecific protein comprises a first (I) polypeptide having an amino acid sequence having at least 90% of sequence identity with the amino acid sequence of SEQ ID NOS: 1 or 66, a second (II) polypeptide having an amino acid sequence having at least 90% of sequence identity with the amino acid sequence of SEQ ID NOS: 6, 67, 70, or 73, and optionally a third (III) polypeptide having an amino acid sequence having at least 90% of sequence identity with the amino acid sequence of SEQ ID NOS: 17 or 74.
  • the multispecific protein comprises all or part of an immunoglobulin Fc region or variant thereof that binds to a human Fc- ⁇ receptor, said all of part of an immunoglobulin Fc region comprising an CH2-CH3 domain having at least 90 % of sequence identity with an amino acid sequence of SEQ ID NO: 6 or 14.
  • the multispecific protein comprises a first and a second antigen binding domains (ABDs), a cytokine moiety and all or part of an immunoglobulin Fc region or variant thereof, wherein the first ABD has a Fab structure and comprises an immunoglobulin heavy chain (VH) and an immunoglobulin light chain variable domain (VL), wherein each VH and VL comprises three complementary determining regions (CDR1, CDR2, CDR3); and wherein: (i) the first ABD binds specifically to human CD20 and comprises: - a VH1 comprising a CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 29 (HCDR1), SEQ ID NO: 32 (HCDR2), SEQ ID NO: 35 (HCDR3), and - a VL1 comprising a CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 38 (LCDR1), SEQ ID NO: 41 (LCDR2), SEQ
  • the cytokine moiety is a variant IL-2.
  • the first and second ABDs of the multispecific protein have a Fab structure.
  • the first ABD of the multispecific protein has a Fab structure and the second ABD of the multispecific protein has an scFv structure.
  • the multispecific protein comprises three polypeptide chains (I), (II) and (III) that form the two ABDs as defined above: V 1A – C 1A – Hinge 1 – (Fc domain) A (I) V 1B – C 1B – Hinge 2 – (Fc domain) B – L 1 – V 2A – C 2A (II) V 2B – C 2B – Hinge 3 – L 2 –IL-2 (III) wherein: V 1A and V 1B form a binding pair V 1 (V H1 /V L1 ) of the first ABD; V 2A and V 2B form a binding pair V 2 (V H2 /V L2 ) of the second ABD; C 1A and C 1B form a pair C 1 (CH1/C L ) and C 2A and C 2B form a pair C 2 (CH1/C L ) wherein CH1 is an immunoglobulin heavy chain constant domain 1 and C L is an immunoglobul
  • the multispecific protein comprises two polypeptide chains (I) and (II) that form two ABDs as defined above: V 1A – C 1A – Hinge 1 – (Fc domain) A (I) V 1B – C 1B – Hinge 2 – (Fc domain) B – L 1 – V 2A – L 2 – V 2B – L 3 – IL-2 (II) wherein: V 1A and V 1B form a binding pair V 1 (V H1 /V L1 ) of the first ABD; V 2A and V 2B form a binding pair V 2 (V H2 /V L2 ) of the second ABD; C1A and C1B form a pair C1 (CH1/CL) wherein CH1 is an immunoglobulin heavy chain constant domain 1 and C L is an immunoglobulin light chain constant domain; Hinge 1 and Hinge 2 are identical or different and correspond to all or part of an immunoglobulin hinge region; (Fc domain)
  • the CH1 domain is an immunoglobulin heavy chain constant domain 1 that comprises the amino acid sequence of SEQ ID NO: 12.
  • the C K domain is an immunoglobulin kappa light chain constant domain (C K ) that comprises the amino acid sequence of SEQ ID NO: 4.
  • the (Fc domain) A comprises a CH2-CH3 domains corresponding to the amino acid sequence of SEQ ID NO: 6.
  • the (Fc domain) B comprises a CH2-CH3 domains corresponding to the amino acid sequence of SEQ ID NO: 14.
  • the Hinge 1 domain has an amino acid sequence of SEQ ID NO: 5.
  • the Hinge 2 domain has an amino acid sequence of SEQ ID NO: 13.
  • the Hinge 3 domain has an amino acid sequence of SEQ ID NO: 19.
  • the linker L 1 has an amino acid sequence of SEQ ID NO: 15.
  • the linker L 2 has an amino acid sequence of any one of SEQ ID NOS: 20-23.
  • the multispecific protein has a residue N297 of the Fc domain or variant thereof according to Kabat numbering that comprises a N-linked glycosylation.
  • the Fc domain or variant thereof of the multispecific protein binds to a human CD16A (Fc ⁇ RIII) polypeptide.
  • the multispecific protein comprises at least two polypeptide chains linked by at least one disulfide bridge.
  • the polypeptide chains (I) and (II) of the multispecific protein are linked by one disulfide bridge between C 1A and Hinge 2 , two disulfide bridges between Hinge 1 and Hinge 2 and wherein the polypeptide chains (II) and (III) are linked by one disulfide bridge between Hinge3 and C2B.
  • the V 1A domain is V L1 and V 1B domain is V H1 .
  • the V 2A domain is V H2 and V 2B domain is V L2 .
  • the C1A domain is CK and C1B domain is CH1.
  • the C 2A domain is C K and C 2B domain is CH1.
  • the C 2A domain is CH1 and C 2B domain is C K .
  • the multispecific protein comprises: (a) V H1 and V L1 corresponds to the amino acid sequences of SEQ ID NO: 11 and 3 respectively, and/or (b) V H2 and V L2 corresponds to the amino acid sequences of SEQ ID NO: 93 and 95 respectively.
  • the variant IL- displays reduced binding to CD25 compared to a wild-type human IL-2 polypeptide.
  • the binding variant IL-2 comprises an amino acid sequence at least 90% identical to a sequence selected from SEQ ID NOS: 24-28 and 65, or to a contiguous sequence of at least 40, 50, 60, 70, 80 or 100 amino acid residues thereof.
  • the multispecific protein comprises a first, second and where present third polypeptide having the amino acid sequence of the respective the first, second and where present, third polypeptides of a multispecific protein used herein, e.g. an NKCE molecule shown in Table 8.
  • the multispecific protein comprises: - A polypeptide (I) consisting of an amino acid sequence of SEQ ID NO: 1; - A polypeptide (II) consisting of an amino acid sequence of SEQ ID NO: 9; and - A polypeptide (III) consisting of an amino acid sequence of SEQ ID NO: 17.
  • the multispecific protein comprises: - A polypeptide (I) consisting of an amino acid sequence of SEQ ID NO: 1; - A polypeptide (II) consisting of an amino acid sequence of SEQ ID NO: 73; and - A polypeptide (III) consisting of an amino acid sequence of SEQ ID NO: 74.
  • the first ABD that binds to CD20 is an Fab and the second ABD that binds to NKp46 is an scFv.
  • the first ABD is a VH/VL pair.
  • the second ABD of the multispecific protein and the cytokine moiety have an arrangement; – L1 –V 2A – L2 – V 2B – L3– IL-2, Wherein V 2A and V 2B form a binding pair V 2 (V H2 /V L2 ) of the second ABD; L 1 , L 2 and L 3 are an amino acid linker, wherein L 1 , L 2 and L 3 can be different or the same; IL-2 is a variant human interleukin-2 polypeptide or portion thereof that binds to CD122 present on NK cells.
  • the V 2A domain of a multispecific protein is V H2 and V 2B domain is V L2 .
  • the multispecific protein is administered between 1 and 4 times per month, optionally once every 2 week, optionally every 3 weeks, optionally once every 4 weeks, optionally further wherein treatment is for a period of at least 3 months, 6 months or 12 months.
  • Figure 1 shows an exemplary multispecific protein in T5 format that binds to NKp46, CD16A and CD122 on an NK cell, and to CD20 on a tumor cell.
  • Figures 2A to 2K show different configurations of multispecific proteins that differ in the number of polypeptide chain, and in the configuration of the domains around an Fc domain dimer.
  • Figure 3 shows individual proliferation curves of NK, total T cells, CD4+ and CD8+ T cells obtained for the 11 R/R B-NHL patients stimulated for 6 days with a concentration range of CD20-2-T13-NKCE4-V2A (from 150 to 0.000015 nM) were plotted.
  • Each dot is the mean of the cell proliferation values of the 11 R/R B-NHL samples included in this study after 6 days of incubation with a concentration range of CD20-2-T13- NKCE4-V2A (from 150 to 0.000015 nM).
  • Figure 5 shows mean of NK proliferation frequencies of the different types of R/R B- NHL samples (R/R DLBCL, R/R MCL, R/R FL, R/R MZL/LPL) after 6 days of incubation with CD20-2-T13-NKCE4-V2A.
  • Figure 6 shows the mean of cell proliferation frequencies for the 11 R/R B-NHL patients and 5 healthy donors, including the mean percentages of proliferating NK cells (Figure 6A), total T cells (Figure 6B) and CD4+ and CD8+ T cells ( Figures 6C and D) were similar in R/R B-NHL patients and healthy donors.
  • Figure 7 shows CD20-2-T13-NKCE4-V2A- and epcoritamab biosimilar-induced B cell depletion in PBMCs from R/R B-NHL patients.
  • PBMCs from B-NHL patients were incubated for 24 hours with a dose range of CD20-2-T13-NKCE4-V2A (NKCE) or epcoritamab biosimilar (TCE), from 9.375 nM to 0.0003 nM, with 8-fold dilutions.
  • the percentage of CD3-CD19+ B cells among lymphocytes was determined by flow cytometry.
  • Sample 7 corresponds to a patient without blood circulating tumoral B cells and samples 10, 9, 12 and 11 were in a leukemic phase and thus PBMCs from these samples included tumoral cells.
  • Black circles correspond to CD20-2-T13-NKCE4-V2A-treated samples; grey triangles correspond to epcoritamab biosimilar-treated samples and red triangles correspond to control (no antibody).
  • Figure 8 shows CD20-2-T13-NKCE4-V2A- and epcoritamab biosimilar-mediated B cell depletion in PBMCs from HDs.
  • Figure 8 (top panel), CD20-2-T13-NKCE4-V2A induced B cell lysis in a dose-dependent manner in the 5 HDs analyzed.
  • Figure 8 (bottom panel) shows B cell lysis by epcoritamab in PBMC from the 5 HDs analyzed.
  • Figure 9 shows representative examples for NKp46 and CD16 expression.
  • NKp46 expression on NK cells was similar in LN and PBMC from R/R B-NHL patients and HD. NKp46 expression was maintained in tumoral LN from RR B-NHL patients while CD16 was largely downmodulated.
  • Figure 10 shows NKp46, CD122 and CD16 expression on PBMC from healthy donors (black dots) or from B-NHL patient who received CAR-T therapy (black dots), either in terms of frequency of positive cells (% positive cells) or expression level (MedFI).
  • NKp46 expression on PBMC was similar in HD and B-NHL patients while CD16 was strongly downmodulated in B-NHL patients.
  • Figure 11 shows depletion of Raji tumor B cells induced by CD20-2-T13-NKCE4-V2A- and epcoritamab biosimilar in PBMCs from B-NHL patients who received prior CAR-T therapy.
  • Figure 12 shows CD20+ B cell depletion in blood of non human primate (NHP) treated with 0.05mg/kg, 0.3mg/kg and 0.05mg/kg Q2W with CD20-2-T13-NKCE4-V2A. Circulating B cell frequencies among leukocytes were analyzed by flow cytometry, and multiplied by the absolute leukocyte counts per volume.
  • NHS non human primate
  • Figure 13 shows CD20+ cell depletion in lymphoid tissues of NHP treated with CD20- 2-T13-NKCE4-V2A.
  • CD20 positive area ratio (% difference to vehicle control), with the bars from left to right representing vehicle (leftmost bar), 0.05 mg/kg, 0.3 mg/kg, 0.5 mg/kg body weight (rightmost bar).
  • Figures 14A and 14B show, respectively, NKp46 staining and CD3 staining, in each case by immunohistochemistry in spleen, axillary lymph nodes (LN Axi) and mandibullary lymph nodes (LN Man) from NHP treated with 0.05, 0.3, and 0.5 mg/kg of CD20-2-T13- NKCE4-V2A.
  • Figure 15 shows IFN- ⁇ , MCP-1, MIP-1 ⁇ , and IL-6 blood levels in NHP treated with 0.05, 0.3, and 0.5 mg/kg of CD20-2-T13-NKCE4-V2A.
  • Figure 16A shows expression levels of NK cell activating receptors NKp30, DNAM-1, and NKG2D on the surface of cells when human PBMC are incubated with CD20-2-T13- NKCE4-V2A for 72 hours.
  • Figure 16B shows expression levels of NK cell activating receptors NKp30, DNAM-1, and NKG2D on the surface of cells when human PBMC are incubated with CD20-2-T13- NKCE4-V2A, control molecule IC-NKCE-IL2v lacking the CD20 binding moiety, recombinant IL-2, rituximab or Obinutuzumab.
  • Figure 16C shows lysis B16F10 cells, B16F10-huMICA cells or B16F10-huCD20 cells after incubation with the CD20-NKCE-IL2v or obinutuzumab, in the presence or absence of a blocking anti-NKG2D antibody.
  • Figure 17 shows structure/function relationships for multispecific NK cell engager (NKCE) protein binding on one side to a tumor antigen on a tumor cell, and on another side to an NK cell via a triple receptor cis-presentation of IL2 ⁇ ⁇ complex, NKp46 and CD16A.
  • NKCE multispecific NK cell engager
  • the words “a” or “an” may mean one or more than one. Where “comprising” is used, this can optionally be replaced by “consisting essentially of”, or optionally by “consisting of”.
  • the term “antigen binding domain” or ”ABD” refers to a domain comprising a three-dimensional structure capable of immunospecifically binding to an epitope.
  • said domain can comprise a hypervariable region, optionally a V H and/or V L domain of an antibody chain, optionally at least a V H domain.
  • the binding domain may comprise at least one complementarity determining region (CDR) of an antibody chain.
  • the binding domain may comprise a polypeptide domain from a non-immunoglobulin scaffold.
  • antibody herein is used in the broadest sense and specifically includes full- length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments and derivatives, so long as they exhibit the desired biological activity.
  • Various techniques relevant to the production of antibodies are provided in, e.g., Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).
  • An “antibody fragment” comprises a portion of a full- length antibody, e.g. antigen-binding or variable regions thereof.
  • antibody fragments include Fab, Fab', F(ab) 2 , F(ab’) 2 , F(ab) 3 , Fv (typically the V L and V H domains of a single arm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the V H and CH1 domain), and dAb (typically a V H domain) fragments; V H , V L , VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 1997;10: 949-57); camel IgG; IgNAR; and multispecific antibody fragments formed from antibody fragments, and one or more isolated CDRs or a functional paratope, where isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment.
  • hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen binding.
  • the hypervariable region generally comprises amino acid residues from a "complementarity-determining region" or "CDR" (e.g.
  • the numbering of amino acid residues in this region is performed by the method described in Kabat et al., supra.
  • Phrases such as “Kabat position”, “variable domain residue numbering as in Kabat” and “according to Kabat” herein refer to this numbering system for heavy chain variable domains or light chain variable domains.
  • the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
  • a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g.
  • residues 82a, 82b, and 82c, etc. according to Kabat after heavy chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard” Kabat numbered sequence.
  • framework or "FR" residues as used herein is meant the region of an antibody variable domain exclusive of those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into the contiguous regions separated by the CDRs (FR1, FR2, FR3 and FR4).
  • constant region as defined herein is meant an antibody-derived constant region that is encoded by one of the light or heavy chain immunoglobulin constant region genes.
  • constant light chain or “light chain constant region” or “CL” as used herein is meant the region of an antibody encoded by the kappa (C ⁇ ) or lambda (C ⁇ ) light chains.
  • the constant light chain typically comprises a single domain, and as defined herein refers to positions 108- 214 of C ⁇ , or C ⁇ , wherein numbering is according to the EU index (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda).
  • constant heavy chain or “heavy chain constant region” as used herein is meant the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon genes to define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively.
  • the constant heavy chain refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus comprising positions 118-447, wherein numbering is according to the EU index.
  • CH1 domain or “C H 1 domain”, or “constant domain 1”
  • CH2 domain or “C H 2 domain”, or “constant domain 2” can be used interchangeably and refer to the corresponding heavy chain immunoglobulin constant domain 2.
  • CH3 domain or “C H 3 domain”, or “constant domain 3” can be used interchangeably and refer to the corresponding heavy chain immunoglobulin constant domain 3.
  • CH2-CH3 as in (CH2-CH3) A and (CH2-CH3) B , thus refers to a polypeptide sequence comprising an immunoglobulin heavy chain constant domain 2 (CH2) and an immunoglobulin heavy chain constant domain 3 (CH3).
  • the terms “pair C (CH1/CL)”, or “paired C (C H 1/C L )” refer to one constant heavy chain domain 1 and one constant light chain domain (e.g. a kappa ( ⁇ or K ) or lamba ( ⁇ ) class of immunoglobulin light chains) bound to one another by covalent or non- covalent bonds, preferably non-covalent bonds; thus forming a heterodimer.
  • the constant chain domains forming the pair are not present on a same polypeptide chain, this term may thus encompass all possible combinations.
  • the corresponding C H 1 and C L domains will thus be selected as complementary to each other, such that they form a stable pair C (CH1/CL).
  • each CH1 and CL domain forming the pairs will be selected so that they are formed between complementary CH1 and CL domains.
  • Examples of complementary C H 1 and C L domains have been previously described in the international patent applications WO2006/064136 or WO2012/089814 or WO2015197593A1.
  • the terms “pair C 1 (C H 1/C L )” or “pair C 2 (C H 1/C L )” may refer to distinct constant pair domains (C 1 and C 2 ) formed by identical or distinct constant heavy 1 domains (C H 1) and identical or distinct constant light chain domains (C L ).
  • the terms “pair C 1 (C H 1/C L )” or “pair C2 (C H 1/CL)” may refer to distinct constant pair domains (C 1 and C 2 ) formed by identical constant heavy 1 domains (C H 1) and identical constant light chain domains (C L ).
  • Fab or "Fab region” as used herein is meant a unit that comprises the V H , CH1, V L , and CL immunoglobulin domains.
  • the term Fab includes a unit that comprises a V H -CH1 moiety that associates with a V L -CL moiety, as well as crossover Fab structures in which there is crossing over or interchange between light- and heavy-chain domains.
  • a Fab may have a V H -CL unit that associates with a V L -CH1 unit.
  • Fab may refer to this region in isolation, or this region in the context of a protein, multispecific protein or ABD, or any other embodiments as outlined herein.
  • single-chain Fv or “scFv” as used herein are meant antibody fragments comprising the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the scFv to form the desired structure for antigen binding.
  • Methods for producing scFvs are well known in the art. For a review of methods for producing scFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.269-315 (1994).
  • Fv or “Fv fragment” or “Fv region” as used herein is meant a polypeptide that comprises the VL and VH domains of a single antibody.
  • Fc or “Fc region”, as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N- terminal to these domains.
  • Fc may include the J chain.
  • Fc comprises immunoglobulin domains C ⁇ 2 (CH2) and C ⁇ 3 (CH3) and optionally the hinge between C ⁇ 1 and C ⁇ 2.
  • the human IgG heavy chain Fc region is usually defined to comprise residues C226, P230 or A231 to its carboxyl-terminus, wherein the numbering is according to the EU index.
  • Fc may refer to this region in isolation, or this region in the context of an Fc polypeptide, as described below.
  • Fc polypeptide or “Fc-derived polypeptide” as used herein is meant a polypeptide that comprises all or part of an Fc region.
  • Fc polypeptides herein include but are not limited to antibodies, Fc fusions and Fc fragments.
  • Fc regions according to the invention include variants containing at least one modification that alters (enhances or diminishes) an Fc associated effector function.
  • Fc regions according to the invention include chimeric Fc regions comprising different portions or domains of different Fc regions, e.g., derived from antibodies of different isotype or species.
  • variant region as used herein is meant the region of an antibody that comprises one or more Ig domains substantially encoded by any of the V L (including V ⁇ (V ⁇ ) and V ⁇ ) and/or V H genes that make up the light chain (including ⁇ and ⁇ ) and heavy chain immunoglobulin genetic loci respectively.
  • a light or heavy chain variable region (V L or V H ) consists of a "framework" or "FR" region interrupted by three hypervariable regions referred to as “complementarity determining regions” or "CDRs".
  • the extent of the framework region and CDRs have been precisely defined, for example as in Kabat (see “Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, (1983)), and as in Chothia.
  • the framework regions of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs, which are primarily responsible for binding to an antigen.
  • domain may be any region of a protein, generally defined on the basis of sequence homologies or identities, which is related to a specific structural or functional entity. Accordingly, the term “region”, as used in the context of the present disclosure, is broader in that it may comprise additional regions beyond the corresponding domain.
  • linker region refers to any amino acid sequence suitable for covalently linking two polypeptide domains, such as two antigen-binding domains together and/or a Fc region to one or more variable regions, such as one or more antigen-binding domains.
  • amino acid linkers are generally less than 50 amino acids in length, preferably less than 30 amino acids in length, for instance 20 or less than 20 amino acids in length, for instance 15 or less than 15 amino acids in length.
  • amino acid linkers may optionally comprise all or part of an immunoglobulin polypeptide chain, such as all or part of a hinge region of an immunoglobulin.
  • the amino acid linker may comprise a polypeptide sequence that is not derived from a hinge region of an immunoglobulin, or even that is not derived from an immunoglobulin heavy or light polypeptide chain.
  • an immunoglobulin hinge region may thus be considered as a particular type of linker, which is derived from an immunoglobulin polypeptide chain.
  • linker which is derived from an immunoglobulin polypeptide chain.
  • the term “hinge region” or “hinge” refers to a generally flexible region and born by the corresponding heavy chain polypeptides, and which separates the Fc and Fab portions of certain isotypes of immunoglobulins, more particularly of the IgG, IgA or IgD isotypes.
  • hinge regions are known in the Art to depend upon the isotype of immunoglobulin which is considered.
  • the hinge region thus separates the C H 1 domain and the C H 2 domain and is generally cleaved upon papain digestion.
  • the region corresponding to the hinge in IgM and IgE heavy chains is generally formed by an additional constant domain with lower flexibility.
  • the hinge region may comprise one or more cysteines involved in interchain disulfide bonds.
  • the hinge region may also comprise one or more binding sites to a Fc ⁇ receptor, in addition to Fc ⁇ R binding sites born by the C H 2 domain, when applicable.
  • the hinge region may comprise one or more post-translational modification, such as one or more glycosylated residues depending on the isotype which is considered.
  • hinge regions which are still particularly considered comprise all or part of a hinge from an immunoglobulin belonging to one isotype selected from: the IgG isotype, the IgA isotype and the IgD isotype; in particular the IgG isotype.
  • the term “specifically binds to” means that an antibody or polypeptide can bind preferably in a competitive binding assay to the binding partner, e.g. NKp46, as assessed using either recombinant forms of the proteins, epitopes therein, or native proteins present on the surface of isolated target cells.
  • an antibody or polypeptide When an antibody or polypeptide is said to “compete with” a particular multispecific protein or a particular monoclonal antibody or a multi-specific protein, it means that the antibody or polypeptide competes with the particular multispecific protein or monoclonal antibody in a binding assay using either recombinant target (e.g. NKp46) molecules or surface expressed target (e.g. NKp46) molecules.
  • recombinant target e.g. NKp46
  • surface expressed target e.g. NKp46
  • the affinity of an antibody is given by the dissociation constant K D , defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody- antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen.
  • K D dissociation constant
  • the affinity constant K A is defined by 1/K D .
  • a “determinant” designates a site of interaction or binding on a polypeptide.
  • epitope refers to an antigenic determinant, and is the area or region on an antigen to which an antibody or protein binds.
  • a protein epitope may comprise amino acid residues directly involved in the binding as well as amino acid residues which are effectively blocked by the specific antigen binding antibody or peptide, i.e., amino acid residues within the "footprint" of the antibody. It is the simplest form or smallest structural area on a complex antigen molecule that can combine with e.g., an antibody or a receptor. Epitopes can be linear or conformational/structural. The term “linear epitope” is defined as an epitope composed of amino acid residues that are contiguous on the linear sequence of amino acids (primary structure).
  • formational or structural epitope is defined as an epitope composed of amino acid residues that are not all contiguous and thus represent separated parts of the linear sequence of amino acids that are brought into proximity to one another by folding of the molecule (secondary, tertiary and/or quaternary structures). A conformational epitope is dependent on the 3-dimensional structure.
  • formational is therefore often used interchangeably with ‘structural’.
  • Epitopes may be identified by different methods known in the art including but not limited to alanine scanning, phage display, X-ray crystallography, array- based oligo-peptide scanning or pepscan analysis, site-directed mutagenesis, high throughput mutagenesis mapping, H/D-Ex Mass Spectroscopy, homology modeling, docking, hydrogen- deuterium exchange, among others.
  • Valent or “valency” denotes the presence of a determined number of antigen-binding moieties in the antigen-binding protein.
  • a natural IgG has two antigen-binding moieties and is bivalent.
  • a molecule having one binding moiety for a particular antigen is monovalent for that antigen.
  • amino acid modification herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence.
  • An example of amino acid modification herein is a substitution.
  • amino acid modification herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence.
  • amino acid substitution or “substitution” herein is meant the replacement of an amino acid at a given position in a protein sequence with another amino acid.
  • substitution Y50W refers to a variant of a parent polypeptide, in which the tyrosine at position 50 is replaced with tryptophan.
  • Amino acid substitutions are indicated by listing the residue present in wild-type protein / position of residue / residue present in mutant protein.
  • variant of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide, typically a native or “parent” polypeptide.
  • the polypeptide variant may possess one or more amino acid substitutions, deletions, and/or insertions at certain positions within the native amino acid sequence.
  • "Conservative” amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties.
  • Families of amino acid residues having similar side chains are known in the art, and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., as
  • identity refers to the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.
  • Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res.12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol.215, 403-410 (1990)).
  • the BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra).
  • NCBI National Center for Biotechnology Information
  • the well-known Smith Waterman algorithm may also be used to determine identity.
  • an “isolated” molecule is a molecule that is the predominant species in the composition wherein it is found with respect to the class of molecules to which it belongs (i.e., it makes up at least about 50% of the type of molecule in the composition and typically will make up at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of the species of molecule, e.g., peptide, in the composition).
  • a composition of a polypeptide will exhibit 98%, 98%, or 99% homogeneity for polypeptides in the context of all present peptide species in the composition or at least with respect to substantially active peptide species in the context of proposed use.
  • treatment refers alleviating, managing, curing or reducing one or more symptoms or clinically relevant manifestations of a disease or disorder, unless contradicted by context.
  • NK cells refers to a sub-population of lymphocytes that is involved in non-conventional immunity.
  • NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD56 and/or NKp46 for human NK cells, the absence of the alpha/beta or gamma/delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to express "self" MHC/HLA antigens by the activation of specific cytolytic machinery, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells, using methods well known in the art. Any subpopulation of NK cells will also be encompassed by the term NK cells.
  • active NK cells designate biologically active NK cells, including NK cells having the capacity of lysing target cells or enhancing the immune function of other cells.
  • NK cells can be obtained by various techniques known in the art, such as isolation from blood samples, cytapheresis, tissue or cell collections, etc. Useful protocols for assays involving NK cells can be found in Natural Killer Cells Protocols (edited by Campbell KS and Colonna M). Humana Press. pp.219-238 (2000).
  • an agent that has “agonist” activity at NKp46 is an agent that can cause or increase "NKp46 signaling".
  • NKp46 signaling refers to an ability of an NKp46 polypeptide to activate or transduce an intracellular signaling pathway. Changes in NKp46 signaling activity can be measured, for example, by assays designed to measure changes in NKp46 signaling pathways, e.g. by monitoring phosphorylation of signal transduction components, assays to measure the association of certain signal transduction components with other proteins or intracellular structures, or in the biochemical activity of components such as kinases, or assays designed to measure expression of reporter genes under control of NKp46-sensitive promoters and enhancers, or indirectly by a downstream effect mediated by the NKp46 polypeptide (e.g. activation of specific cytolytic machinery in NK cells).
  • assays designed to measure changes in NKp46 signaling pathways e.g. by monitoring phosphorylation of signal transduction components, assays to measure the association of certain signal transduction components with other proteins or intracellular structures, or in the biochemical activity of components such as kinases, or
  • Reporter genes can be naturally occurring genes (e.g. monitoring cytokine production) or they can be genes artificially introduced into a cell. Other genes can be placed under the control of such regulatory elements and thus serve to report the level of NKp46 signaling.
  • NKp46 refers to a protein or polypeptide encoded by the Ncr1 gene or by a cDNA prepared from such a gene. Any naturally occurring isoform, allele, ortholog or variant is encompassed by the term NKp46 polypeptide (e.g., an NKp46 polypeptide 90%, 95%, 98% or 99% identical to SEQ ID NO 1, or a contiguous sequence of at least 20, 30, 50, 100 or 200 amino acid residues thereof).
  • NKp46 (isoform a) is shown below: Table 1: S N SEQ ID NO: 88 corresponds to NCBI accession number NP_004820, the disclosure of which is incorporated herein by reference.
  • the human NKp46 mRNA sequence is described in NCBI accession number NM_004829, the disclosure of which is incorporated herein by reference.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the term “subject” or “individual” or “patient” are used interchangeably and may encompass a human or a non-human mammal, rodent or non-rodent.
  • the term includes, but is not limited to, mammals, e.g., humans including man, woman and child, other primates (monkey), pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats.
  • administerable includes but is not limited to delivery of a drug by an injectable form, such as, for example, an intravenous, intramuscular, intradermal or subcutaneous route.
  • the administration is by an injectable form.
  • Multispecific proteins can be conveniently configured and produced using well known immunoglobulin-derived domains, notably heavy and light chain variable domains, hinge regions, CH1, CL, CH2 and CH3 constant domains, and wild-type or variant cytokine polypeptides. Domains placed on a common polypeptide chain can be fused to one another either directly or connected via linkers, depending on the particular domains concerned.
  • the immunoglobulin-derived domains will preferably be humanized or of human origin, thereby providing decreased risk of immunogenicity when administered to humans.
  • advantageous protein formats are described that use minimal non-immunoglobulin linking amino acid sequences (e.g.
  • Immunoglobulin variable domains are commonly derived from antibodies (immunoglobulin chains), for example in the form of associated V L and V H domains found on two polypeptide chains, or a single chain antigen binding domain such as an scFv, a VH domain, a V L domain, a dAb, a V-NAR domain or a V H H domain.
  • the antigen binding domain e.g., ABD 1 and ABD 2
  • the antigen binding domain can also be readily derived from antibodies as a Fab or scFv.
  • the term “antigen-binding protein” can be used to refer to an immunoglobulin derivative with antigen binding properties.
  • the multispecific protein can be specified as being an antigen-binding protein that comprises an immunologically functional immunoglobulin portion capable of binding to a target antigen.
  • the immunologically functional immunoglobulin portion may comprise immunoglobulins, or portions thereof, fusion peptides derived from immunoglobulin portions or conjugates combining immunoglobulin portions that form an antigen binding site.
  • Each antigen binding moiety comprises at least the necessarily one, two or three CDRs of the immunoglobulin heavy and/or light chains from which the antigen binding moiety was derived.
  • an antigen-binding protein can consist of a single polypeptide chain (a monomer).
  • the antigen-binding protein comprises at least two polypeptide chains, e.g. a multimeric protein, optionally specified as being a dimeric protein trimeric protein.
  • an antigen binding domain can conveniently comprise a VH and a VL (a VH/VL pair).
  • the VH/VL pair can be integrated in a Fab structure further comprising a CH1 and CL domain (a CH1/CL pair).
  • a VH/VL pair refers to one VH and one VL domain that associate with one another to form an antigen binding domain.
  • a CH1/CL pair refers to one CH1 and one CL domain bound to one another by covalent or non-covalent bonds, preferably non-covalent bonds, thus forming a heterodimer (e.g., within a protein such as a heterotrimer that can comprise one or more further polypeptide chains).
  • a binding protein comprises: (i) a first antigen-binding domain (ABD) comprising a variable region which binds specifically to a human CD20 polypeptide, (ii) a second antigen-binding domain (ABD) comprising a variable region which binds specifically to a human NKp46 polypeptide, (iii) all or part of an immunoglobulin Fc region or variant thereof which binds to a human Fc- ⁇ receptor (CD16), and optionally a cytokine (e.g., variant IL-2) moiety.
  • Multispecific polypeptides for use in the treatments of the disclosure are described in PCT patent publication no. WO2022/258673, the disclosure of which is incorporated herein by reference.
  • Figure 17 shows structure/function relationships for a multispecific NKCE protein comprising a variant IL2 (IL2v) binding on one side to a tumor antigen on a tumor cell, and on another side to an NK cell via a triple receptor cis-presentation of IL2 ⁇ ⁇ complex, NKp46 and CD16A.
  • Multimeric, multispecific proteins such as heterodimers and heterotrimers can be produced according to a variety of formats. Different domains onto different polypeptide chain that associate to form a multimeric protein.
  • an Fc moiety is derived from a human IgG1 isotype constant region.
  • a protein comprises a first and a second polypeptide chain each comprising a variable domain fused to a human Fc domain monomer (i.e. a CH2-CH3 unit), optionally a Fc domain monomer comprising a CH3 domain capable of undergoing preferential CH3-CH3 hetero-dimerization, wherein the first and second chain associate via CH3-CH3 dimerization and the protein consequently comprises a Fc domain dimer.
  • the variable domains of each chain can be part of the same or different antigen binding domains.
  • Multispecific proteins can thus be conveniently constructed using VH and VL pairs arranged as scFv or Fab structures, together with CH1 domains, CL domain, Fc domains and cytokines, and domain linkers.
  • the proteins will use minimal non-natural sequences, e.g. minimal use of non-Ig linkers, optionally no more than 5, 4, 3, 2 or 1 domain linker(s) that is not an antibody-derived sequence, optionally wherein domain linker(s) are no more than 15, 10 or 5 amino acid residues in length.
  • the protein comprises a CD16 ABD embodied as a Fc domain dimer.
  • the multispecific proteins e.g.
  • dimers, trimers may comprise a domain arrangement of any of the following in which domains can be placed on any of the 2 or 3 polypeptide chains, wherein the NKp46 ABD is interposed between the Fc domain and the cytokine moiety (e.g. the protein has a terminal or distal cytokine receptor ABD at the C- terminal end and a terminal or distal CD20 ABD at the topological N-terminal end), wherein the NKp46 ABD is connected to one of the polypeptide chains of the Fc domain dimer via a hinge polypeptide or a flexible linker, and wherein the ABD that binds the cytokine receptor is connected to NKp46 ABD (e.g.
  • a flexible linker e.g. a linker comprising G and S residues
  • the cytokine moiety can be an IL2 polypeptide or variant thereof.
  • the Fc domain dimer can be specified to be a Fc domain dimer that binds human FcRn and/or Fc ⁇ receptors.
  • one or both of the CD20 ABD and NKp46 ABD is formed from two variable regions present, wherein the variable regions that associate to form a particular ABD can be on the same polypeptide chain or on different polypeptide chains.
  • one or both of CD20 ABD and NKp46 ABD comprises a tandem variable region (scFv) and the other comprises a Fab structure.
  • both of the antigen of interest and NKp46 ABD comprises a Fab structure.
  • the CD20 ABD comprises a Fab structure and the NKp46 ABD comprises an scFv structure.
  • the multispecific protein is heterotrimeric and comprises three polypeptide chains (I), (II) and (III) that form ABDs, as defined above: V 1A – C 1A – Hinge 1 – (Fc domain) A (I) V 1B – C 1B – Hinge 2 – (Fc domain) B – L 1 – V 2A – C 2A (II) V 2B – C 2B – Hinge 3 – L 2 –IL-2 (III) wherein: V 1A and V 1B form a binding pair V 1 (V H1 /V L1 ); V 2A and V 2B form a binding pair V 2 (V H2 /V L2 ); C 1A and C 1B form a pair C 1 (CH1/C L ) and C 2A and C 2B form a pair C 2 (CH1/C L ) wherein CH1 is an immunoglobulin heavy chain constant domain 1 and C L is an immunoglobulin light chain constant domain; Hing
  • binding pair V 1 binds CD20 and binding pair V 2 binds NKp46.
  • Each of V 1A , V 1B, V 2A , V 2B are an immunoglobulin VH or VL domain, wherein one of V 1A and V 1B is a VH and the other is a VL, and wherein one of V 2A and V 2B is a VH and the other is a VL.
  • the multispecific protein has a residue N297 of the Fc domain or variant thereof according to Kabat numbering that comprises a N-linked glycosylation.
  • the multispecific protein comprises an Fc domain that binds to human CD16A polypeptide.
  • V1A is VL1 and V1B is VH1.
  • V2A is VH2 and V2B is VL2.
  • C1A is CK and C1B is CH1.
  • C2A is CK and C2B is CH1.
  • VH1 comprises a CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 29 (HCDR1), SEQ ID NO: 32 (HCDR2), SEQ ID NO: 35 (HCDR3);
  • VL1 comprises a CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 38 (LCDR1), SEQ ID NO: 41 (LCDR2), SEQ ID NO: 44 (LCDR3);
  • VH2 comprises a CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 47 (HCDR1), SEQ ID NO: 50 (HCDR2), SEQ ID NO: 53 (HCDR3), and
  • VL2 comprises a CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 56 (LCDR1), SEQ ID NO: 59 (LCDR2), SEQ ID NO: 62 (LCDR3).
  • the multispecific protein comprises (a) V H1 and V L1 corresponds to the amino acid sequences of SEQ ID NOS: 11 and 3 respectively, and/or (b) V H2 and V L2 corresponds to the amino acid sequences of SEQ ID NOS: 93 and 95 respectively, as shown hereinafter.
  • V H1 (SEQ ID NO: 11) EVQLVESGGG LVQPDRSLRL SCAASGFTFH DYAMHWVRQA PGKGLEWVST ISWNSGTIGY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TALYYCAKDI QYGNYYYGMD VWGQGTTVTVTV SS V L1 (SEQ ID NO: 3) EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPITFGQ GTRLEIK V H2 (SEQ ID NO: 93) QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYVINWVRQA PGQGLEWMGE IYPGSGTNYY NEKFKAKATI TADKSTSTAY MELSSLRSED TAVYYCARRG RY
  • the multispecific protein comprises (a) VH1 and VL1 corresponds to the amino acid sequences of SEQ ID NOS: 11 and 3 respectively or a variant thereof with at least 90% of sequence identity, and/or (b) V H2 and V L2 corresponds to the amino acid sequences of SEQ ID NOS: 93 and 95 respectively or a variant thereof with at least 90% of sequence identity.
  • CH1 is an immunoglobulin heavy chain constant domain 1 that comprises the amino acid sequence of SEQ ID NO: 12;
  • CK is an immunoglobulin kappa light chain constant domain (CK) that comprises the amino acid sequence of SEQ ID NO: 4;
  • (Fc domain)A comprises a CH2-CH3 domains corresponding to the amino acid sequence of SEQ ID NO: 6;
  • (Fc domain)B comprises a CH2-CH3 domains corresponding to the amino acid sequence of SEQ ID NO: 14;
  • Hinge1 corresponds to the amino acid sequence of SEQ ID NO: 5;
  • Hinge2 corresponds to the amino acid sequence of SEQ ID NO: 13;
  • Hinge3 corresponds to the amino acid sequence of SEQ ID NO: 19;
  • the ABD that binds to CD20 and the ABD that binds to NKp46 each have a Fab structure.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 1, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 9, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17, as disclosed hereinafter.
  • First polypeptide chain (I) (SEQ ID NO: 1): EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPITFGQ GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGECDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 1, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 9, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17, or a variant thereof with at least 90% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 1, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 9, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17, or a variant thereof with at least 80% of sequence identity.
  • the multispecific protein having the above first, second and third polypeptide chains are in a protein format shown in Figures 1 and 2A (CD20-2-T5-NKCE4).
  • C2A is CH1 and C2B is CK.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 1, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 73, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 1, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 73, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74, or a variant thereof with at least 95% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 1, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 73, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74, or a variant thereof with at least 90% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 1, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 73, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74, or a variant thereof with at least 80% of sequence identity.
  • the multispecific proteins having the above first, second and third polypeptide chains are arranged in format shown in Figure 2G (CD20-2-T25- NKCE4).
  • the multispecific protein has a residue N297 (according to Kabat numbering) of Fc domains mutated to prevent said residue from be glycosylated. A substitution at N297 can thus substantially abolish CD16A binding where the Fc domain is desired to be configured without CD16A binding.
  • C2A is CK and C2B is CH1.
  • An exemplary multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 66, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 67, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17.
  • Another exemplary multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 66, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 67, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17, or a variant thereof with at least 95% of sequence identity.
  • Another exemplary multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 66, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 67, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17, or a variant thereof with at least 90% of sequence identity.
  • Another exemplary multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 66, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 67, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17, or a variant thereof with at least 80% of sequence identity.
  • An example of such proteins is represented in Figure 2B (CD20-2-T6-NKCE4).
  • C 2A is CH1
  • C 2B is C K .
  • a multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 66, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 75, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74.
  • a multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 66, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 75, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74, or a variant thereof with at least 95% of sequence identity.
  • a multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 66, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 75, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74, or a variant thereof with at least 90% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 66, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 75, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74, or a variant thereof with at least 80% of sequence identity.
  • the multispecific protein has Fc domains comprising L234A, L235E, G237A, A330S and/or P331S substitutions according to kabat numbering.
  • C2A is CK and C2B is CH1.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 68, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 69, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 68, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 69, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17, or a variant thereof with at least 95% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 68, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 69, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17, or a variant thereof with at least 90% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 68, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 69, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17, or a variant thereof with at least 80% of sequence identity.
  • a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 68
  • a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 69
  • a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17, or a variant thereof with at least 80% of sequence identity.
  • An example of such protein format is presented in Figure 2C (CD20-2-T6B3-NKCE4).
  • C2A is CH1
  • C2B is CK.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 68, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 76, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 68 or a variant thereof with at least 95% of sequence identity, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 76 or a variant thereof with at least 95% of sequence identity, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74 or a variant thereof with at least 95% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 68 or a variant thereof with at least 90% of sequence identity, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 76 or a variant thereof with at least 90% of sequence identity, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74 or a variant thereof with at least 90% of sequence identity, or a variant thereof with at least 90% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 68 or a variant thereof with at least 80% sequence identity, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 76 or a variant thereof with at least 80% of sequence identity, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74 or a variant thereof with at least 80% sequence identity.
  • An example of such proteins format is represented in Figure 2I (CD20-2-T26B3- NKCE4).
  • the multispecific protein comprises an ABD that binds to CD20 that is a VH/VL pair and an ABD that binds to NKp46 that is a Fab.
  • C2A is CK and C2B is CH1.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 77, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 79, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 77 or a variant thereof with at least 90% of sequence identity, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 79 or a variant thereof with at least 95% of sequence identity, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17 or a variant thereof with at least 95% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 77 or a variant thereof with at least 90% of sequence identity, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 79 or a variant thereof with at least 90% of sequence identity, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17 or a variant thereof with at least 90% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 77 or a variant thereof with at least 80% of sequence identity, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 79 or a variant thereof with at least 80% of sequence identity, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 17 or a variant thereof with at least 80% of sequence identity, or a variant thereof with at least 80% of sequence identity.
  • An example of such proteins is represented in Figure 2K (CD20-2-T195-NKCE4).
  • C 2A is CH1
  • C 2B is C K .
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 77, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 78, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 77, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 78, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74, or a variant thereof with at least 95% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 77 or a variant thereof with at least 90% of sequence identity, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 78 or a variant thereof with at least 90% of sequence identity, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74 or a variant thereof with at least 90% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 77 or a variant thereof with at least 80% of sequence identity, a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 78 or a variant thereof with at least 80% of sequence identity, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 74 or a variant thereof with at least 80% of sequence identity.
  • An example of such proteins is represented in Figure 2J (CD20-2-T175-NKCE4).
  • the multispecific protein is heterodimeric and comprises two polypeptide chains (I) and (II) that form two ABDs, as defined above: V 1A – C 1A – Hinge 1 – (Fc domain) A (I) V 1B – C 1B – Hinge 2 – (Fc domain) B – L1 –V 2A – L2 – V 2B – L3– IL-2 (II) wherein: V 1A and V 1B form a binding pair V 1 (V H1 /V L1 ); V 2A and V 2B form an scFv; C1A and C1B form a pair C1 (CH1/CL) and C2A and C2B form a pair C2 (CH1/CL) wherein CH1 is an immunoglobulin heavy chain constant domain 1 and C L is an immunoglobulin light chain constant domain; Hinge1, Hinge2 and Hinge3 are identical or different and correspond to all or part of an immunoglobulin hinge
  • binding pair V 1 binds CD20 and binding pair V 2 binds NKp46.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 1 (disclosed hereinafter), and a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 70 (disclosed hereinafter.
  • First polypeptide chain (I) (SEQ ID NO: 1): EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPITFGQ GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGECDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 1 or a variant thereof with at least 90% of sequence identity, and a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 70, or a variant thereof with at least 90% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 1 or a variant thereof with at least 80% of sequence identity, and a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 70, or a variant thereof with at least 80% of sequence identity.
  • the multispecific protein has a residue N297 (according to Kabat numbering) of Fc domains.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 66, and a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 71.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 66 or a variant thereof with at least 95% of sequence identity, and a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 71 or a variant thereof with at least 95% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 6 or a variant thereof with at least 90% of sequence identity, and a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 71 or a variant thereof with at least 90% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 66 or a variant thereof with at least 80% of sequence identity, and a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 71 or a variant thereof with at least 80% of sequence identity.
  • a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 66 or a variant thereof with at least 80% of sequence identity
  • a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 71 or a variant thereof with at least 80% of sequence identity.
  • An example of such protein format is presented in Figure 2E (CD20-2-T14-NKCE4).
  • the multispecific protein has Fc domains comprising L234A, L235E, G237A, A330S and/or P331S substitutions according to kabat numbering.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 68, and a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 72.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 68 or a variant thereof with at least 95% of sequence identity, and a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 72 or a variant thereof with at least 95% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 68 or a variant thereof with at least 90% of sequence identity, and a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 72, or a variant thereof with at least 90% of sequence identity.
  • the multispecific protein comprises a first polypeptide chain (I) comprising an amino acid sequence of SEQ ID NO: 68 or a variant thereof with at least 80% of sequence identity, and a second polypeptide chain (II) comprising an amino acid sequence of SEQ ID NO: 72, or a variant thereof with at least 80% of sequence identity.
  • the ABD that binds to a CD20 polypeptide of a binding protein of the disclosure comprises a VH and VL pair presented hereinafter in Table 2: Table 2: S N S N
  • the ABD that binds to a CD20 polypeptide of a binding protein of the disclosure comprises a VH comprising three CDRs (HCDR1, HCDR2 and HCDR3) and a VL comprising three CDRs (LCDR1, LCDR2 and LCDR3).
  • any of the CDR1, CDR2 and CDR3 of the heavy and light chains may be characterized by a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, and/or as having an amino acid sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR or set of CDRs listed in the corresponding SEQ ID NO or table hereinafter, that summarize the sequences of the CDRs, according to IMGT, Kabat and Chothia definitions systems: Table 3: C
  • the first ABD of the multispecific protein specifically binds to a human CD20 polypeptide and comprises: - a VH1 comprising a CDR1, CDR2 and CDR3 corresponding to the amino acid sequences of SEQ ID NO: 29 (HCDR1), SEQ ID NO: 32 (HCDR2), SEQ ID NO: 35 (HCDR3), and - a VL1 comprising a CDR1, CDR
  • the ABD that binds to a human NKp46 polypeptide (e.g., the second ABD) of the multispecific protein comprises a VH comprising CDR 1, 2 and 3 of amino acid sequences of SEQ ID NO: 47 (HCDR1), SEQ ID NO: 50 (HCDR2), SEQ ID NO: 53 (HCDR3), optionally wherein one, two, three or more amino acids in a CDR may be substituted by a different amino acid; and a VL comprising CDR 1, 2 and 3 of amino acid sequences of SEQ ID NO: 56 (LCDR1), SEQ ID NO: 59 (LCDR2), SEQ ID NO: 62 (LCDR3) optionally wherein one, two, three or more amino acids in a CDR may be substituted by a different amino acid.
  • said second ABD of the multispecific protein can bind a region spanning the D1 and D2 domains (at the border of the D1 and D2 domains, the D1/D2 junction), of the NKp46 polypeptide of SEQ ID NO: 1.
  • the VH/VL pair of the second ABD of the multispecific protein have an affinity for human NKp46, as a full-length IgG antibody, characterized by a K D of less than 10 -8 M, less than 10 -9 M, or less than 10 -10 M.
  • the multispecific proteins have an affinity (KD) for human NKp46 of between 1 and 100 nM, optionally between 1 and 50 nM, optionally between 1 and 20 nM, optionally about 10 or 15 nM, as determined by SPR.
  • the multispecific protein (or a NKp46-binding ABD or VH/VL pair thereof, for example as when configured in the multispecific protein or as a conventional full- length antibody) binds NKp46 at substantially the same region, site or epitope on NKp46 as antibody NKp46-1.
  • all key residues of the epitope are in a segment corresponding to domain D1 or D2.
  • the antibody or multispecific protein binds a residue present in the D1 domain as well as a residue present in in the D2 domain. In one embodiment, the antibodies bind an epitope comprising 1, 2, 3, 4, 5, 6, 7 or more residues in the segment corresponding to D1/D2 junction of the NKp46 polypeptide of SEQ ID NO: 88. In one embodiment, the antibodies or multispecific proteins bind NKp46 at the D1/D2 domain junction and bind an epitope comprising or consisting of 1, 2, 3, 4 or 5 of the residues K41, E42, E119, Y121 and/or Y194.
  • N N A NKp46-binding multispecific protein that binds essentially the same epitope or determinant as monoclonal antibody NKp46-1, optionally the antibody comprises a hypervariable region of antibody NKp46-1.
  • antibody NKp46- 1 can be characterized by its amino acid sequence and/or nucleic acid sequence encoding it.
  • the antibody comprises the Fab or F(ab') 2 portion of NKp46-1.
  • the NKp46 binding ABD comprises humanized VH/VL of antibody NKp46-1.
  • NKp46-1 CDRs and human frameworks were designed that included NKp46-1 CDRs and human frameworks, produced as human IgG1 antibodies, and tested for binding to cynomolgus NKp46.
  • Two combinations of heavy and light chains were able to bind to both human and cynomolgus NKp46: the heavy chain variable region “H1” and the heavy chain “H3”, in each case combined with the light chain “L1”.
  • These cross-binding variable regions included, for the heavy chain variable region: the NKp46-1 heavy chain CDRs (shown below, underlined), human IGHV1-69*06 gene framework 1, 2 and 3 regions and a human IGHJ6*01 gene framework 4 region.
  • the light chain variable region the NKp46-1 light chain CDRs (shown below, underlined), human IGKV1-33*01 gene framework 1, 2 and 3 regions and a human IGKJ4*01 gene framework 4 region.
  • CDRs were chosen according to Kabat numbering.
  • the H1, H3 and L1 chain had the specific amino acid substitutions (shown in bold and underlining below).
  • L1 had a phenylalanine at Kabat light chain residue 87.
  • H1 had a tyrosine at Kabat heavy chain residue 27 and a lysine and alanine at Kabat residues 66 and 67, respectively.
  • an antibody comprises the three CDRs of the heavy chain variable region of NKp46-1, or humanized version thereof (NKp46-1H1 or NKp46-1H3). Also provided is a polypeptide that further comprises one, two or three of the CDRs of the light chain variable region of NKp46-1 or humanized version thereof (NKp46-1L1 or NKp46-1L1).
  • any one or more of said light or heavy chain CDRs may contain one, two, three, four or five or more amino acid modifications (e.g. substitutions, insertions or deletions).
  • a multispecific protein or NKp46-binding ABD can for example comprise: (a) the heavy chain variable region of NKp46-1 (SEQ ID NO: 16) optionally wherein one, two, three or more amino acids may be substituted by a different amino acid; (b) the light chain variable region NKp46-1 (SEQ ID NO: 18), optionally wherein one, two, three or more amino acids may be substituted by a different amino acid; or, (a) the heavy chain variable region of NKp46-1H1 (SEQ ID NO: 93) optionally wherein one, two, three or more amino acids may be substituted by a different amino acid; (b) the light chain variable region NKp46-1L1 (SEQ ID NO: 95), optionally wherein one, two, three or more amino acids may be substituted by a different amino acid;
  • the multispecific protein or NKp46-binding ABD can comprise: (a) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequence of NKp46-1, as shown in the table hereinafter, optionally wherein one, two, three or more amino acids in a CDR may be substituted by a different amino acid; (b) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequence of NKp46-1 as shown in the table hereinafter, optionally wherein one, two, three or more amino acids in a CDR may be substituted by a different amino acid;
  • the aforementioned CDRs are according to Kabat, e.g.
  • any of the CDR1, CDR2 and CDR3 of the heavy and light chains may be characterized by a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, and/or as having an amino acid sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR or set of CDRs listed in the corresponding SEQ ID NO or table hereinafter.
  • the ABD that binds CD122 is a variant interleukin-2 polypeptide.
  • the multispecific protein and/or the ABD that binds CD122 binds to the human IL-2 receptor (IL-2R) without binding to the CD25 subunit thereof.
  • the multispecific protein and/or the ABD that binds CD122 can thus bind the IL-2R complex when it is made up of CD122 and CD132 and lacks CD25.
  • the cytokine moiety can be a fragment comprising at least 20, 30, 40, 50, 60, 70, 80 or 100 contiguous amino acids of a human interleukin-2 polypeptide.
  • the IL- 2 polypeptide is a variant of a human cytokine comprising one or more amino acid modifications (e.g. amino acid substitutions) compared to the wild-type IL-2, for example to decrease binding affinity to a receptor present on non-NK cells, for example Treg cells, CD4 T cells, CD8 T cells.
  • IL2R signaling is assessed by bringing the IL-2 (e.g.
  • the IL-2 or CD122-specific ABD binds its receptor, as determined by SPR, with a binding affinity (KD) between about 1 nm and about 200 nm, optionally between about 1 nm and about 100 nm optionally between about 10 nM and about 200 nM, optionally between about 10 nM and about 100 nM optionally between about 15 nM and about 100 nM.
  • KD binding affinity
  • the CD122-binding ABD is advantageously a variant or modified IL-2 polypeptide that has reduced binding to CD25 (IL-2R ⁇ ) (e.g. reduced or abolished binding affinity, for example as determined by SPR) compared to a wild-type human interleukin-2.
  • IL-2R ⁇ IL-2R ⁇
  • Such a variant or modified IL-2 polypeptide is also referred herein to as an “IL2v” or a “not-alpha IL-2”.
  • the CD122-binding ABD can optionally be specified to have a binding affinity for human CD122 that is substantially equivalent to that of wild-type human IL-2.
  • the CD122-binding ABD can optionally be specified to have an ability to induce CD122 signaling and/or binding affinity for CD122 that is substantially equivalent to that of wild-type human IL-2.
  • the CD122-binding ABD has a reduction in binding affinity for CD25 that is greater than the reduction in binding affinity for CD122, for example a reduction of at least 1-log, 2-log or 3-log in binding affinity for CD25 and a reduction in binding affinity for CD122 that is less than 1-log (in each case compared to binding affinity observed for wild-type IL-2).
  • IL-2 is believed to bind IL-2R ⁇ (CD122) in its form as a monomeric IL-2 receptor (IL- 2R), followed by recruitment of the IL-2R ⁇ (CD132; also termed common ⁇ chain) subunit.
  • binding e.g. reduced binding
  • CD122 can therefore optionally be specified as being binding in or to a CD122:CD132 complex.
  • the CD122 can optionally be specified as being present at the surface of an NK cell.
  • IL-2 is believed to bind CD25 (IL- 2R ⁇ ) in its form as a monomeric IL-2 receptor, followed by association of the subunits IL-2R ⁇ and IL-2R ⁇ . Binding (e.g. reduced binding, partially reduced binding) to CD25 can therefore optionally be specified as being binding in or to a CD25:CD122 complex or a CD25:CD122:CD132 complex.
  • the multispecific protein can optionally be specified as being configured and/or in a conformation (or capable of adopting a conformation) in which the CD122 ABD (e.g. IL2v) is capable of binding to CD122 at the surface of a cell (e.g.
  • the multispecific protein:CD122 complex is capable of binding to CD132 at the surface of said cell.
  • the multispecific protein is specified as being configured and/or in a conformation (or capable of adopting a conformation) in which is can induce signaling by each of NKp46, CD16A and CD122 in an NK cell.
  • the CD122 ABD or IL2v can be a modified IL-2 polypeptide, for example a monomeric IL-2 polypeptide modified by introducing one more amino acid substitutions, insertions or deletions that decrease binding to CD25.
  • a IL-2 polypeptide can be modified by binding or associating it with one or more other additional molecules such as polymers or (poly)peptides that result in a further decrease of or abolished binding to CD25.
  • additional molecules such as polymers or (poly)peptides that result in a further decrease of or abolished binding to CD25.
  • a wild-type or mutated IL-2 polypeptide can be modified or further modified by binding to it another moiety that shields, masks, binds or interacts with CD25-binding site of human IL-2, thereby decreasing binding to CD25.
  • molecules such as polymers (e.g.
  • an IL-2 polypeptide is conjugated to an IL-2 polypeptide to shield or mask the epitope on IL-2 that is bound by CD25, for example by introduction (e.g. substitution) to install an amino acid containing a dedicated chemical hook at a specific site on the IL-2 polypeptide.
  • a wild-type or variant IL-2 polypeptide is bound to anti-IL-2 monoclonal antibody or antibody fragment that binds or interacts with CD25-binding site of human IL-2, thereby decreasing binding to CD25.
  • an IL2 polypeptide can be a full-length IL-2 polypeptide or it can be an IL-2 polypeptide fragment, so long as the fragment or IL2v that comprises it retains the specified activity (e.g.
  • an IL2v polypeptide can advantageously comprise an IL-2 polypeptide comprising one or more amino acid mutations designed to reduce its ability to bind to human CD25 (IL-2R ⁇ ), while retaining at least at least some, or optionally substantially full, ability to bind human CD122.
  • IL-2R ⁇ human CD25
  • IL2v or not-alpha IL-2 moieties have been described which reduce the activation bias of IL-2 on CD25+ cells. Such IL2v reduce binding to IL-2R ⁇ and maintain at least partial binding to IL-2R ⁇ .
  • IL2v polypeptides have been described, many having mutations in amino acid residue regions 35-72 and/or 79-92 of the IL-2 polypeptide.
  • decreased affinity to IL-2R ⁇ may be obtained by substituting one or more of the following residues in the sequence of a wild-type IL-2 polypeptide: R38, F42, K43, Y45, E62, P65, E68, V69, and L72 (amino acid residue numbering is with reference to the mature IL-2 polypeptide shown in SEQ ID NO: 27).
  • Wild-type mature human IL-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ CLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRW ITFCQSIISTLT (SEQ ID NO: 27).
  • IL-2p wild-type mature IL-2 with optional deletion of the three N-terminal residues APA: SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE ELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFC QSIISTLT (SEQ ID NO: 28).
  • An exemplary IL2v can have the amino acid of wild-type IL-2 with the five amino acid substitutions T3A, F42A, Y45A, L72G and C125A, as shown below, optionally further with deletion of the three N-terminal residues APA: APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQ CLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFAQSIISTLT (SEQ ID NO: 24). As few as one or two mutations can reduce binding to IL-2R ⁇ and L-2R ⁇ .
  • IL2v polypeptide having two amino acid substitutions R38A and F42K in the wild-type human IL-2 amino acid sequence displayed suitable reduced binding to IL-2R ⁇ , with retention of binding to IL-2R ⁇ resulting in highly active multispecific proteins, referred to herein as IL2v2.
  • IL2v2 R38A/F42K substitutions: SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF CQSIISTLT (SEQ ID NO: 25).
  • an IL2v2 polypeptide can further encompass substitution C125A (with reference to the wild-type mature human IL-2 of SEQ ID NO: 27), referred to herein as IL2v2A.
  • IL2V2A R38A/F42K/C125A substitutions: SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF AQSIISTLT (SEQ ID NO: 65).
  • an IL2v polypeptide has the wild-type IL-2p amino acid sequence with the three amino acid substitutions R38A, F42K and T41A (with reference to the wild-type mature human IL-2 of SEQ ID NO: 27), as shown below, referred to herein as IL2v3.
  • IL2v3 R38A/T41A/F42K substitutions: SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLAKKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF CQSIISTLT (SEQ ID NO: 26).
  • an IL2 variant comprises at least one or at least two amino acid modifications (e.g. substitution, insertion, deletion) compared to a human wild type IL-2 polypeptide.
  • an IL2v comprises a R38 substitution (e.g. R38A) and an F42 substitution (e.g., F42K), compared to a human wild type IL-2 polypeptide.
  • an IL2v comprises a R38 substitution (e.g. R38A), an F42 substitution (e.g., F42K) and a T41 substitution (e.g. T41A), compared to a human wild type IL-2 polypeptide.
  • an IL2v comprises a T3 substitution (e.g.
  • the IL2v comprises an amino acid sequence identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to the polypeptide of SEQ ID NOS: 24-26 or 65.
  • the IL2v comprises a fragment of a human IL-2 polypeptide, wherein the fragment has an amino sequence identical to or at least 70%, 80%, 90%, 95%, 98% or 99% identical to a contiguous sequence of 40, 50, 60, 70 or 80 amino acids of the polypeptide of SEQ ID NOS: 24-26 or 65. Any combination of the positions can be modified.
  • the IL-2 variant comprises two or more modification.
  • the IL-2 variant comprises three or more modification.
  • the IL-2 variant comprises four, five, or six or more modifications.
  • IL2 variant polypeptides can for example comprise two, three, four, five, six or seven amino acid modifications (e.g. substitutions).
  • residue Y45 e.g., Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R and Y45K
  • WO2020/057646 relates to amino acid sequence of IL-2v polypeptides comprising amino acid substitutions in various combinations among amino acid residues K35, T37, R38, F42, Y45, E61 and E68.
  • WO2020252418 relates to amino acid sequence of IL-2v polypeptides having at least one amino acid residues position R38, T41, F42, F44, E62, P65, E68, Y107, or C125 substituted with another amino acid, for example wherein the amino acid substitution is selected from the group consisting of: the substitution of L19D, L19H, L19N, L19P, L19Q, L19R, L19S, L19Y at position 19, the substitution of R38A, R38F, R38G at position 38, the substitution of T41A, T41G, and T41V at position 41, the substitution of F42A at position 42, the substitution of F44G and F44V at position 44, the substitution of E62A, E62F, E62H, and E62L at position 62, the substitution of P65A, P65E, P65G, P65H, P65K, P65N, P65Q, P65R at
  • a modified IL-2 can have a lower binding affinity for its receptor(s), optionally the modified IL-2 can be specified as exhibiting a KD for binding to CD25 or to a CD25:CD122:CD132 complex that is within 1-log, optionally 2-log, optionally 3-log, of the KD of a wild-type human IL-2 polypeptide (e.g. comprising the amino acid sequence of SEQ ID NO: 27).
  • a modified IL-2 can optionally be specified as exhibiting less than 20%, 30%, 40% or 50% of binding affinity to CD25 or to a CD25:CD122:CD132 complex compared to a wild- type human IL-2 polypeptide.
  • An IL2 can optionally be specified as exhibiting at least 50%, 70%, 80% or 90% of binding affinity to CD122 or to a CD122:CD132 complex compared to a wild-type human IL-2 polypeptide. In some embodiments, an IL2 exhibits at least 50%, 60%, 70% or 80% but less than 100% of binding affinity to CD122 or to a CD122:CD132 complex compared to a wild-type human IL-2 polypeptide. In some embodiments, an IL2v exhibits less than 50% of binding affinity to CD25 and at least 50%, 60%, 70% or 80% of binding affinity to CD122, compared to wild-type IL-2 polypeptide.
  • IL2 variant polypeptides have one or more, two or more, or three or more CD25-affinity-reducing amino acid substitutions relative to the wild-type mature IL-2 polypeptide having an amino acid sequence of SEQ ID NO: 27.
  • the exemplary IL2v polypeptides comprise one or more, two or more, or three or more substituted residues selected from the following group: Q11, H16, L18, L19, D20, D84, S87, Q22, R38, T41, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127, I129, and S130.
  • the exemplary IL2 variant polypeptide has one, two, three, four, five or more of amino acid residues position R38, T41, F42, F44, E62, P65, E68, Y107, or C125 substituted with another amino acid.
  • decreased affinity to CD25 or a protein complex comprising such may be obtained by substituting one or more of the following residues in the sequence of the wild-type mature IL-2 polypeptide: R38, F42, K43, Y45, E62, P65, E68, V69, and L72.
  • an IL-2 polypeptide is modified by connecting, fusing, binding or associating it with one or more other additional compounds, chemical compounds, polymer (e.g. PEG), or polypeptides or polypeptide chains that result in a decrease of binding to CD25.
  • a wild-type IL-2 polypeptide or fragment thereof can be modified by binding to it a CD25 binding peptide or polypeptide, including but not limited to an anti-IL-2 monoclonal antibody or antibody fragment thereof that binds or interacts with CD25-binding site of human IL-2, thereby decreasing binding to CD25.
  • an IL-2 polypeptide or fragment thereof can be modified by binding to it a moiety of interest (e.g. a compound, chemical compounds, polymer, linear or branched PEG polymer), covalently attached to a natural amino acid or to an unnatural amino acid installed at a selected position.
  • Such a modified interleukin 2 (IL-2) polypeptide can comprise at least one unnatural amino acid at a position on the polypeptide that reduces binding between the modified IL-2 polypeptide and CD25 but retains significant binding to the CD122:CD132 signaling complex, wherein the reduced binding to CD25 is compared to binding between a wild-type IL-2 polypeptide and CD25.
  • An unnatural amino acid can be positioned at any one or more of residues K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107 of IL-2.
  • the unnatural amino acid can be incorporated into the modified IL-2 polypeptide by an orthogonal tRNA synthetase/tRNA pair.
  • the unnatural amino acid can for example comprise a lysine analogue, an aromatic side chain, an azido group, an alkyne group, or an aldehyde or ketone group.
  • the modified IL-2 polypeptide can then be covalently attached to a water-soluble polymer, a lipid, a protein, or a peptide through the unnatural amino acid.
  • suitable polymers include polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N- acryloylmorpholine), or a combination thereof, or a polysaccharide such as dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES).
  • PEG polyethylene glycol
  • PPG poly(propylene glycol)
  • PPG poly(propylene glycol
  • Constant region domains can be derived from any suitable human antibody, particularly human antibodies of gamma isotype, including, the constant heavy (CH1) and light (CL, C ⁇ or C ⁇ ) domains, hinge domains, CH2 and CH3 domains.
  • CH1 generally refers to positions 118- 220 according to the EU index as in Kabat.
  • a CH1 domain e.g. as shown in the domain arrangements, can optionally comprise residues that extend into the hinge region such that the CH1 comprises at least part of a hinge region.
  • the CH1 domain when positioned C-terminal on a polypeptide chain and/or or C-terminal to the Fc domain, and/or within a Fab structure that is or C-terminal to the Fc domain, can optionally comprise at least part of a hinge region, for example CH1 domains can comprise at least an upper hinge region, for example an upper hinge region of a human IgG1 hinge, optionally further in which the terminal threonine of the upper hinge can be replaced by a serine.
  • a CH2 domain can therefore comprise at its C-terminus the amino acid sequence: EPKSCDKTHS.
  • Exemplary human CH1 domain amino acid sequences includes: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV (SEQ ID NO: 12).
  • Exemplary human C ⁇ domain amino acid sequences include: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4).
  • the multispecific protein can be a heterodimer or a heterotrimer comprising one or two Fabs (e.g. one Fab binding NKp46 and the other binding CD20), in which variable regions, CH1 and/or CL domains are engineered by introducing amino acid substitutions in a knob-into-holes or electrostatic steering approach to promote the desired chain pairings of CH1 domains with CK domains.
  • the multispecific protein can be a heterodimer, or a heterotrimer comprising one or two Fabs (e.g.
  • CH2 and CH3 domains can be derived from any suitable antibody. Such CH2 and CH3 domains can be used as wild-type domains or may serve as the basis for a modified CH2 or CH3 domain.
  • the CH2 and/or CH3 domain is of human origin or may comprise that of another species (e.g., rodent, rabbit, non-human primate) or may comprise a modified or chimeric CH2 and/or CH3 domain, e.g., one comprising portions or residues from different CH2 or CH3 domains, e.g., from different antibody isotypes or species antibodies.
  • exemplary human IgG1 CH2 domain amino acid sequence includes: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 7).
  • Exemplary human IgG1 CH3 domain amino acid sequence includes: GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8).
  • the Fc domain monomer may comprise a CH2- CH3 unit (a full length CH2 and CH3 domain or a fragment thereof).
  • heterodimers or heterotrimers comprising two chains with Fc domain monomers i.e. the heterodimers or heterotrimers comprise a Fc domain dimer
  • the CH3 domain will be capable of CH3-CH3 dimerization (e.g.
  • An Fc domain may optionally further comprise a C-terminal lysine (K) (See SEQ ID NO: 6).
  • Exemplary human IgG1 CH2-CH3 (Fc) domain amino acid sequences include: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6).
  • the multispecific protein can be a heterodimer, a heterotrimer or a heterotetramer, wherein the polypeptide chains are engineered for heterodimerization among each other so as to produce the desired protein.
  • the chains may comprise constant or Fc domains with amino acid modifications (e.g., substitutions) that favor the preferential hetero-dimerization of the two different chains over the homo-dimerization of two identical chains.
  • a “knob-into-holes” approach is used in which the domain interfaces (e.g.
  • CH3 domain interface of the antibody Fc region are mutated so that the antibodies preferentially heterodimerize.
  • These mutations create altered charge polarity across the interface (e.g. Fc dimer interface) such that co-expression of electrostatically matched chains (e.g. Fc-containing chains) support favorable attractive interactions thereby promoting desired heterodimer (e.g. Fc heterodimer) formation, whereas unfavorable repulsive charge interactions suppress unwanted heterodimer (e.g., Fc homodimer) formation.
  • one heavy chain comprises a T366W substitution and the second heavy chain comprises a T366S, L368A and Y407V substitution, see, e.g. Ridgway et al (1996) Protein Eng., 9, pp. 617–621; Atwell (1997) J. Mol. Biol., 270, pp.26–35; and WO2009/089004, the disclosures of which are incorporated herein by reference.
  • one heavy chain comprises a F405L substitution and the second heavy chain comprises a K409R substitution, see, e.g., Labrijn et al. (2013) Proc. Natl. Acad. Sci. U.S.A., 110, pp.5145–5150.
  • one heavy chain comprises T350V, L351Y, F405A, and Y407V substitutions and the second heavy chain comprises T350V, T366S, K392L, and T394W substitutions, see, e.g. Von Kreudenstein et al., (2013) mAbs 5:646-654.
  • one heavy chain comprises both K409D and K392D substitutions and the second heavy chain comprises both D399K and E356K substitutions, see, e.g. Gunasekaran et al., (2010) J. Biol. Chem.285:19637-19646.
  • one heavy chain comprises D221E, P228E and L368E substitutions and the second heavy chain comprises D221R, P228R, and K409R substitutions, see, e.g. Strop et al., (2012) J. Mol. Biol. 420: 204-219.
  • one heavy chain comprises S364H and F405A substitutions and the second heavy chain comprises Y349T and, T394F substitutions, see, e.g. Moore et al., (2011) mAbs 3: 546-557.
  • one heavy chain comprises a H435R substitution and the second heavy chain optionally may or may not comprise a substitution, see, e.g. US Patent no. 8,586,713.
  • the Fc regions of these antibodies can be engineered to contain amino acid modifications that permit CD16 binding.
  • the antibody may comprise mammalian antibody-type N-linked glycosylation at residue N297 (Kabat EU numbering).
  • one or more pairs of disulfide bonds such as A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C are introduced into the Fc region to increase stability, for example further to a loss of stability caused by other Fc modifications.
  • Additional example includes introducing K338I, A339K, and K340S mutations to enhance Fc stability and aggregation resistance (Gao et al, 2019 Mol Pharm. 2019;16:3647).
  • a multispecific protein is intended to have reduced binding to a human Fc gamma receptor.
  • a multispecific protein is intended to have reduced binding to a human CD16A polypeptide (and optionally further reduced binding to CD32A, CD32B and/or CD64)
  • the Fc domain is a human IgG4 Fc domain, optionally further wherein the Fc domain comprises a S228P mutation to stabilize the hinge disulfide.
  • a CH2 and/or CH3 domain may comprise a modification to decrease or abolish binding to Fc ⁇ RIIIA (CD16).
  • CD16 Fc ⁇ RIIIA
  • CH2 mutations in a Fc domain dimer proteins at reside N297 can substantially eliminate CD16A binding.
  • substitutions into human IgG1 or IgG2 residues at positions 234-237 and/or residues at positions 327, 330 and 331 were shown to greatly reduce binding to Fc ⁇ receptors and thus ADCC and complement-dependent cytotoxicity (CDC).
  • Idusogie et al. (2000) J. Immunol. 164(8):4178-84 demonstrated that alanine substitution at different positions, including K322, significantly reduced complement activation.
  • the asparagine (N) at Kabat heavy chain residue 297 can be substituted by a residue other than an asparagine (e.g. a sérine).
  • an Fc domain modified to reduce binding to CD16A comprises a substitution in the Fc domain at Kabat residues 234, 235, 237, 330 and 331.
  • the Fc domain is of human IgG1 subtype. Amino acid residues are indicated according to EU numbering according to Kabat.
  • an Fc domain modified to reduce binding to CD16A comprises an amino acid modification (e.g. substitution) at one or more of Kabat residue(s) 233-237, and an amino acid modification (e.g. substitution) at Kabat residue(s) 330 and/or 331.
  • an Fc domain comprises substitutions at Kabat residues L234, L235, G237, A330 and P331 (e.g., L234A/L235E/G237A/A330S/P331S).
  • an Fc domain that has low or reduced binding to CD16A comprises a human IgG1 Fc domain, wherein the CH2-CH3 domain has the amino acid sequence below (human IgG1 with N297S substitution), or an amino acid sequence at least 90%, 95% or 99% identical thereto.
  • an Fc domain modified to reduce binding to CD16A comprises a CH2-CH3 domain with the amino acid sequence below, or an amino acid sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235, 237, 330 and 331:
  • any of the above Fc domain sequences can optionally further comprise a C-terminal lysine (K), i.e. as in the naturally occurring sequence.
  • a CH2 and/or CH3 domain may be a wild-type domain or may comprise one or more amino acid modifications (e.g. amino acid substitutions) which increase binding to human CD16 and optionally another receptor such as FcRn.
  • the modifications will not substantially decrease or abolish the ability of the Fc-derived polypeptide to bind to neonatal Fc receptor (FcRn), e.g. human FcRn.
  • Typical modifications include modified human IgG1-derived constant regions comprising at least one amino acid modification (e.g. substitution, deletions, insertions), and/or altered types of glycosylation, e.g., hypofucosylation.
  • Fc receptors Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), and Fc ⁇ RIII (CD16).
  • Fc ⁇ RI (CD64), Fc ⁇ RIIA (CD32A) and Fc ⁇ RIII (CD 16) are activating (i.e., immune system enhancing) receptors while Fc ⁇ RIIB (CD32B) is an inhibiting (i.e., immune system dampening) receptor.
  • a modification may, for example, increase binding of the Fc domain to Fc ⁇ RIIIa on effector (e.g. NK) cells and/or decrease binding to Fc ⁇ RIIB.
  • effector e.g. NK
  • modifications are provided in PCT publication no. WO2014/044686, the disclosure of which is incorporated herein by reference. Specific mutations (in IgG1 Fc domains) which affect (enhance) Fc ⁇ RIIIa or FcRn binding are also set forth below.
  • the multispecific protein comprises a variant Fc region comprise at least one amino acid modification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH2 and/or CH3 domain of the Fc region, wherein the modification enhances binding to a human CD16 polypeptide.
  • the multispecific protein comprises at least one amino acid modification (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH2 domain of the Fc region from amino acids 237-341, or within the lower hinge-CH2 region that comprises residues 231-341.
  • the multispecific protein comprises at least two amino acid modifications (for example, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications), wherein at least one of such modifications is within the CH3 region and at least one such modifications is within the CH2 region.
  • amino acid modifications in the hinge region encompassed are amino acid modifications in the CH1 domain, optionally in the upper hinge region that comprises residues 216-230 (Kabat EU numbering). Any suitable functional combination of Fc modifications can be made, for example any combination of the different Fc modifications which are disclosed in any of United States Patents Nos.
  • WO2011/109400 WO 2008/105886; WO 2008/002933; WO 2007/021841; WO 2007/106707; WO 06/088494; WO 05/115452; WO 05/110474; WO 04/1032269; WO 00/42072; WO 06/088494; WO 07/024249; WO 05/047327; WO 04/099249 and WO 04/063351; and/or in Lazar et al. (2006) Proc. Nat. Acad. Sci. USA 103(11): 405-410; Presta, L.G. et al. (2002) Biochem. Soc. Trans. 30(4):487-490; Shields, R.L.
  • the multispecific protein comprises an Fc domain comprising at least one amino acid modification (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to a wild-type Fc region, such that the molecule has an enhanced binding affinity for human CD16 relative to the same molecule comprising a wild-type Fc region, optionally wherein the variant Fc region comprises a substitution at any one or more of positions 221, 239, 243, 247, 255, 256, 258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 308, 309, 310, 311, 312, 316, 320, 322, 326, 329, 330, 332, 331, 332, 333, 334, 335, 337, 338, 339, 340, 359, 360, 370, 373, 376, 378, 39
  • the multispecific protein comprises an Fc domain comprising at least one amino acid modification (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to a wild-type Fc region, such that the molecule has enhanced binding affinity for human CD16 relative to a molecule comprising a wild-type Fc region, optionally wherein the variant Fc region comprises a substitution at any one or more of positions 239, 298, 330, 332, 333 and/or 334 (e.g. S239D, S298A, A330L, I332E, E333A and/or K334A substitutions), optionally wherein the variant Fc region comprises a substitution at residues S239 and I332, e.g.
  • amino acid modification for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications
  • the multispecific protein comprises an Fc domain comprising N-linked glycosylation at Kabat residue N297. In some embodiments, the multispecific protein comprises an Fc domain comprising altered glycosylation patterns that increase binding affinity for human CD16.
  • carbohydrate modifications can be accomplished by, for example, by expressing a nucleic acid encoding the multispecific protein in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery are known in the art and can be used as host cells in which to express recombinant antibodies to thereby produce an antibody with altered glycosylation. See, for example, Shields, R.L. et al. (2002) J.
  • the multispecific protein contains one or more hypofucosylated constant regions.
  • Such multispecific protein may comprise an amino acid alteration or may not comprise an amino acid alteration and/or may be expressed or synthesized or treated under conditions that result in hypofucosylation.
  • a multispecific protein composition comprises a multispecific protein described herein, wherein at least 20, 30, 40, 50, 60, 75, 85, 90, 95% or substantially all of the antibody species in the composition have a constant region comprising a core carbohydrate structure (e.g. complex, hybrid and high mannose structures) which lacks fucose.
  • a multispecific protein composition which is free of N- linked glycans comprising a core carbohydrate structure having fucose.
  • the core carbohydrate will preferably be a sugar chain at Asn297.
  • a multispecific protein comprising a Fc domain dimer can be characterized by having a binding affinity to a human CD16A polypeptide that is within 1-log of that of a conventional human IgG1 antibody, e.g., as assessed by surface plasmon resonance.
  • the multispecific protein comprising a Fc domain dimer in which an Fc domain is engineered to enhance Fc receptor binding can be characterized by having a binding affinity to a human CD16A polypeptide that is at least 1-log greater than that of a conventional or wild-type human IgG1 antibody, e.g., as assessed by surface plasmon resonance.
  • a multispecific protein comprising a Fc domain dimer can be characterized by having a binding affinity to a human FcRn (neonatal Fc receptor) polypeptide that is within 1-log of that of a conventional human IgG1 antibody, e.g., as assessed by surface plasmon resonance.
  • a multispecific protein comprising a Fc domain dimer can be characterized by a Kd for binding (monovalent) to a human Fc receptor polypeptide (e.g., CD16A) of less than 10 -5 M (10 ⁇ molar), optionally less than 10 -6 M (1 ⁇ molar), as assessed by surface plasmon resonance (e.g.
  • linker is a "domain linker”, used to link any two domains as outlined herein together. Adjacent protein domains can be specified as being connected or fused to one another by a domain linker.
  • An exemplary domain linker is a (poly)peptide linker, optionally a flexible (poly)peptide linker.
  • Peptide linkers or polypeptide linkers, used interchangeably herein, may have a subsequence derived from a particular domain such as a hinge, CH1 or CL domain, or may predominantly include the following amino acid residues: Gly, Ser, Ala, or Thr.
  • the linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity.
  • the linker is from about 1 to 50 amino acids in length, preferably about 2 to 30 amino acids in length.
  • linkers of 4 to 20 amino acids in length may be used, with from about 5 to about 15 amino acids finding use in some embodiments.
  • linkers e.g. flexible linkers
  • linkers can utilize a glycine-serine polypeptide or polymer, including for example comprising (GS) n , (GSGGS) n , (GGGGS) n , (GSSS) n , (GSSSS) n and (GGGS) n , where n is an integer of at least one (optionally n is 1, 2, 3 or 4), glycine-alanine polypeptide, alanine-serine polypeptide, and other flexible linkers.
  • Linkers comprising glycine and serine residues generally provides protease resistance.
  • a (GS) 1 linker is a linker having the amino acid sequence STGS; such a linker can be useful to fuse a domain to the C-terminus of an Fc domain (or a CH3 domain thereof).
  • a domain linker comprises a (G 4 S) n peptide, wherein, for example, n is an integer from 1-10, optionally 1-6, optionally 1-4.
  • a domain linker comprises a (GS 4 ) n peptide, wherein, for example, n is an integer from 1-10, optionally 1-6, optionally 1-4.
  • a domain linker comprises a C- terminal GS dipeptide, e.g., the linker comprises (GS 4 ) and has the amino acid sequence a GSSSS (SEQ ID NO: 20), GSSSSGSSSS (SEQ ID NO: 21), GSSSSGSSSSGS (SEQ ID NO: 22) or GSSSSGSSSSGSSSS (SEQ ID NO: 23).
  • Any of the peptide or domain linkers may be specified to comprise a length of at least 4 residues, at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues, or more.
  • the linkers comprise a length of between 2-4 residues, between 2-4 residues, between 2-6 residues, between 2-8 residues, between 2-10 residues, between 2-12 residues, between 2-14 residues, between 2-16 residues, between 2-18 residues, between 2- 20 residues, between 2-22 residues, between 2-24 residues, between 2-26 residues, between 2-28 residues, between 2-30 residues, between 2 and 50 residues, or between 10 and 50 residues.
  • Examples of polypeptide linkers may include sequence fragments from CH1 or CL domains; for example the first 4-12 or 5-12 amino acid residues of the CL/CH1 domains are particularly useful for use in linkages of scFv moieties.
  • Linkers can be derived from immunoglobulin light chains, for example CK or C ⁇ .
  • Linkers can be derived from immunoglobulin heavy chains of any isotype, including for example Cy1, Cy2, Cy3, Cy4 and C ⁇ .
  • Linker sequences may also be derived from other proteins such as Ig-like proteins (e.g. TCR, FcR, KIR), hinge region-derived sequences, and other natural sequences from other proteins.
  • V H and V L domains are linked to another in tandem separated by a linker peptide (e.g. an scFv) and in turn be fused to the N- or C-terminus of an Fc domain (or CH2 domain thereof).
  • tandem variable regions or scFv can be connected to the Fc domain via a hinge region or a portion thereof, an N-terminal fragment of a CH1 or CL domain, or a glycine- and serine-containing flexible polypeptide linker.
  • Fc domains can be connected to other domains via immunoglobulin-derived sequence or via non-immunoglobulin sequences, including any suitable linking amino acid sequence.
  • immunoglobulin-derived sequences can be readily used between CH1 or CL domains and Fc domains, in particular, where a CH1 or CL domain is fused at its C-terminus to the N-terminus of an Fc domain (or CH2 domain).
  • An immunoglobulin hinge region or portion of a hinge region can and generally will be present on a polypeptide chain between a CH1 domain and a CH2 domain.
  • a hinge or portion thereof can also be placed on a polypeptide chain between a CL (e.g. C ⁇ ) domain and the CH2 domain of an Fc domain when a CL is adjacent to an Fc domain on the polypeptide chain.
  • a hinge region can optionally be replaced for example by a suitable linker peptide, e.g. a flexible polypeptide linker.
  • the NKp46 ABD and CD122 ABD e.g., a cytokine
  • the multispecific protein may have a structure or a flexible linker between the NKp46 ABD and constant domain or Fc domain that permits an increased range of domain motion compared to the two ABDs in a conventional (e.g. wild-type full length human IgG) antibody.
  • the structure or a flexible linker can be configured to confer on the antigen binding sites greater intrachain domain movement compared to antigen binding sites in a conventional human IgG1 antibody.
  • Rigidity or domain motion/interchain domain movement can be determined, e.g., by computer modeling, electron microscopy, spectroscopy such as Nuclear Magnetic Resonance (NMR), X-ray crystallography, or Sedimentation Velocity Analytical ultracentrifugation (AUC) to measure or compare the radius of gyration of proteins comprising the linker or hinge.
  • a test protein or linker may have lower rigidity relative to a comparator protein if the test protein has a value obtained from one of the tests described in the previous sentence differs from the value of the comparator, e.g., an IgG1 antibody or a hinge, by at least 5%, 10%, 25%, 50%, 75%, or 100%.
  • a cytokine can for example be fused to the C-terminus of a CH3 domain by a linker of any of SEQ ID NOS: 20- 23.
  • the multispecific protein may have a structure or a flexible linker between the NKp46 ABD and Fc domain that permits the NKp46 ABD and the ABD which binds to CD20 to have a spacing between said ABDs comprising less than about 80 angstroms, less than about 60 angstroms or ranges from about 40-60 angstroms.
  • an Fc domain (or a CH3 domain thereof) can be connected to the N- terminus of a NKp46 ABD or a cytokine polypeptide via a polypeptide linker, for example a glycine-serine-containing linker, optionally a linker having the amino acid sequence STGS (SEQ ID NO: 15).
  • a CH1 or CL domain of a Fab (e.g. of an NKp46 ABD) is fused at its C-terminus to the N-terminus of the cytokine via a flexible polypeptide linker, for example a glycine-serine-containing linker.
  • the linker will have a chain length of at least 4 amino acid residues, optionally the linker has a length of 5, 6, 7, 8, 9 or 10 amino acid residues.
  • the NKp46 ABD is placed C-terminal to the Fc domain, and the NKp46 is positioned between an Fc domain and the cytokine polypeptide in the multispecific protein.
  • the NKp46 ABD will be connected or fused at its N-terminus (at the N- terminus of a VH or a VL domain) to the C-terminus of the Fc domain via a linker (e.g. a glycine and serine containing linker, a linker having the sequence STGS, a flexible polypeptide linker) of sufficient length to enable the NKp46 binding ABD to fold and/or adopt an orientation in such a way as to permit binding to Nkp46 at the surface of an NK cell, while at the same time possesses a sufficient distance and range of motion relative to the adjacent Fc domain (or more generally to rest of the multispecific protein) such that the Fc domain can also simultaneously be found by CD16 expressed at the surface of the same NK cell.
  • a linker e.g. a glycine and serine containing linker, a linker having the sequence STGS, a flexible polypeptide linker
  • the C-terminus of a VH or VL of an scFv NKp46 ABD, or the CH1 or CL domain of a Fab NKp46 ABD will be connected or fused to the N-terminus of the cytokine polypeptide via a flexible linker (e.g.
  • the linker will have a chain length of at least 4 amino acid residues, optionally the linker has a length of 5, 6, 7, 8, 9 or 10 amino acid residues.
  • tandem variable regions e.g. scFv
  • two V domains e.g.
  • a VH domain and VL domains are generally linked together by a linker of sufficient length to enable the ABD to fold in such a way as to permit binding to the antigen for which the ABD is intended to bind.
  • linkers include linkers comprising glycine and serine residues, e.g., the amino acid sequence GEGTSTGSGGSGGSGGAD (SEQ ID NO: 96).
  • the V H domain and V L domains of an scFv are linked together by the amino acid sequence (G 4 S) 3 .
  • a (poly)peptide linker used to link a VH or VL domain of an scFv to a CH2 domain of an Fc domain comprises a fragment of a CH1 domain or CL domain and/or hinge region.
  • an N-terminal amino acid sequence of CH1 can be fused to a variable domain in order to mimic as closely as possible the natural structure of a wild-type antibody.
  • the linker comprises an amino acid sequence from a hinge domain or an N-terminal CH1 amino acid.
  • the linker peptide mimics the regular VK-CK elbow junction, e.g., the linker comprises or consists of the amino acid sequence RTVA.
  • the hinge region used to connect the C-terminal end of a CH1 or CK domain (e.g. of a Fab) with the N-terminal end of a CH2 domain will be a fragment of a hinge region (e.g. a truncated hinge region without cysteine residues) or may comprise one or more amino acid modifications which remove (e.g. substitute by another amino acid, or delete) a cysteine residue, optionally both cysteine residues in a hinge region. Removing cysteines can be useful to prevent undesired disulfide bond formation, e.g., the formation of disulfide bridges in a monomeric polypeptide.
  • a hinge can generally include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), wherein the numbering is according to the EU index as in Kabat.
  • References to specific amino acid residues within constant region domains found within the polypeptides shall be, unless otherwise indicated or as otherwise dictated by context, be defined according to Kabat, in the context of an IgG antibody.
  • a hinge domain may comprise the amino acid sequences: DKTHTCPPCP (SEQ ID NO: 5), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto; EPKSCDKTHTCPPCP (SEQ ID NO: 13), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto; or EPKSCDKTHS (SEQ ID NO: 19), or an amino acid sequence at least 60%, 70%, 80% or 90% identical thereto.
  • Polypeptide chains that dimerize and associate with one another via non-covalent bonds may or may not additionally be bound by an interchain disulfide bond formed between respective CH1 and C ⁇ domains, and/or between respective hinge domains on the chains.
  • CH1, C ⁇ and/or hinge domains can optionally be configured such that interchain disulfide bonds are formed between chains such that the desired pairing of chains is favored and undesired or incorrect disulfide bond formation is avoided.
  • the polypeptide chains can be configured such that the number of available cysteines for interchain disulfide bond formation between respective CH1/C ⁇ - hinge segments is reduced (or is entirely eliminated).
  • the amino acid sequences of respective CH1, C ⁇ and/or hinge domains can be modified to remove cysteine residues in both the CH1/C ⁇ and the hinge domain of a polypeptide; thereby the CH1 and C ⁇ domains of the two chains that dimerize will associate via non-covalent interaction(s).
  • the CH1 or C ⁇ domain adjacent to (e.g., N-terminal to) a hinge domain comprises a cysteine capable of interchain disulfide bond formation
  • the hinge domain which is placed at the C-terminus of the CH1 or C ⁇ comprises a deletion or substitution of one or both cysteines of the hinge (e.g.
  • the CH1 or C ⁇ domain adjacent (e.g., N-terminal to) a hinge domain comprises a deletion or substitution at a cysteine residue capable of interchain disulfide bond formation, and the hinge domain placed at the C-terminus of the CH1 or C ⁇ comprises one or both cysteines of the hinge (e.g. Cys 239 and Cys 242, as numbered for human IgG1 hinge according to Kabat).
  • a hinge region is derived from an IgM antibody. In such embodiments, the CH1/CK pairing mimics the C ⁇ 2 domain homodimerization in IgM antibodies.
  • the CH1 or C ⁇ domain adjacent (e.g., N-terminal to) a hinge domain comprises a deletion or substitution at a cysteine capable of interchain disulfide bond formation
  • an IgM hinge domain which is placed at the C-terminus of the CH1 or C ⁇ comprises one or both cysteines of the hinge.
  • a multispecific protein can be assessed for biological activity, e.g., antigen binding, ability to elicit proliferation of NK cells, ability to elicit target cell lysis by NK and/or elicit activation of NK cells, including any specific signaling activities elicited thereby, for example cytokine production or cell surface expression of markers of activation.
  • the multispecific format can be produced in a suitable format which allows for assessment of the component (e.g. domain) of interest.
  • the multispecific protein can be produced as a protein having the cytokine and another protein in which the cytokine is modified to delete it or otherwise modulate its activity (e.g., wherein the two multispecific proteins otherwise have the same or comparable structure), and tested in an assay of interest.
  • the multispecific protein can be produced as a protein having the ABD and another protein in which the ABD is absent or is replaced by an ABD that does not bind NKp46 (e.g., an ABD that binds an antigen not present in the assay system), wherein the two multispecific proteins otherwise have the same or comparable structure, and the two multispecific proteins are tested in an assay of interest.
  • the multispecific protein can be produced as a protein having the ABD and another protein in which the ABD is absent or is replaced by an ABD that does not bind CD20 (e.g., an ABD that binds an antigen not present in the assay system, an ABD that bind to a different tumor antigen), wherein the two multispecific proteins otherwise have the same or comparable structure, and the two multispecific proteins are tested in an assay of interest.
  • the multispecific protein is capable of inducing activation of an NKp46- expressing cell (e.g. an NK cell, a reporter cell) when the protein is incubated in the presence of the NKp46-expressing cell (e.g.
  • the multispecific protein is capable of inducing NKp46 signaling in an NKp46-expressing cell (e.g. an NK cell, a reporter cell) when the protein is incubated in the presence of an NKp46-expressing cell (e.g. purified NK cells) and a target cell that expresses the antigen of interest).
  • the multispecific protein is capable of inducing CD16A signaling in an CD16A and NKp46-expressing cell (e.g.
  • NK cell activation or signaling in characterized by the increased expression of a cell surface marker of activation, e.g. CD107, CD69, Sca-1 or Ly-6A/E, KLRG1, etc.
  • the multispecific protein is capable of inducing an increase of CD137 present on the cell surface of an NKp46- and/or a CD16-expressing cell (e.g.
  • the multispecific protein is capable of activating or enhancing the proliferation of NK cells by at least 10-fold, at least 50-fold, or at least 100-fold compared to the same multispecific protein lacking the cytokine receptor ABD (e.g. the CD122 ABD).
  • the multispecific protein displays an EC50 for activation or enhancing the proliferation of NK cells that is at least 10-fold, 50-fold or 100-fold lower than its EC50 for activation or enhancing the proliferation of CD25-expressing T cells.
  • the multispecific protein is capable of activating or enhancing the proliferation of NK cells over CD25-expressing T cells, by at least 10-fold, at least 50-fold, or at least 100-fold.
  • the CD25 expressing T cells are CD4 T cells, optionally Treg cells, or CD8 T cells.
  • Activation or enhancement of proliferation via cytokine receptor in cells e.g. NK cells, CD4 T cells, CD8 Tcells or Treg cells
  • cytokine receptor ABD-containing protein can be determined by measuring the expression of pSTAT or the cell proliferation markers (e.g. Ki67) in said cells following the treatment with the multispecific protein.
  • Activation or enhancement of proliferation via the IL-2R pathway in cells e.g.
  • NK cells, CD4 T cells, CD8 Tcells or Treg cells) by the CD122 ABD-containing protein can be determined by measuring the expression of pSTAT5 or the cell proliferation marker Ki67 in said cells following the treatment with the multispecific protein.
  • IL-2 and IL-15 lead to the phosphorylation of the STAT5 protein, which is involved in cell proliferation, survival, differentiation and apoptosis.
  • Phosphorylated STAT5 (pSTAT5) translocates into the nucleus to regulate transcription of the target genes including the CD25. STAT5 is also required for NK cell survival and NK cells are tightly regulated by the JAK-STAT signaling pathway.
  • the multispecific protein is capable of inducing STAT5 signaling in an NKp46-expressing cell (e.g. an NK cell) when the protein is incubated in the presence of an NKp46-expressing cell (e.g. purified NK cells). In one aspect of any embodiment described herein, the multispecific protein is capable of causing an increase of expression of pSTAT5 in NK cells over CD25-expressing T cells, by at least 10-fold, at least 50-fold, or at least 100-fold.
  • the multispecific protein displays an EC 50 for induction of expression of pSTAT5 in NK cells that is at least 10- fold, 50-fold or 100-fold lower than its EC50 for induction of expression of pSTAT5 in CD25- expressing T cells.
  • Activity can be measured for example by bringing NKp46-expressing cells (or CD25- expressing cells, depending on the assay) into contact with the multispecific polypeptide, optionally further in presence of target cells (e.g. tumor cells).
  • target cells e.g. tumor cells
  • activity is measured for example by bringing target cells and NK cells (i.e. NKp46-expressing cells) into contact with one another, in presence of the multispecific polypeptide.
  • the NKp46- expressing cells may be employed either as purified NK cells or NKp46-expressing cells, or as NKp46-expressing cells within a population of peripheral blood mononuclear cell (PBMC).
  • the target cells can be cells expressing the antigen of interest, optionally tumor cells.
  • the multispecific protein can be assessed for the ability to cause a measurable increase in any property or activity known in the art as associated with NK cell activity, respectively, such as marker of cytotoxicity (CD107) or cytokine production (for example IFN- ⁇ or TNF- ⁇ ), increases in intracellular free calcium levels, the ability to lyse target cells, for example in a redirected killing assay, etc.
  • the multispecific protein will be capable of causing an increase in a property or activity associated with NK cell activity (e.g. activation of NK cell cytotoxicity, CD107 expression, IFN ⁇ production, killing of target cells) in vitro.
  • a property or activity associated with NK cell activity e.g. activation of NK cell cytotoxicity, CD107 expression, IFN ⁇ production, killing of target cells
  • a multispecific protein can be selected based on its ability to increase an NK cell activity by more than about 20%, preferably by least about 30%, at least about 40%, at least about 50%, or more compared to that achieved with the same effector: target cell ratio with the same NK cells and target cells that are not brought into contact with the multispecific protein, as measured by an assay that detects NK cell activity, e.g., an assay which detects the expression of an NK activation marker or which detects NK cell cytotoxicity, e.g., an assay that detects CD107 or CD69 expression, IFN ⁇ production, or a classical in vitro chromium release test of cytotoxicity.
  • an assay that detects NK cell activity e.g., an assay which detects the expression of an NK activation marker or which detects NK cell cytotoxicity, e.g., an assay that detects CD107 or CD69 expression, IFN ⁇ production, or a classical in vitro chromium release test of cyto
  • a multispecific protein according to the invention can be selected for or characterized by its ability to have greater ability to induce NK cell activity towards target cells, i.e., lysis of target cells compared to a conventional human IgG1 antibody that binds to the same antigen of interest, as measured by an assay of NK cell activity (e.g. an assay that detects NK cell-mediated lysis of target cells that express the antigen of interest).
  • the different ABDs contribute to the overall activity of the multispecific protein that ultimately manifests itself in potent anti-tumor activity in vivo.
  • Testing methods exemplified in PCT publication no. WO2022/258673 allows the in vitro assessment of the activities of the different individual ABDs of the multispecific protein by making variants of the multispecific protein that lack a particular ABD and/or using cells that lack receptors for the particular ABD.
  • a multispecific protein when it does not comprise the cytokine receptor ABD (e.g.
  • the CD122 ABD when it possesses an Fc domain that does not bind CD16, does not, substantially induce NKp46 signaling (and/or NK activation that results therefrom) of NK cells when the protein is not bound to the antigen of interest on target cells (e.g. in the absence of the antigen of interest and/or target cells).
  • the monovalent NKp46 binding component of the multispecific protein does not itself cause NKp46 signaling.
  • such multispecific protein can be produced in a configuration where the cytokine receptor ABD (e.g. CD122 ABD) is inactivated (e.g.
  • the multispecific protein can optionally be characterized as not substantially causing (or increasing) NKp46 signaling by an NKp46-expressing, CD16- negative cell (e.g. a NKp46 + CD16- NK cell, a reporter cell) when the multispecific protein is incubated with such NKp46-expressing, CD16-negative cells (e.g., purified NK cells or purified reporter cells) in the absence of target cells.
  • a multispecific protein can for example be characterized by: (a) capable of inducing cytokine receptor (e.g. CD122) signaling (e.g., as determined by assessing STAT signaling, for example assessing STAT phosphoylation) in an NKp46-expressing cell (e.g. an NK cell) when the multispecific protein is incubated in the presence of an NKp46-expressing cell (e.g.
  • cytokine receptor e.g. CD122
  • STAT signaling for example assessing STAT phosphoylation
  • NK cells purified NK cells
  • cytokine receptor ABD e.g. CD122 ABD
  • Non-Hodgkin’s lymphoma (“NHL”) is a heterogeneous malignancy originating from lymphocytes. NHL is characterized by a clonal proliferation of lymphocytes that accumulate in the lymph nodes, blood, bone marrow and spleen, although any major organ may be involved.
  • the current classification system used by pathologists and clinicians is the World Health Organization (WHO) Classification of Tumours, which organizes NHL into precursor and mature B-cell or T-cell neoplasms.
  • WHO World Health Organization
  • the multispecific proteins are particularly suited to treat B- cell NHL, referred to as B-NHL.
  • a NHL can be specified to be a B- NHL.
  • the NHL or B-NHL may optionally be specified as being indolent or aggressive.
  • NHL may be divided into indolent or aggressive.
  • the indolent NHL group is comprised primarily of follicular subtypes, small lymphocytic lymphoma, MALT (mucosa-associated lymphoid tissue), and marginal zone.
  • Indolent NHL includes many newly diagnosed B-cell NHL patients.
  • Aggressive NHL includes patients with histologic diagnoses of primarily diffuse large B cell lymphoma (DLBL, “DLBCL”, or DLCL) (40% of all newly diagnosed patients have diffuse large cell lymphoma), Burkitt's, and mantle cell lymphoma (“MCL”).
  • DLBL diffuse large B cell lymphoma
  • DLBCL diffuse large B cell lymphoma
  • MCL mantle cell lymphoma
  • the multispecific proteins are advantageously used to treat patients having R/R NHL who have received one or at least one prior line of treatment with an immunotherapy, for example an immunotherapy that comprises an antigen binding domain (e.g. antibody or antibody fragment) that binds B-NHL cells (e.g. an anti-CD20, anti-CD19, anti-CD30 or anti-CD79b antibody or antibody fragment).
  • an immunotherapy that comprises an antigen binding domain (e.g. antibody or antibody fragment) that binds B-NHL cells (e.g. an anti-CD20, anti-CD19, anti-CD30 or anti-CD79b antibody or antibody fragment).
  • the multispecific proteins are advantageously used to treat patients having R/R NHL who have received one or at least one prior treatment with anti-CD20 agent; in one embodiment the anti-CD20 agent is a full-length antibody that mediates ADCC; in one embodiment the anti-CD20 agent is a bispecific antibody that binds CD20 and CD3; in one embodiment the anti-CD20 agent is a CAR-T cell wherein the CAR comprises an anti-CD20 or anti-CD19 antibody fragment.
  • the prior immunotherapy is a full-length antibody that mediates ADCC (e.g. having a human IgG1 isotype, optionally modified to enhance ADCC). In one embodiment the antibody induces ADCC mediated by NK cells.
  • the prior immunotherapy is or comprises rituximab or more generally a full-length anti-CD20 antibody that competes for binding to CD20 with rituximab.
  • Rituximab includes any rituximab including reference rituximab (e.g. MabtheraTM, RituxanTM) or biosimilar rituximab, e.g. TruximaTM, RitemviaTM, BlitzimaTM, RixathonTM, RiximyoTM or RuxienceTM or generally any antibody having the same amino acid sequence as rituximab.
  • the prior immunotherapy can comprise an antibody that specifically binds CD20.
  • the antibody is rituximab, ofatumumab, veltuzumab, or ocrelizumab.
  • the prior immunotherapy is or comprises an anti-CD19 antibody, an anti-CD79b antibody or an anti-CD30 antibody, optionally wherein the antibody is a full-length antibody.
  • the prior immunotherapy is a multispecific antigen binding protein (e.g. bispecific antibody, a T cell engager) comprising an antigen binding domain that binds an antigen present at the surface of B-NHL cells (e.g.
  • CD19, CD20, CD79b or CD30 an antigen binding domain that binds an antigen present at the surface of an immune effector cell.
  • the antigen binding domain that binds CD19, CD20, CD79b or CD30 is an antibody fragment, for example a fragment comprising a VH and VL pair.
  • the antigen binding domain that binds an antigen present at the surface of immune effector cells binds to an antigen present at the surface of effector T cells, for example CD8 T cells.
  • the antigen binding domain that binds an antigen present at the surface of immune effector cells specifically binds to CD3, e.g., the antigen binding domain can be an anti-CD3 antibody fragment comprising a VH and VL pair.
  • the multispecific antigen binding protein is an anti-CD20 agent, optionally an anti-CD20 x anti- CD3 agent, optionally epcoritamab (approved in DLBCL), odronextamab (FL), glofitamab (approval pending in DLBCL), mosunetuzumab (approved in FL) or plamotamab.
  • the prior immunotherapy is a cell that expresses a chimeric antigen receptor comprising an antibody fragment that binds to an antigen at the surface of B- NHL cells.
  • the CAR can for example comprise an anti-CD19, anti-CD20, anti-CD79b or anti- CD30 antibody fragment, optionally an anti-CD19 and/or an CD20 scFv.
  • Examples of CAR-T cell therapies include lisocabtagene maraleucel (BreyanziTM), tisagenlecleucel (KymriahTM), lisocabta seemingly maraleucel (BreyanziTM) and axicabtagene ciloleucel (YescartaTM).
  • the multispecific proteins that specifically bind NKp46 and CD20 can be used advantageously to treat R/R NHL in patients who have experienced prior treatment with an anti-CD20 agent (e.g. rituximab) in combination with a chemotherapy, e.g. a chemotherapy that includes at least one, two, three or four of the agents cyclophosphamide, adriamycin, vincristine or prednisone.
  • a chemotherapy e.g. a chemotherapy that includes at least one, two, three or four of the agents cyclophosphamide, adriamycin, vincristine or prednisone.
  • the most commonly used agents for rituximab combination chemotherapy include cyclophosphamide, vincristine and prednisone (CVP); or cyclophosphamide, adriamycin, vincristine, prednisone (CHOP).
  • Other chemotherapy treatments may include variants of CHOP, for example that substitute one agent of the regimen by a different agent or that add an additional agent.
  • CHOP includes variants that use the agents in different treatment regimens, for example CHOP-21 (3 weekly), CHOP-14 (2 weekly, optionally further with G-CSF administration), CHOEP-14.
  • Most patients will respond to their initial chemotherapy, duration of remissions last about 2- 3 years, but the majority of patients relapse at some point.
  • Anti-CD20 antibody, rituximab has provided significant improvements in response and survival rate.
  • rituximab is often used as rituximab + CHOP (R-CHOP) or rituximab + CVP (R-CVP) in immunochemotherapy regimens for remission induction of indolent non-Hodgkin lymphomas (iNHLs), and as ritixumab along for maintenance therapy.
  • Rituximab maintenance (RM) significantly improves progression- free survival (PFS) in patients with complete/partial remission (CR/PR).
  • PFS progression- free survival
  • CR/PR complete/partial remission
  • Rituximab is currently approved as a first line treatment for both indolent (follicular lymphoma) and aggressive NHL (diffuse large B cell lymphoma). Relapse involves the reappearance of disease (e.g.
  • a response can involve the absence of detectable MRD (minimal residual disease).
  • the initial period of responsiveness lasts at least 1, 2, 3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years.
  • Refractory involves disease (e.g., NHL), that does not respond to a treatment.
  • a refractory cancer can be resistant to a treatment before or at the beginning of the treatment.
  • the refractory cancer can become resistant during a treatment.
  • a refractory cancer is also called a resistant cancer.
  • the prior immunotherapy e.g. an anti-CD20 agent, optionally further together with chemotherapy (e.g. CHOP), a CAR-T cell
  • CHOP chemotherapy
  • a CAR-T cell a CAR-T cell
  • the multispecific protein that specifically binds NKp46 and CD20 will be administered sequentially thereafter.
  • Rituximab combined with CHOP is typically referred to as R-CHOP.
  • the multispecific protein that specifically binds NKp46 and CD20 can for example be administered when the individual has been determined to have R/R disease.
  • the multispecific protein that specifically binds NKp46 and CD20 can be optionally specified as being a second or third line or therapy for B-NHL (e.g. after a first line therapy that includes the prior immunotherapy).
  • Sequential administration refers to the administration of a different therapy after a previous therapy was completed. Additional therapies may be included between the two therapies.
  • a sequential combination is the treatment of a patient with a multispecific protein that specifically binds NKp46 and CD20 after the patient has relapsed from or is refractory to a previous therapy comprising a prior immunotherapy (e.g. comprising an anti-CD20 agent).
  • the previous or first therapy of a sequential combination was completed for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer before the second different therapy is administered.
  • a pharmaceutical composition containing a multispecific protein that specifically binds NKp46 and CD20 to provide a liquid formulation for administration (e.g., by subcutaneous or intravenous injection) for the treatment of R/R B-NHL who has been previously treated with an immunotherapy (e.g., an anti-CD20 agent (e.g.
  • Such a method or process at least comprises the step of mixing the compound with a pharmaceutically acceptable carrier.
  • the multispecific protein can be advantageously administered at a dose of 1 ⁇ g/kg to 1 mg/kg body weight, optionally 0.05 - 0.5 mg/kg body weight, optionally between 0.03 and 0.5 mg/kg body weight, optionally between 0.03 and 1 mg/kg body weight, optionally between 0.05 and 0.5 mg/kg body weight, optionally between 0.05 and 1 mg/kg body-weight, optionally between 0.05 and 0.1 mg/kg body-weight.
  • the multispecific protein can be advantageously administered 1-4 times per month, preferably 1-2 times per month, for instance once per week, once every two weeks, once every three weeks or once every four weeks.
  • administration is by intravenous infusion of subcutaneous administration.
  • a method to treat a B-NHL e.g. R/R B-NHL
  • an immunotherapy e.g., an anti-CD20 agent
  • a multispecific protein that specifically binds NKp46 and CD20 described herein or a (pharmaceutical) composition comprising such.
  • the invention provides a method of restoring or potentiating the activity and/or proliferation of NKp46-expressing cells, particularly NKp46 + NK cells (e.g.
  • NKp46 + CD16 + NK cells, NKp46 + CD16- NK cells) in a patient having a B-NHL optionally the NKp46-expressing cells are in a lymph tissue or lymph done of such patient, wherein the patient has received prior treatment with an immunotherapy (e.g. an anti-CD20 agent, a CAR-T cell), comprising the step of administering a multispecific protein that specifically binds NKp46, CD20 and CD122 (e.g. a multispecific protein described herein) to said patient.
  • an immunotherapy e.g. an anti-CD20 agent, a CAR-T cell
  • a multispecific protein that specifically binds NKp46, CD20 and CD122 e.g. a multispecific protein described herein
  • the invention provides a method of selectively restoring or potentiating the activity and/or proliferation of NK cells of over CD25-expressing lymphocytes, e.g.
  • the method is directed at increasing the activity of NKp46 + lymphocytes (e.g. NKp46 + CD16 + NK cells, NKp46 + CD16- NK cells).
  • NKp46 + lymphocytes e.g. NKp46 + CD16 + NK cells, NKp46 + CD16- NK cells.
  • the subject multispecific protein that specifically binds NKp46 and CD20 is used or administered as a single agent.
  • the subject multispecific protein that specifically binds NKp46 and CD20 is used or administered without combined or concurrent administration of immune cells, particularly NK cells.
  • the B-NHL is characterized by malignant CD20 expressing cells.
  • CD20 is a transmembrane protein expressed in most B-cell lymphomas with levels of expression that vary among subtypes and patients within the same subtype.
  • the individual to be treated has a B-NHL that is characterized by or determined to have lymphoma cells in lymphoid tissue (e.g. a lymph node).
  • the individual to be treated has a B-NHL that is characterized by or determined to have lymphoma cells in circulation or peripheral blood (e.g. CD20-expressing lymphoma cells in circulation).
  • lymphoma cells in circulation or peripheral blood e.g. CD20-expressing lymphoma cells in circulation.
  • Such an individual can be considered as being or having disease that is in the leukemic phase.
  • the methods of treatment comprise administering, to an individual who has a R/R NHL and who has received a prior course of treatment with an immunotherapy, e.g., an anti-CD20 agent), a multispecific protein described herein in a therapeutically effective amount.
  • a therapeutically effective amount may be any amount that has a therapeutic effect in a patient having the disease order (or promotes, enhances, and/or induces such an effect in at least a substantial proportion of patients with the disease or disorder and substantially similar characteristics as the patient).
  • the individual has received prior treatment with rituximab in combination with chemotherapy, optionally CHOP (R-CHOP).
  • the multispecific protein may be used with our without a prior step of detecting the expression of CD20 on the NHL cells in a biological sample obtained from an individual (e.g. a biological sample comprising cancer cells or tissues).
  • the disclosure provides a method for the treatment of an NHL in an individual in need thereof, the method comprising: a) assessing whether the individual has a R/R B-NHL, and b) upon a determination that the individual has a R/R B-NHL, administering to the individual a multispecific protein that specifically binds NKp46 and CD20, e.g. a multispecific protein as described herein.
  • the disclosure provides a method for the treatment of an NHL in an individual in need thereof, the method comprising: a) assessing whether an individual who has received prior treatment with an immunotherapy for B-NHL has a R/R B-NHL, and b) upon a determination that the individual has a R/R B-NHL, administering to the individual a multispecific protein that specifically binds NKp46 and CD20, preferably wherein the protein binds NKp46, CD20 and CD122, e.g. a multispecific protein as described herein.
  • the disclosure provides a method for the treatment of an NHL in an individual in need thereof, the method comprising: a) assessing whether an individual who has received prior treatment with an immunotherapy for B-NHL has B-NHL cells in lymph tissues (e.g. lymph node(s)), and b) upon a determination that the individual has B-NHL cells in lymph tissues, administering to the individual a multispecific protein that specifically binds NKp46 and CD20, preferably wherein the protein binds NKp46, CD20 and CD122, e.g. a multispecific protein as described herein.
  • lymph tissues e.g. lymph node(s)
  • the disclosure provides a method for the treatment of an NHL in an individual in need thereof, the method comprising: a) determining whether an individual having an R/R B-NHL has received prior treatment with an anti-CD20 agent, optionally a full-length anti-CD20 antibody (e.g. rituximab), optionally a multispecific protein (e.g. bispecific antibody) that specifically binds CD20 and CD3, optionally a CAR-T cell, and b) upon a determination that the individual has received prior treatment with an anti- CD20 agent, administering to the individual a multispecific protein that specifically binds NKp46 and CD20, preferably wherein the protein binds NKp46, CD20 and CD122, e.g.
  • the disclosure provides a method for the treatment of an NHL in an individual in need thereof, the method comprising: a) determining whether an individual having an R/R B-NHL has received prior treatment with an anti-CD20 antibody (e.g. rituximab) in combination with chemotherapy (e.g. R-CHOP), and b) upon a determination that the individual has received prior treatment with an anti- CD20 antibody in combination with chemotherapy, administering to the individual a multispecific protein that specifically binds NKp46 and CD20, preferably wherein the protein binds NKp46, CD20 and CD122, e.g. a multispecific protein as described herein.
  • an anti-CD20 antibody e.g. rituximab
  • chemotherapy e.g. R-CHOP
  • the disclosure provides a method for the sequential treatment of an NHL in an individual in need thereof, the method comprising: a) treating an individual having an B-NHL with a course of therapy comprising an immunotherapy, optionally an anti-CD20 agent, optionally a full-length anti-CD20 antibody (e.g. rituximab optionally further in combination with chemotherapy (e.g. R-CHOP)), optionally a multispecific protein (e.g.
  • a method may comprise a step of assessing whether the individual has a R/R NHL.
  • assessing or determining whether an individual has a R/R NHL comprises assessing symptoms, performing a radiographic evaluation (e.g. by CT or PET-CT imaging methods) and/or performing a non-radiographic evaluation.
  • non- radiographic evaluation may comprising obtaining a biological sample (e.g. peripheral blood) from the individual and assessing (e.g.
  • NHL cells for example by absolute lymphocyte count, assessing lactate dehydrogenase (LDH), assessing minimal residual disease (MRD) for example via MRD detection methods such as multicolor flow cytometry, end point PCR, quantitative PCR and next-generation sequencing, particularly detecting or sequencing circulating tumor DNA (ctDNA; tumor-specific DNA sequences found in either the plasma or serum of the blood).
  • LDH lactate dehydrogenase
  • MRD minimal residual disease
  • MRD detection methods such as multicolor flow cytometry, end point PCR, quantitative PCR and next-generation sequencing, particularly detecting or sequencing circulating tumor DNA (ctDNA; tumor-specific DNA sequences found in either the plasma or serum of the blood).
  • ctDNA tumor-specific DNA sequences found in either the plasma or serum of the blood.
  • the multispecific protein may be used with our without a prior step of detecting or characterizing NK cells from an individual to be treated.
  • kits which include: (i) a pharmaceutical composition containing a multispecific protein as described herein, (ii) a pharmaceutical composition containing a multispecific protein as described herein, and optionally instructions to administer said multispecific protein for the treatment of an NHL (e.g. an R/R NHL), optionally in an individual who has received a prior treatment with an immunotherapy (e.g. anti-CD20, anti- CD19, anti-CD79b, anti-CD30 agent).
  • a pharmaceutical composition may optionally be specified as comprising a pharmaceutically-acceptable carrier.
  • An multispecific protein may optionally be specified as being present in a therapeutically effective amount adapted for use in any of the methods herein.
  • kits optionally can be specified to include instructions, e.g., comprising administration schedules, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition contained therein to a patient having a cancer.
  • a kit optionally can include instructions to administer said multispecific protein, optionally other therapeutic agent.
  • the kit also can include a syringe.
  • the kits include multiple packages of the single-dose pharmaceutical compositions each containing an effective amount of a multispecific protein and optionally another therapeutic agent, for a single administration. Instruments or devices necessary for administering the pharmaceutical composition(s) also may be included in the kits. For instance, a kit may provide one or more pre-filled syringes containing an amount of the multispecific protein.
  • the present invention provides a kit for treating a cancer or a tumor in a human patient afflicted with R/R B-NHL, optionally after a first line of treatment with an immunotherapy, the kit comprising: (a) a dose of a multispecific protein that binds specifically to human CD20, human NKp46, human CD122, and optionally CD16A, wherein said protein comprises a first (I) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 1, and a second (II) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70; and (b) instructions for using said multispecific protein for the treatment of an NHL (e.g.
  • the multispecific protein comprises a first (I) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 1, and a second (II) polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70.
  • the multimeric protein is administered at a dose comprised between 1 ⁇ g/kg body weight and 1 mg/kg body weight every one, two, three or four weeks.
  • a multispecific protein and optionally another compound may be administered in purified form together with a pharmaceutical carrier as a pharmaceutical composition.
  • the form depends on the intended mode of administration and therapeutic or diagnostic application.
  • the pharmaceutical carrier can be any compatible, nontoxic substance suitable to deliver the compounds to the patient.
  • Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as (sterile) water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters, alcohol, fats, waxes, and inert solids.
  • a pharmaceutically acceptable carrier may further contain physiologically acceptable compounds that act for example to stabilize or to increase the absorption of the compounds
  • physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients
  • carbohydrates such as glucose, sucrose or dextrans
  • antioxidants such as ascorbic acid or glutathione
  • chelating agents such as ascorbic acid or glutathione
  • low molecular weight proteins or other stabilizers or excipients include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients
  • chelating agents such ascorbic acid or glutathione
  • Multispecific proteins according to the invention can be administered parenterally. Pre
  • Sterilization is readily accomplished by filtration through sterile filtration membranes, optionally prior to or following lyophilization and reconstitution.
  • the parenteral route for administration of compounds is in accord with known methods, preferably by injection or infusion.
  • the compounds may be administered for example continuously by infusion or by bolus injection.
  • Methods for preparing parenterally administrable compositions are well known in the art. Examples Preparation of multispecific proteins
  • the domain structure of an exemplary “T5” format multispecific protein is shown in Figures 1 and 2A.
  • Figure 1 shows domain linkers such as hinge and glycine-serine linkers, and interchain disulfide bridges.
  • the domain structure of the exemplary “T6” format, having a N297S mutation to substantially abolish CD16A binding but otherwise equivalent to format T5, is shown in Figure 2B.
  • T5 chain L also referred to as chain 3
  • the CK domain normally associated with the NKp46-1 VK domain in the NKp46-binding ABD was replaced by a CH1 domain (cross-mab version).
  • the T25 ( Figure 2G) format differs from the T5 format by replacement of the CH1 and CK of the NKp46-binding ABD such that the CK domain normally associated with the NKp46-1 VK domain and the CH1 normally associated with the VH remain associated with therewith.
  • CD20-2-T13-NKCE4-v2A is a heterodimer that comprises a first polypeptide chain of SEQ ID NO: 1 and a second polypeptide chain of SEQ ID NO: 70.
  • CD20-2-T13-NKCE4-v2A contains from N- to C-terminus, anti-CD20 VH/VL pair (Fab), Fc domain dimer that binds CD16, NKp46 scFv, IL2v2A.
  • CD20-2-T5A-NKCE4-v2A is a heterotrimer that comprises a first polypeptide chain of SEQ ID NO: 91, a second polypeptide chain of SEQ ID NO: 9, a third polypeptide chain of SEQ ID NO: 17.
  • CD20-2-T5A-NKCE4-v2A contains from N- to C-terminus, anti-CD20 VH/VL pair (Fab), Fc domain dimer that binds CD16, NKp46 VH/VL pair (Fab), IL2v2A.
  • CD20-2-T6AB3-NKCE4-v2A is a heterotrimer hat comprises a first polypeptide chain of SEQ ID NO: 92, a second polypeptide chain of SEQ ID NO: 69, a third polypeptide chain of SEQ ID NO: 17, and contains from N- to C-terminus, anti-CD20 VH/VL pair (Fab), Fc domain dimer that lacks CD16 binding, NKp46 VH/VL pair (Fab), IL2v2A.
  • Fab anti-CD20 VH/VL pair
  • Fab Fc domain dimer that lacks CD16 binding
  • NKp46 VH/VL pair IL2v2A.
  • CD20-2-T5-NKCE4-v2 is a heterotrimer that comprises a first polypeptide chain of the amino acid sequence of SEQ ID NO: 1, a second polypeptide chain of the amino acid sequence of SEQ ID NO: 9, and a third polypeptide chain of the amino acid sequence of SEQ ID NO: 98.
  • CD20-2-T5-NKCE4-v2 contains from N- to C-terminus, anti-CD20 VH/VL pair (Fab), Fc domain dimer that binds CD16, NKp46 VH/VL pair (Fab), IL2v2.
  • CD20-2-T5A-NKCE4-v2 is a heterotrimeric protein that comprises a first polypeptide chain of the amino acid sequence of SEQ ID NO: 91, a second polypeptide chain of the amino acid sequence of SEQ ID NO: 9, and a third polypeptide chain of the amino acid sequence of SEQ ID NO: 98.
  • CD20-2-T5A-NKCE4-v2 contains from N- to C-terminus, anti-CD20 VH/VL pair (Fab), Fc domain dimer that binds CD16, NKp46 VH/VL pair (Fab), IL2v2.
  • CD20-2-T13A-NKCE4-v2 is a heterodimeric protein that comprises a first polypeptide chain of the amino acid sequence of SEQ ID NO: 91, and a second polypeptide chain of the amino acid sequence of SEQ ID NO: 99.
  • CD20-2-T13A-NKCE4-v2 contains from N- to C- terminus, anti-CD20 VH/VL pair (Fab), Fc domain dimer that binds CD16, NKp46 scFv, IL2v2.
  • CD20-2-T6AB3-NKCE4-v2 is a heterotrimeric protein that comprises a first polypeptide chain of the amino acid sequence of SEQ ID NO: 92, a second polypeptide chain of the amino acid sequence of SEQ ID NO: 69, and a third polypeptide chain of the amino acid sequence of SEQ ID NO: 98.
  • CD20-2-T13AB3-NKCE4-v2 contains from N- to C-terminus, anti-CD20 VH/VL pair (Fab), Fc domain dimer mutated to abolish CD16 binding, NKp46 (Fab), IL2v2.
  • CD20-2-T14A-NKCE4-v2A is a heterodimeric protein that comprises a first polypeptide chain of the amino acid sequence of SEQ ID NO: 92, and a second polypeptide chain of the amino acid sequence of SEQ ID NO: 71.
  • CD20-2-T14A-NKCE4-v2A contains from N- to C- terminus, anti-CD20 VH/VL pair (Fab), Fc domain dimer mutated to abolish CD16 binding, NKp46 scFv, IL2v2A.
  • CD20-2-T175-NKCE4-v2 is a heterotrimeric protein that comprises a first polypeptide chain of the amino acid sequence of SEQ ID NO: 77, a second polypeptide chain of the amino acid sequence of SEQ ID NO: 78, and a third polypeptide chain of the amino acid sequence of SEQ ID NO: 100.
  • CD20-2-T175-NKCE4-v2 contains from N- to C-terminus, anti-CD20 VH/VL pair, Fc domain dimer that binds to CD16, NKp46 (Fab), IL2v2A.
  • CD20-2-T195-NKCE4-v2 is a heterotrimeric protein that comprises a first polypeptide chain of the amino acid sequence of SEQ ID NO: 77, a second polypeptide chain of the amino acid sequence of SEQ ID NO: 79, and a third polypeptide chain of the amino acid sequence of SEQ ID NO: 98.
  • CD20-2-T195-NKCE4-v2 contains from N- to C-terminus, anti-CD20 VH/VL pair, Fc domain dimer that binds to CD16, NKp46 (Fab), IL2v2.
  • Table 8 N C C C C C C C C C C C C Characterization of numerous multispecific proteins is shown in PCT publication nos.
  • WO2022/258673 and WO2022/200525 are incorporated herein by reference, including result showing ability to promote IL2R activation selectively in NK cells over CD4 T cells, CD8 T cells and regulatory T cells (Treg), In vitro binding to RAJI tumor cells and induction of NK-cell mediated cytotoxicity toward Raji tumor cells, selective binding to CD122, binding affinity on CD122, anti-tumor activity in vivo in mice, ability of different formats the multispecific proteins to induce cytotoxicity toward RAJI tumor cells, comparison of multispecific proteins for induction of IL2R signaling in NK cells, administration of the multispecific proteins to non-human primates.
  • NK cell engagers that comprise an anti-CD20 VH/VL pair (Fab), a Fc domain dimer that binds CD16, an NKp46 binding domain (scFv or Fab) and an optional a cytokine (variant IL-2) induce NK cell accumulation and an inflammatory microenvironment in solid tumor.
  • NK cell engagers CD20-F5-NKp46 (without cytokine) and CD20-T5-NKp46-IL2v (with variant IL- 2) were assessed for the ability to induce NK cell accumulation in tumors, in an in vivo murine model of human cancer in comparison to obinutuzumab.
  • mice treated by the CD20-T5-NKp46-IL2v NK cell engager protein that bound CD20, NKp46, CD16A and CD122 showed high expression of the ncr1 transcript (encoding for NKp46 protein and highly specific for NK cells), demonstrating an increase of NK cell infiltration in tumor.
  • tumors harvested in mice treated by the CD20-F5-NKp46 protein or obinutuzumab showed only minor increase of ncr1 (NKp46) transcripts revealing a much lower NK cell infiltrate in tumors.
  • CD20-NKCE-IL2v mediates NK and T cell proliferation in PBMC from R/R B- NHL patient samples
  • the ability of CD20-2-T13-NKCE4-V2A to induce the proliferation of NK cells, total T cells, CD4+ and CD8+ T cells was evaluated in PBMCs from R/R non-Hodgkin’s B cell lymphoma (B-NHL) patients relapsing or refractory after 1-3 lines of treatment including anti- CD20 therapy.
  • B-NHL B cell lymphoma
  • B-NHL diffuse large B-cell lymphoma
  • FL follicular lymphoma
  • MCL mantle cell lymphoma
  • MZL marginal zone lymphoma
  • LPL lymphoplasmacytic lymphoma
  • R/R Relapsed/Refractory
  • B-NHL Non-Hodgkin B-cell Lymphoma
  • DLBCL Diffuse large B-cell Lymphoma
  • FL Follicular Lymphoma
  • MCL Mantle Cell lymphoma
  • MZL Marginal Zone Lymphoma
  • LPL Lymphoplasmacytic Lymphoma
  • R-CHOP Rituximab- Cyclophosphamide Hydroxydaunomycin Oncovin Prednisone
  • R-DHAC Rituximab- Dexamethasone High dose Ara-C Carboplatin. % of blood circulating tumor cells are expressed among total lymphocyte population.
  • PBMC from R/R B-NHL patients and healthy donors were incubated with a concentration range of molecules, and proliferation of NK cells, total T cells, CD4+ and CD8+ T cells was assessed at day 6 using the CellTraceTM Violet (CTV) proliferation kit (Invitrogen). This kit was used for in vitro labeling of cells to trace multiple generations using dye dilution by flow cytometry. The frozen PBMC were thawed and labelled with the CellTraceTM Violet following the provider’s guidelines. Finally, molecules were added in the plate containing the cells (final concentrations from 150 to 0.000015 nM with 10-fold serial dilutions) and the plate was incubated for 6 days at +37 ⁇ 1°C, 5 ⁇ 1% CO2.
  • CTV CellTraceTM Violet
  • a gate including all the cells that proliferated was drawn (respectively “proliferating T”, “proliferation CD4 T”, “proliferating CD8 T” and “proliferating NK”) by selecting all the cells that shifted on the left of the non-proliferating cells (based on the unstimulated condition). Analysis of the percentages of proliferating cells was done using PrismTM (GraphPad). Molecule concentrations required to induce proliferation of 50% (EC50) of NK or T cells were calculated. EC50 values of each molecule were expressed in nM and were converted in ⁇ g/mL according to the molecular weight of each molecule.
  • Results The individual proliferation curves of NK, total T cells, CD4+ and CD8+ T cells obtained for the 11 R/R B-NHL patients stimulated for 6 days with a concentration range of CD20-2-T13-NKCE4-V2A (from 150 to 0.000015 nM) were plotted. Results are shown in Figure 3. These data show CD20-2-T13-NKCE4-V2A was a potent and efficient molecule to induce NK cell proliferation in PBMC from R/R B-NHL patients. In addition, all R/R B-NHL patients presented NK cell proliferation following incubation with CD20-2-T13-NKCE4-V2A ( Figure 3A).
  • Example 2 CD20-NKCE-IL2v mediates NK and T cell proliferation in PBMC from R/R B- NHL patient samples similarly to that for healthy donors
  • the 11 R/R B-NHL patient samples of Example 1 were subdivided into four groups according to their lymphoma subtype: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) or lymphoplasmacytic lymphoma (LPL).
  • LLBCL diffuse large B-cell lymphoma
  • FL follicular lymphoma
  • MCL mantle cell lymphoma
  • MZL marginal zone lymphoma
  • LPL lymphoplasmacytic lymphoma
  • FIG. 5 shows mean of NK proliferation frequencies of the different types of R/R B- NHL samples (R/R DLBCL, R/R MCL, R/R FL, R/R MZL/LPL) after 6 days of incubation with a concentration range of CD20-2-T13-NKCE4-V2A.
  • Figure 6 shows the mean of cell proliferation frequencies for the 11 R/R B-NHL patients and 5 healthy donors.
  • Graphs represent the percentages of proliferating NK cells after 6 days of incubation with a concentration range of CD20-2-T13-NKCE4-V2A (from 150 to 0.000015 nM). Cell proliferation was assessed by looking at the dye dilution by flow cytometry (CellTraceTM violet kit). The mean of proliferation for the 4 R/R DLBCL, the 3 R/R MCL, the 2 R/R FL and the 2 R/R MZL/LPL samples are shown. Error bars represent SD of the mean of proliferation for the 4 R/R DLBCL, the 3 R/R MCL, the 2 R/R FL and the 2 R/R MZL/LPL samples.
  • Proliferation for the R/R B-NHL samples was compared to proliferation observed for healthy donors.
  • the mean percentages of proliferating NK cells Figure 6A
  • total T cells Figure 6B
  • CD4+ and CD8+ T cells Figure 6C and D
  • CD20-2-T13-NKCE4-V2A is able to induce NK cell proliferation (and T cell proliferation to a lower extent) in B-NHL patients relapsing or refractory after at least one line of treatment including anti-CD20 therapy with a similar potency and efficacy than in healthy donors.
  • Example 3 Effective concentration of CD20-2-T13-NKCE4-V2A to induce NK cell, total T cell, CD4+ or CD8+ T cell proliferation
  • the effective concentration (EC) of molecules required to induce the proliferation of 20%, 50% and 90% of the cells were calculated.
  • the Geomean EC20, EC50 and EC90 of the proliferation of NK cells, total T cells and CD4+ and CD8+ T cells upon incubation with CD20- 2-T13-NKCE4-V2A for 6 days were calculated in nM for the 11 R/R B-NHL patients and 5 HD analyzed, shown in Table 10 below.
  • Table 10 Abbreviations: EC: Effective Concentration, CI: Confidence Interval and nd: not defined.
  • Geomean EC50 of the proliferation of NK cells upon incubation with CD20-2-T13-NKCE4-V2A for 6 days were calculated in nM and ⁇ g/ml for the 4 R/R DLBCL, 3 R/R MCL, 2 R/R FL, 2 R/R MZL/LPL patients, shown in Table 11 below.
  • Table 11 N Example 4: Maximum of proliferation for NK cells, total T cells, CD4+ or CD8+ T cells after incubation with CD20-2-T13-NKCE4-V2A The means of the top of proliferation from the R/R B-NHL patients and the healthy donors after 6 days of incubation with CD20-2-T13-NKCE4-V2A were calculated, shown in Table 12 below.
  • CD20-2-T13-NKCE4-V2A mainly induced the proliferation of the NK cells with 92 ⁇ 6.3% of the cell proliferating at maximum, followed by the proliferation of the T cells with 60 ⁇ 11% of the total T cell, 28 ⁇ 19% of the CD4+ T cell and 77 ⁇ 11% of the CD8+ T cell proliferating at maximum.
  • CD20-2-T13- NKCE4-V2A induced proliferation in the same range as in R/R B-NHL patients: 87 ⁇ 13% of NK cell, 74 ⁇ 8.0% of total T cell, 50 ⁇ 5.9% of CD4+ T cell and 79 ⁇ 10% of CD8+ T cell proliferating at maximum.
  • Table 12 T C C means of the top of proliferation were determined for the 4 B-NHL subtypes and were found in similar range, shown in Table 13 below, indicating the means of the top of proliferation from 3 R/R DLBCL, 3 R/R MCL, 2 R/R FL, 2 R/R MZL/LPL patients, as % of proliferating cells.
  • Example 5 CD20-NKCE-IL2v-mediated B cell depletion in PBMCs from R/R B-NHL patients
  • PBMC depletion in PBMC from healthy donors was assessed in parallel and compared. Additionally, cytotoxic activity was studied for CD20-2- T13-NKCE4-V2A and epcoritamab biosimilar. Briefly, human PBMCs were incubated with a dose range of CD20-2-T13-NKCE4-V2A or epcoritamab biosimilar from 9.375 nM to 0.0003 nM with an 8-fold dilution (in simplicate) or without molecule. Plates were centrifuged at 300g for 2 minutes and incubated for 24 h at +37 ⁇ 1°C, 5 ⁇ 1% CO2.
  • FIG. 7 shows CD20-2-T13-NKCE4-V2A (NKCE) induced B cell depletion in a dose- dependent manner in PBMC from R/R B-NHL patients with (leukemic phase) or without blood circulating tumoral B cells.
  • NKCE CD20-2-T13-NKCE4-V2A
  • TCE Epcoritamab biosimilar
  • the EC50 of B cell depletion for CD20-2-T13-NKCE4-V2A was calculable for three out of five patients (these three patients were in a leukemic phase) and were respectively 0.0227 nM (95% CI of EC50 [0.0070, 0.0612]), 0.4732 nM (95% CI of EC50 [0.2123, 3.5760]) and 0.0564 nM (95% CI of EC50 [0.0125, 0.5954]).
  • Sample 7 corresponds to a patient without blood circulating tumoral B cells and samples 10, 9, 12 and 11 were in a leukemic phase.
  • SD standard deviation.
  • CD20-2-T13-NKCE4-V2A was more efficient than epcoritamab biosimilar to induce B cell depletion in PBMCs from the R/R B-NHL patients who had received prior anti- CD20 therapy.
  • CD20-2-T13-NKCE4-V2A- and epcoritamab biosimilar-mediated B cell depletion in PBMCs from HD patients was evaluated. Results are shown in Figures 8 and 9.
  • FIG 8 shows B cell lysis by epcoritamab biosimilar.
  • EC50 values of B cell depletion could be calculated for three out of five healthy donors and were respectively 0.0046 nM (95% CI of EC50 [0.0033, 0.0064]), 0.0081 nM (95% CI of EC50 [0.0077, 0.0086]) and 0.0320 nM (95% CI of EC50 [0.0158, 0.0644]).
  • EC50 values of B cell depletion could be calculated for two out of five HDs, and were slightly higher than CD20-2-T13-NKCE4-V2A (0.0380 nM (95% CI of EC50 [0.0186, 0.0907]) and 0.0607 nM (95% CI of EC50 [0.0475, 0.0772]).
  • CD20-2-T13-NKCE4-V2A Finally, efficacy values for maximal dose of CD20-2-T13- NKCE4-V2A and epcoritamab biosimilar (9.375 nM) to induce B cell depletion were calculated and reported in Table 15, below.
  • CD20-2-T13-NKCE4-V2A Table 15 Ef do N Ef do bi Percentage of B cells of all patients were converted into percentage of depletion, considering initial percentage of B cells as 0% depletion, and 0% of B cells as 100% depletion. Then, efficacy values were calculated for each patient with GraphPad Prism.
  • CD20-2-T13-NKCE4-V2A is a potent and efficient molecule to induce CD20+ B cell depletion in R/R B-NHL patients with an in vitro activity in PBMC which compares favorably to the activity of the epcoritamab T cell engager.
  • the findings are particularly remarkable considering that the R/R B-NHL patients studied all had relapsed after prior therapy that relies on NK cell activity (the ADCC-inducing antibody rituximab), and yet further the rituximab therapy was together with chemotherapy which itself can have a negative effect on NK cells.
  • Example 6 NK cell population and expression of NCKE targets in post anti-CD20 R/R B-NHL patient samples
  • PBMC peripheral blood mononuclear cells
  • LN lymph node
  • % of blood circulating tumor cells are expressed among total lymphocyte population.
  • NKp46, CD16, CD122 and CD20 expression in PBMC The expression of NKp46, an activating receptor targeted by CD20-2-T13-NKCE4- V2A, was assessed in the main lymphocyte populations and NKp46 was confirmed to be expressed specifically on NK cells.
  • CD122 IL-2R ⁇ receptor
  • the mean MedFI of CD122 was approximatively ten times lower than on NK cells.
  • NKp46 expression level was assessed only one 1 LN sample from B-NHL (sample 15) because it was the only one with more than 200 events in the NK cell gate, our prerequisite for MedFI analysis.
  • the expression frequency of CD16 on NK cells was lower in lymph nodes than in PBMC of R/R B-NHL patients (86.7 ⁇ 8.4%) and has been already observed by others (see Ferlazzo et al. (2004) J Immunol.1;172(3):1455-62).
  • Figure 9 shows NKp46 and CD16 expression.
  • the left hand panel shows NKp46 expression on NK cells was similar in LN and PBMC from R/R B-NHL patients and HD.
  • the right panel shows that CD16 expression was lower in LN from B-NHL patient compared to PBMC from R/R B-NHL patient or HD.
  • CD122 expression frequency on NK cells was similar in LN and in PBMC from B-NHL patients and in PBMC from HD.
  • CD20 expression levels CD20 expression level was similar on B cells in B-NHL patients (both in PBMC and LN) and in healthy donors (PBMC).
  • PBMC and LN B-NHL patients
  • NK cells were the only population that co-expressed all targets of the multispecific protein (NKp46, CD16 and CD122).
  • NKp46 expression level was evaluated in a single LN sample from B-NHL patient and was found at similar level than in PBMC.
  • Example 7 Expression of NCKE targets in PBMC from post-CAR-T cell therapy B-NHL patient samples
  • PBMC peripheral blood mononuclear cells
  • CAR-T cells CAR-T cells in which the chimeric antigen receptor specifically binds CD19
  • HDs heathy donors
  • Figure 10 shows NKp46, CD122 and CD16 expression, either in terms of frequency of cells expressing the target (% positive cells) or the expression level (MedFI), in each of the NK and T cell subsets.
  • NKp46 expression levels in NK cells was similar in PBMC from post CAR-T B-NHL patients and HD samples.
  • the frequency of cells expressing CD16 was somewhat lower in NK cells from post CAR-T B-NHL samples compared to HD, while expression levels of CD16 were strongly lower in NK cells from post CAR-T B-NHL patients compared to HD.
  • Example 8 CD20-NKCE-IL2v outperforms a CD20xTCE in PBMC samples from B-NHL patients post CAR-T cell therapy
  • the ability of CD20-2-T13-NKCE4-V2A to induce lysis of CD20-positive Raji B cells was assessed in the presence of peripheral blood mononuclear cells (PBMCs) from three B- NHL patients who had received an approved CD19-targeting CAR-T cell therapy.
  • Fresh PBMC were cultured with Raji cells loaded with Chromium-51 radionuclide, at an effector target ratio of 50:1, in a round-bottom 96-well plate.
  • Effector and target cells were incubated with a concentration range of CD20-NKCE-IL2V or a CD3xCD20 T cell engager (epcoritamab biosimilar).
  • the maximal release control was performed by adding 4% of Tergitol in wells containing only target cells in complete RPMI, while the spontaneous release was measured in wells containing only target cells in complete RPMI. Chromium release in supernatant was detected with a Microbeta2 (PerkinElmer).
  • CD20-2-T13-NKCE4-V2A was able to induce the depletion of the Raji B cells in presence of PBMCs from each of the three post CAR-T B-NHL patients in a dose-dependent manner.
  • the results showed that the mean of efficacy at the maximal dose of CD20-2-T13-NKCE4-V2A was higher than the mean of efficacy at the maximal doses of epcoritamab biosimiliar.
  • the epcoritamab biosimilar did not significantly mediate tumor cell lysis at all.
  • CD20-NKCE-IL2v mediated tumor cell lysis in presence of PBMC for all post-CAR-T samples suggesting that the CD20-NKCE-IL2v may permit elimination of tumor cells where there is resistance to the T cell engager.
  • CD20-2-T13-NKCE4-V2A is a potent and efficient molecule to induce CD20+ B cell depletion in post-CAR-T B-NHL patients with an in vitro activity in PBMC which compares favorably to the activity of the epcoritamab T cell engager.
  • Example 9 CD20-NKCE-IL2v increased NK cell counts and eliminates CD20+ B cells in blood and lymphoid organs
  • the efficacy of CD20-2-T13-NKCE4-V2A to mediate depletion of CD20+ cells and induced pharmacodynamics were evaluated in non-human primates.
  • a GLP-compliant repeat- dose toxicity study of CD20-2-T13-NKCE4-V2A was performed in cynomolgus monkeys with CD20-2-T13-NKCE4-V2A administered by IV bolus every 2 weeks during 4 weeks (3 total doses, on days 1, 15 and 29), with 5 weeks of recovery.
  • CD20-2-T13-NKCE4-V2A induced B-cell depletion in blood in a dose dependent manner as well as NK cell and CD8 T cell expansion.
  • Repetition of CD20-2-T13- NKCE4-V2A injections induced the depletion of B cells and expansion of NK cells and CD8 T cells after the second cycle of administration. Results are shown in Figure 12. Circulating B cell frequencies among leukocytes were analyzed by flow cytometry, and multiplied by the absolute leukocyte counts per volume measured by ADVIA 2120 hematology system to obtain the absolute count per volume.
  • the baseline was defined as the mean of the predose values (day 1 predose, and either day -10 for males or day -11), and the % of baseline of each sample as (sample - baseline)/baseline.
  • the Y axis indicates the % of baseline of the B cell counts per volume, and the X axis indicates the time in days (first treatment on day 1).
  • the dotted horizontal line shows the 0% level (no change to baseline), and the vertical dotted lines show the CD20-2-T13-NKCE4-V2A administrations. Symbols represent the arithmetic mean of the values, and the bars represent the standard deviation.
  • CD20-2-T13-NKCE4-V2A-induced modulation of circulating CD20+ B cells in NHP Consistent with CD20-2-T13-NKCE4-V2A’s mode of action CD20+ B cells were depleted in blood, as seen by decrease in B cells absolute counts in blood samples in a dose dependent manner.
  • the median maximal effect on B cells at day 4 is mean depletion of 57% (min-max 43-65).
  • NK cell and T cell modulation induced by CD20-2-T13- NKCE4-V2A in lymphoid tissues immunohistochemistry staining against CD20, NKp46 and CD3 were performed on mandibular, mesenteric, inguinal, axillary lymph nodes and spleen from cynomolgus monkeys treated with repeated intravenous injections of CD20-2-T13- NKCE4-V2A given once every 2 weeks for 4 weeks 3 total doses.
  • Positive staining was quantified as ratio of positive area over total tissue area by digital pathology.
  • the cellularity of follicles and germinal centers was decreased in the lymph nodes (mandibular, mesenteric, inguinal and/or axillary) at doses ⁇ 0.05 mg/kg, spleen at doses ⁇ 0.3 mg/kg, and GALT at 0.5 mg/kg.
  • This observation was characterized by decreased size and number of follicles and germinal centers and correlated with decreased area and number of CD20- positive cells (i.e.
  • the NKp46 staining positive area and counts were increased in the spleen in a dose dependent manner already at the dose of 0.05 mg/kg few animals, and in the axillary lymph nodes and the mandibular lymph node at 0.3 mg/kg and 0.5 mg/kg.
  • the mean CD3-positive staining area ratio was also increased in the spleen as compared to controls at doses ⁇ 0.3 mg/kg and a marked increase was observed at 0.5 mg/kg in the spleen. No relevant changes in the mean CD3-positive staining area and count (ratio to total tissue area) were observed in the lymph nodes.
  • Figure 14A and 14B respectively show NKp46 staining and CD3 staining, in each case by immunohistochemistry in spleen, axillary lymph nodes (LN Axi) and mandibullary lymph nodes (LN Man) from NHP treated with 0.05, 0.3, and 0.5 mg/kg of CD20-2-T13-NKCE4-V2A administered by IV bolus every 2 weeks during 4 weeks (3 total doses). Positive staining were quantified as a ratio of positive area over total tissue area by digital pathology, and expressed as a percentage of difference as compared to vehicle treated animal.
  • Results are shown as % difference to vehicle control, with the bars from left to right representing vehicle (leftmost bar), 0.05 mg/kg, 0.3 mg/kg, 0.5 mg/kg body weight (rightmost bar).
  • CD20-2-T13-NKCE4-V2A induced CD20+ B-cell depletion within lymphoid tissues as well as NK cell expansion. T cell number was increased in the spleen (in 3/6 animals) as compared to vehicle control animals at the dose of 0.5 mg/kg but not in the lymph nodes.
  • CD20-2-T13-NKCE4-V2A-induced systemic cytokine production in NHP Blood cytokine concentrations (IFN- ⁇ , IL-1 ⁇ , IL-5, IL-6, IL-10, MCP-1, MIP-1 ⁇ , and TNF- ⁇ ) evaluations were conducted at different time points by Luminex after CD20-2-T13- NKCE4-V2A injection, repeated once every two weeks for 3 total intravenous administrations of CD20-2-T13-NKCE4-V2A at 0.05, 0.3, 0.5 mg/kg to cynomolgus monkey for up to 4 weeks.
  • Results showed CD20-2-T13-NKCE4-V2A-related increases in IFN- ⁇ , IL-6, MCP-1 and MIP- 1 ⁇ concentrations in animals treated at 0.05, 0.3, 0.5 mg/kg within 2 to 4 hours of after dose administration. These effects tended to resolve within 24 hours after each dose administration and remained constant after a 5-week recovery period. No impact of CD20-2-T13-NKCE4- V2A injected at 0.05, 0.3, 0.5 mg/kg was identified for IL-1 ⁇ , IL-5, IL-10 and TNF- ⁇ animals serum concentrations.
  • the cytokine content of serum samples was analyzed by Luminex assay.
  • Example 10 Activity of CD20-NKCE-IL2v towards CD20-negative cells
  • CD20-NKCE-IL2V influences the innate ability of NK cells to recognize and target tumor cells that lack CD20. It has been reported that relapse following treatment with CD20 targeting immunotherapies such as rituximab and mosunetuzumab (anti- CD20 TCE) are associated with CD20 editing. Reduced transcription or gain of truncating mutations have been reported to explain cases of CD20 loss following anti-CD20 TCE treatment.
  • Part A Effect of CD20-NKCE-IL2v on NK cell activating receptors
  • PBMCs were treated for 72h with 10nM of CD20-2-T13-NKCE4-V2A, its control devoid of CD20 binding, IL-2 recombinant (Miltenyi Biotec), obinutuzumab (Roche) or rituximab (Roche) in RPMI 1640 medium (Gibco) supplemented with 1% heat-inactivated fetal bovine serum (FBS, Gibco), 2mM L-glutamine (Gibco), 1% non-essential amino acids (Gibco) and 1mM sodium pyruvate (Gibco) and maintained at 37°C under an atmosphere containing 5% CO2.
  • IL-2 recombinant Miltenyi Biotec
  • obinutuzumab obinutuzumab
  • rituximab Roche
  • RPMI 1640 medium Gibco
  • FBS heat-inactivated fetal bovine serum
  • Gibco 2mM L-glutamine
  • NKp30, DNAM-1, and NKG2D receptors were increased, indicating enhanced activation potential of the NK cells (Figure 16A).
  • This enhancement in receptor expression was also observed with the control molecule IC-NKCE-IL2v, which lacks the CD20 binding moiety, and recombinant IL-2, but not with the therapeutic antibodies rituximab and obinutuzumab ( Figure 16B).
  • IC-NKCE-IL2v which lacks the CD20 binding moiety
  • recombinant IL-2 but not with the therapeutic antibodies rituximab and obinutuzumab
  • NK cells were purified from healthy donor PBMC with EasySepTM Human NK Cell Enrichment Kit (Stemcell) according to manufacturer’s instructions, and cultured for 72 hours with 10nM of CD20-2-T13-NKCE4-V2A or obinutuzumab (Roche) at 37 °C 5% CO2.
  • B16F10-huMICA cells cells made to express human MHC class I chain- related protein A (MICA) or B16F10-huCD20 cells (cells made to express human CD20 loaded with Chromium-51 radionuclide, at an effector target ratio of 10:1, in a round-bottom 96-well plate.
  • MICA human MHC class I chain- related protein A
  • B16F10-huCD20 cells cells made to express human CD20 loaded with Chromium-51 radionuclide, at an effector target ratio of 10:1, in a round-bottom 96-well plate.
  • NK cells displayed a significantly enhanced capacity to lyse B16F10-huMICA cells, a genetically engineered cell line devoid of CD20 but modified to overexpress the NKG2D ligand MICA (Figure 16C), thereby indicating a potentiation of an NKG2D-dependent cytotoxic response.
  • This augmented cytotoxicity was reversed by the addition of an anti-NKG2D blocking antibody.
  • NK cells treated with the therapeutic antibody obinutuzumab did not exhibit such increased lytic proficiency, indicating that CD20-NKCE-IL2V invokes a distinct mechanism of action that conventional therapeutic antibodies do not engage.

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Abstract

La divulgation concerne des protéines de liaison multispécifiques comprenant un premier et un second domaines de liaison à l'antigène (ABD) et un fragment de cytokine, le premier ABD se liant spécifiquement au CD20 humain et le second ABD se liant spécifiquement au NKp46 humain, pour une utilisation dans le traitement d'un lymphome non hodgkinien à lymphocytes B réfractaire et/ou récidivant.
PCT/EP2024/065622 2023-06-09 2024-06-06 Protéines d'activation de cellules nk comprenant un anticorps anti-cd20 et ant-nkp46, liées à il-2 dans le traitement de lnh-b r/r WO2024251884A1 (fr)

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