[go: up one dir, main page]

WO2025045250A1 - Anti-human factor d antibody constructs and uses thereof - Google Patents

Anti-human factor d antibody constructs and uses thereof Download PDF

Info

Publication number
WO2025045250A1
WO2025045250A1 PCT/CN2024/116356 CN2024116356W WO2025045250A1 WO 2025045250 A1 WO2025045250 A1 WO 2025045250A1 CN 2024116356 W CN2024116356 W CN 2024116356W WO 2025045250 A1 WO2025045250 A1 WO 2025045250A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
acid sequence
cdr2
cdr1
Prior art date
Application number
PCT/CN2024/116356
Other languages
French (fr)
Inventor
Jianjun Zhang
Ping Tsui
Damodar GULLIPALLI
Sayaka Sato
Takashi Miwa
Wenchao Song
Original Assignee
Kira Pharmaceuticals (Us) Llc
The Trustees Of The University Of Pennsylvania
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kira Pharmaceuticals (Us) Llc, The Trustees Of The University Of Pennsylvania filed Critical Kira Pharmaceuticals (Us) Llc
Publication of WO2025045250A1 publication Critical patent/WO2025045250A1/en

Links

Classifications

    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/60—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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • This invention relates to anti-human factor D (FD) antibody constructs and uses thereof.
  • the complement system is part of innate immunity that plays a key role in host defense. Complement also plays a pathogenic role in human inflammatory diseases. Activation of the complement system occurs via three different pathways, the classical pathway (CP) , the lectin pathway (LP) and the alternative pathway (AP) .
  • the CP is initiated by antigen-antibody binding.
  • the LP is triggered when mannose-binding lectins (MBL) interact with surface sugar molecules on microorganisms. Activation of both pathways leads to the assembly of the CP C3 convertase C4b2a, although direct cleavage of C3 by MBL-associated serine proteases can also occur.
  • the AP is a self-amplification loop driven by the AP C3 convertase, C3bBb.
  • AP activation can occur secondary to CP or LP activation, or is initiated independently. In the latter case, a low level spontaneous C3 “tick-over” generates the initial C3bBb, which rapidly propagates the AP in the absence of adequate regulation.
  • AP activation on non-self surfaces with no or insufficient negative regulation is considered a default process, whereas autologous cells typically avoid this outcome with the help of multiple membrane-bound and fluid phase complement inhibitory proteins. Under certain conditions, altered, damaged or stressed autologous cells and tissues can also activate AP and cause inflammatory injury.
  • Factor D is an essential enzyme for AP complement activation. It cleaves factor B after the latter is bound to C3b to produce an active C3 convertase, C3bBb.
  • Factor D is a serine protease of approximately 24 kDa in size and it circulates in the blood as a constitutively active enzyme after being generated from pro-factor D by enzymatic action of mannose binding lectin-associated serine protease-3 (MASP-3) .
  • MASP-3 mannose binding lectin-associated serine protease-3
  • the concentration of factor D in blood is rather low (approximately 2 ⁇ g/ml) . While the latter fact may suggest that therapeutic inhibition of factor D activity in blood is possible and might be easily achieved, previous studies have shown that factor D has a fast turnover, and accordingly the consensus in the complement research field is that it would not be feasible to block factor D systemically.
  • the present application provides anti-human FD antibody constructs, including anti-human FD antibody constructs having pH-dependent binding to factor D and/or affinity maturation.
  • an isolated antibody construct comprising an antibody moiety specifically bindings to human factor D (anti-human FD antibody moiety) , wherein the anti-human FD antibody moiety comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL) , wherein: (i) the VH comprises a heavy chain CDR1 ( “H-CDR1” ) comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to 3 amino acid variations; a heavy chain CDR2 ( “H-CDR2” ) comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to 3 amino acid variations; and a heavy chain CDR3 (“H-CDR3” ) comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to 3 amino acid variations; and (ii) the VL comprises a light chain CDR1 ( “L-CDR1” )
  • the amino acid residue at position 24 of the VH is A or T; ii) the amino acid residue at position 54 of the VH is A or H; iii) the amino acid residue at position 55 of the VH is N, S, or Y; iv) the amino acid residue at position 57 of the VH is H or L; v) the amino acid residue at position 61 of the VH is D, H, L, or V; vi) the amino acid residue at position 74 of the VH is K or T; vii) the amino acid residue at position 26 of the VL is N, R, or S; viii) the amino acid residue at position 31 of the VL is D, V, or Y; ix) the amino acid residue at position 49 of the VL is D or H; x) the amino acid residue at position 70 of the VL is F or Y; xi) the amino acid residue at position 93 of the VL is H or Y; and/or xii) the amino acid residue at position
  • the anti-human FD antibody moiety cross-reacts with a cynomolgus monkey factor D (cyno FD) .
  • the isolated anti-human FD antibody moiety is pH-dependent, wherein the anti-human FD antibody moiety binds more strongly to human FD at a neutral pH than it does at an acidic pH.
  • the anti-human FD antibody moiety is an scFv (anti-human FD scFv) .
  • the anti-human FD scFv comprises the amino acid sequence of SEQ ID NO: 219 or 220.
  • the isolated anti-human FD antibody construct further comprises a second antibody moiety specifically recognizing a component of the complement pathway.
  • the second antibody moiety is selected from the group consisting of: a full-length antibody, Fab, Fab’ , F (ab) 2 , F (ab’ ) 2 , scFv, sdAb, and a combination thereof.
  • the component of the complement pathway is complement component 2 (C2) .
  • the component of the complement pathway is complement component 5 (C5) .
  • the anti-human FD antibody moiety is an scFv (scFv1)
  • the second antibody moiety is an scFv (scFv2)
  • the isolated anti-human FD antibody construct comprises from N-terminus to C-terminus: (i) scFv1-optional linker-scFv2; (ii) scFv2-optional linker-scFv1; (iii) scFv1-optional linker-Fc domain-optional linker-scFv2; or (iv) scFv2-optional linker-Fc domain-optional linker-scFv1.
  • the linker comprises the amino acid sequence of any of SEQ ID NOs: 221-229
  • the Fc domain comprises the amino acid sequence of SEQ ID NO: 230.
  • isolated nucleic acids encoding any one of the isolated anti-human FD antibody constructs described above.
  • vectors comprising any of the isolated nucleic acids described herein.
  • the vector is a viral vector, such as an adeno-associated virus (AAV) vector or a lentiviral vector.
  • host cells comprising any of the isolated nucleic acids described herein, or any of the vectors described herein.
  • an isolated anti-human FD antibody construct e.g., any one of the anti-human FD antibody constructs described herein
  • a method for making an isolated anti-human FD antibody construct comprising i) culturing a host cell comprising any of the isolated nucleic acids described herein or any of the vectors described herein, or any of the host cells described herein, under a condition suitable for the expression of the isolated anti-human FD antibody construct; and ii) obtaining the expressed isolated anti-human FD antibody construct from said host cell.
  • composition comprising any one of the isolated anti-human FD antibody constructs described above, any of the isolated nucleic acids described above, or any of the vectors described above, and a pharmaceutically acceptable carrier.
  • a method for reducing the activity of a complement system in an individual comprising administering to the individual an effective amount of any of the pharmaceutical compositions described above.
  • FIG. 1 depicts binding activities of representative anti-FD scFv variants to human FD.
  • z16-p2 was the parental humanized anti-FD scFv.
  • Clone z16-p2 served as reference anti-human FD control.
  • An irrelevant antibody clone served as negative control.
  • FIGs. 2A and 2B depict binding activities of representative anti-FD scFv variants to cynomolgus monkey FD.
  • z16-p2 was the parental humanized anti-FD scFv.
  • Clone z16-p2 served as reference anti-human FD control.
  • An irrelevant antibody clone served as negative control.
  • FIG. 4 depicts Bio-Layer Interferometry (BLI) sensorgrams of pH-binding-engineered anti-FD variants to human and cynomolgus monkey FD.
  • FIG. 5 depicts inhibition of 50%NHS (normal human serum) -induced rabbit RBC lysis by affinity-matured anti-FD mAb variants.
  • Parental humanized z16-p2 clone served as control.
  • Distilled water served as positive control.
  • EDTA served as negative control.
  • FIG. 6 depicts inhibition of LPS-induced C3b deposition (in 10%NHS) by affinity-matured anti-FD mAb variants.
  • Parental humanized z16-p2 clone served as control.
  • FIG. 7 depicts inhibition of 30%NHS-induced rabbit RBC lysis by pH-binding-engineered anti-FD mAbs. Parental affinity-matured mAb-42 served as control.
  • FIG. 9 depicts FcRn binding optimization mutations for enhancement of IgG recycling and reduction of antigen serum levels.
  • FIG. 10 depicts optimization of FcRn binding for antigen sweeping using BLI. The tests were conducted in duplicates.
  • FIG. 11A depicts a graph of total plasma human IgG4 levels across time in hFD Scid/FcRn mice injected with 40 mg/kg of mAb42-IgG4P (WT) or mAb42pH FcRn binding variants (Mut, YEY, YPY, N3E, and YTE) .
  • FIG. 11B depicts a graph of total plasma hFD levels across time in hFD Scid/FcRn mice injected with 40 mg/kg of mAb42-IgG4P (WT) or mAb42pH FcRn binding variants (Mut, YEY, YPY, N3E, and YTE) .
  • FIG. 11A depicts a graph of total plasma human IgG4 levels across time in hFD Scid/FcRn mice injected with 40 mg/kg of mAb42-IgG4P (WT) or mAb42pH FcR
  • 11C depicts a graph of the ratio of total plasma IgG4 mAb to total plasma hFD across time in hFD Scid/FcRn mice injected with 40 mg/kg of mAb42-IgG4P (WT) or mAb42pH FcRn binding variants (Mut, YEY, YPY, N3E, and YTE) . The tests were conducted in duplicates.
  • FIG. 12A depicts a graph of the total plasma IgG4 levels of mAb42-IgG4P-LA (42WT) and mAb42pH-IgG4P-N3E (N3E) in hFD Scid/FcRn mice injected with 40 mg/kg of either mAb.
  • FIG. 12B depicts a graph of the total free plasma IgG4 levels of 42WT and N3E in hFD Scid/FcRn mice injected with 40 mg/kg of either mAb.
  • FIG. 12C depicts a graph of the total hFD levels across time in the hFD Scid/FcRn mice injected with 40 mg/kg of either 42WT or N3E mAb.
  • FIG. 12A depicts a graph of the total plasma IgG4 levels of mAb42-IgG4P-LA (42WT) and mAb42pH-IgG4P-N3E (N3E) in hFD
  • the present invention provides various anti-human FD antibody constructs comprising antibody moieties specifically binding to human factor D (anti-human FD antibody moiety) .
  • the anti-human FD antibody constructs described herein have one or more superior properties: i) have reduced immunogenicity (e.g., compared to parental mouse Ab) ; ii) have reduced N-linked glycosylation potential; iii) have improved cross-reactivity to cynomolgus monkey FD (e.g., affinity-matured variants) , enabling the extrapolation of monkey study results to human; iv) retain similar binding affinity or have improved binding affinity to human FD (e.g., affinity-matured variant, humanized variant, or pH-dependent FD binding variant) ; v) some variants have stronger binding to FD under neutral pH (e.g., about pH 7.4; such as in plasma) but less binding to FD under acidic pH (e.g., about pH 5.8; such as in endosomes)
  • antibody herein is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) , full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.
  • antibody moiety refers to a full-length antibody or an antigen-binding fragment thereof.
  • an “antibody” may refer an immunoglobulin molecule or a fragment thereof which is able to specifically bind to a specific epitope of an antigen (including the basic 4-chain antibody unit) .
  • Antibodies can be intact immunoglobulins derived from natural sources, or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ( “intrabodies” ) , antigen-binding fragments (such as Fv, Fab, Fab’ , F (ab) 2 and F (ab’ ) 2 ) , as well as single chain antibodies (scFv) , heavy chain antibodies, such as camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Bird et al., 1988, Science 242: 423-426) .
  • intracellular antibodies “intrabodies”
  • antigen-binding fragments such as Fv, Fab, Fab’ , F (ab)
  • a full-length antibody comprises two heavy chains and two light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable domains of the heavy chain and light chain may be referred to as “VH” and “VL” , respectively.
  • the variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3) .
  • CDRs complementarity determining regions
  • CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991) .
  • the three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • FRs framework regions
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ heavy chains, respectively.
  • Several of the major antibody classes are divided into subclasses such as lgG1 ( ⁇ 1 heavy chain) , lgG2 ( ⁇ 2 heavy chain) , lgG3 ( ⁇ 3 heavy chain) , lgG4 ( ⁇ 4 heavy chain) , lgA1 ( ⁇ 1 heavy chain) , or lgA2 ( ⁇ 2 heavy chain) .
  • antigen-binding fragment refers to an antibody fragment including, for example, a diabody, a Fab, a Fab’ , a F (ab’ ) 2 , an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv’ ) , a disulfide stabilized diabody (ds diabody) , a single-chain Fv (scFv) , an scFv dimer (bivalent diabody) , a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a single domain antibody (sdAb) (e.g., a camelized single domain antibody) , a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure
  • sdAb
  • an antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds.
  • an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
  • “Fv” is the minimum antibody fragment, which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy-and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single-chain Fv also abbreviated as “sFv” or “scFv, ” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994) .
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain.
  • the 4-chain unit is generally about 150,000 Daltons.
  • Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the ⁇ and ⁇ chains and four CH domains for ⁇ and ⁇ isotypes.
  • Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1) . Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site.
  • L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes or isotypes.
  • immunoglobulins There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , respectively.
  • the ⁇ and ⁇ classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
  • the Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides.
  • the effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.
  • variable region refers to the amino-terminal domains of the heavy or light chain of the antibody.
  • the variable domains of the heavy chain and light chain may be referred to as “VH” and “VL” , respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.
  • Heavy-chain only antibodies from the Camelidae species have a single heavy chain variable region, which is referred to as “VHH” . VHH is thus a special type of VH.
  • variable refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies.
  • the V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains.
  • HVRs hypervariable regions
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991) ) .
  • the constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes) , each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present application may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256: 495-97 (1975) ; Hongo et al., Hybridoma, 14 (3) : 253-260 (1995) , Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd ed.
  • the hybridoma method e.g., Kohler and Milstein., Nature, 256: 495-97 (1975) ; Hongo et al., Hybridoma, 14 (3) : 253-260 (1995)
  • Harlow et al. Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd ed.
  • full-length antibody ” “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment.
  • full-length 4-chain antibodies include those with heavy and light chains including an Fc region.
  • the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.
  • the intact antibody may have one or more effector functions.
  • diabodies refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10) residues) between the VH and V L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites.
  • Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and V L domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are described in greater detail in, for example, EP 404, 097; WO 93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) .
  • the monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is (are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984) ) .
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is (are) identical
  • Chimeric antibodies of interest herein include PRIMATTZFACTOR antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest.
  • “humanized antibody” is used a subset of “chimeric antibodies. ”
  • CDR complementarity determining region
  • CDR complementarity determining region
  • variable-domain residue-numbering as in Kabat or “amino-acid-position numbering as in Kabat, ” and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or hypervariable region (HVR) of the variable domain.
  • HVR hypervariable region
  • a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of 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.
  • the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra with minor modification.
  • the “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody.
  • Framework or “FR” residues are those variable-domain residues other than the CDR residues as herein defined.
  • an “immunoassay” refers to any binding assay that uses an antibody capable of binding specifically to a target molecule to detect and quantify the target molecule.
  • “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
  • donor antibody such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a suitable human acceptor antibody may be one selected from a conventional database, e.g., the KABAT database, Los Alamos database, the AbM, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody.
  • a human antibody characterized by a homology to the framework regions of the donor antibody may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs.
  • a suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody.
  • the prior art describes several ways of producing such humanized antibodies (see, for example, EP-A-0239400 and EP-A-054951) .
  • a “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991) ; Marks et al., J. Mol. Biol., 222: 581 (1991) . Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE TM technology) . See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
  • donor antibody refers to an antibody (monoclonal, and/or recombinant) which contributes the amino acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner, so as to provide the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralizing activity characteristic of the donor antibody.
  • acceptor antibody refers to an antibody (monoclonal and/or recombinant) heterologous to the donor antibody, which contributes all (or any portion, but in some embodiments all) of the amino acid sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the first immunoglobulin partner.
  • a human antibody is the acceptor antibody.
  • attach, refers to connecting or uniting by a bond, link, force or tie in order to keep two or more components together, which encompasses either direct or indirect attachment such that, for example, where a first polypeptide is directly bound to a second polypeptide or material, and, for example, where one or more intermediate compounds (e.g., amino acids, peptides, polypeptides, etc. ) are disposed between the first polypeptide and the second polypeptide or material.
  • intermediate compounds e.g., amino acids, peptides, polypeptides, etc.
  • Percent (%) amino acid sequence identity or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) , or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
  • %amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32 (5) : 1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5 (1) : 113, 2004) .
  • “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60%homologous.
  • the DNA sequences ATTGCC and TATGGC share 50%homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
  • constant domain refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site.
  • the constant domain contains the C H 1, C H 2 and C H 3 domains (collectively, C H ) of the heavy chain and the CHL (or C L ) domain of the light chain.
  • the “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ( “ ⁇ ” ) and lambda ( “ ⁇ ” ) , based on the amino acid sequences of their constant domains.
  • CH1 domain (also referred to as “C1” of “H1” domain) usually extends from about amino acid 118 to about amino acid 215 (EU numbering system) .
  • Hinge region is generally defined as a region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985) ) . Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-Sbonds in the same positions.
  • the “CH2 domain” of a human IgG Fc domain usually extends from about amino acid 231 to about amino acid 340.
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain.
  • CH3 domain (also referred to as “C3” domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc domain (i.e., from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG) .
  • Fc domain or “fragment crystallizable region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc domains and variant Fc domains.
  • the boundaries of the Fc domain of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc domain is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc domain may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.
  • composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • Suitable native-sequence Fc domains for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B) , IgG3 and IgG4.
  • epitope refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds. Two antibodies or antibody moieties may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.
  • a first antibody or fragment thereof “competes” for binding to a target antigen with a second antibody or fragment thereof when the first antibody or fragment thereof inhibits the target antigen binding of the second antibody of fragment thereof by at least about 50%(such as at least about any one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%or 99%) in the presence of an equimolar concentration of the first antibody or fragment thereof, or vice versa.
  • a high throughput process for “binning” antibodies based upon their cross-competition is described in PCT Publication No. WO 03/48731.
  • the terms “specifically binds, ” “specifically recognizing, ” and “is specific for” refer to measurable and reproducible interactions, such as binding between a target and an antibody or antibody moiety, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules.
  • an antibody or antibody moiety that specifically recognizes a target is an antibody or antibody moiety that binds this target with greater affinity, avidity, more readily, and/or with greater duration than its bindings to other targets.
  • the extent of binding of an antibody to an unrelated target is less than about 10%of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA) .
  • an antibody that specifically binds a target has a dissociation constant (K D ) of ⁇ 10 -5 M, ⁇ 10 -6 M, ⁇ 10 -7 M, ⁇ 10 -8 M, ⁇ 10 -9 M, ⁇ 10 -10 M, ⁇ 10 -11 M, or ⁇ 10 -12 M.
  • K D dissociation constant
  • an antibody specifically binds an epitope on a protein that is conserved among the protein from different species.
  • specific binding can include, but does not require exclusive binding.
  • Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE TM -tests and peptide scans.
  • the term “specificity” refers to selective recognition of an antigen binding protein or antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific.
  • the term “multispecific” as used herein denotes that an antigen binding protein or an antibody has two or more antigen-binding sites of which at least two bind a different antigen or a different epitope of the same antigen.
  • Bispecific as used herein denotes that an antigen binding protein or an antibody has two different antigen-binding specificities.
  • the term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind the same epitope of the same antigen.
  • Binding affinity generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen) .
  • binding affinity refers to intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen) .
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd) . Affinity can be measured by common methods known in the art, including those described herein.
  • Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
  • a variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present application. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
  • an “on-rate, ” “rate of association, ” “association rate, ” or “k on ” as used herein can also be determined as described above using methods such as biolayer interferometry and surface plasmon resonance (SPR) .
  • an “isolated” antibody is one that has been identified, separated and/or recovered from a component of its production environment (e.g., natural or recombinant) .
  • the isolated polypeptide is free of association with all other components from its production environment.
  • Contaminant components of its production environment such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the polypeptide will be purified: (1) to greater than 95%by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99%by weight; (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.
  • an “isolated” nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment.
  • the isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies described herein existing naturally in cells.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in its normal context in a living subject is not “isolated, ” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is “isolated. ”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • hybridoma refers to a cell resulting from the fusion of a B-lymphocyte and a fusion partner such as a myeloma cell.
  • a hybridoma can be cloned and maintained indefinitely in cell culture and is able to produce monoclonal antibodies.
  • a hybridoma can also be considered to be a hybrid cell.
  • nucleic acid molecule refers to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or unnatural nucleotides, and include, but are not limited to, DNA, RNA, and PNA.
  • Nucleic acid sequence refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.
  • isolated nucleic acid refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • the term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • “Complementary” as used herein to refer to a nucleic acid refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ( “base pairing” ) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and or at least about 75%, or at least about 90%, or at least about 95%of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • a vector may be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
  • a vector may be a DNA or RNA vector.
  • a vector may be either a self-replicating extrachromosomal vector or a vector which integrates into a host genome.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors. ”
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting there from.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • polypeptide and “peptide” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or unnatural amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • a “polypeptide” includes modifications, such as deletions, additions, and substitutions (generally conservative in nature) , to the native sequence, as long as the polypeptide maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • conjugated refers to covalent attachment of one molecule to a second molecule.
  • “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.
  • the variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%identical to the reference sequence.
  • regulating can mean any method of altering the level or activity of a substrate.
  • Non-limiting examples of regulating with regard to a protein include affecting expression (including transcription and/or translation) , affecting folding, affecting degradation or protein turnover, and affecting localization of a protein.
  • Non-limiting examples of regulating with regard to an enzyme further include affecting the enzymatic activity.
  • “Regulator” refers to a molecule whose activity includes affecting the level or activity of a substrate.
  • a regulator can be direct or indirect.
  • a regulator can function to activate or inhibit or otherwise modulate its substrate.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • a “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to a subject.
  • a pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.
  • diluent of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, such as a formulation reconstituted after lyophilization.
  • exemplary diluents include sterile water, bacteriostatic water for injection (BWFI) , a pH buffered solution (e.g., phosphate-buffered saline) , sterile saline solution, Ringer’s solution or dextrose solution.
  • BWFI bacteriostatic water for injection
  • a pH buffered solution e.g., phosphate-buffered saline
  • sterile saline solution e.g., Ringer’s solution or dextrose solution.
  • diluents can include aqueous solutions of salts and/or buffers.
  • a “preservative” is a compound which can be added to the formulations herein to reduce bacterial activity.
  • the addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.
  • potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (amixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds) , and benzethonium chloride.
  • preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.
  • aromatic alcohols such as phenol, butyl and benzyl alcohol
  • alkyl parabens such as methyl or propyl paraben
  • catechol resorcinol
  • cyclohexanol 3-pentanol
  • m-cresol m-cresol
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient (s) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • Such formulations may be sterile.
  • a “sterile” formulation is aseptic or essentially free from living microorganisms and their spores.
  • a “stable” formulation is one in which the protein therein essentially retains its physical and chemical stability and integrity upon storage.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993) .
  • Stability can be measured at a selected temperature for a selected time period. For rapid screening, the formulation may be kept at 40°C. for 2 weeks to 1 month, at which time stability is measured. Where the formulation is to be stored at 2-8°C., generally the formulation should be stable at 30°C. or 40°C.
  • a “stable” formulation may be one wherein less than about 10%and preferably less than about 5%of the protein are present as an aggregate in the formulation. In other embodiments, any increase in aggregate formation during storage of the formulation can be determined.
  • a “reconstituted” formulation is one which has been prepared by dissolving a lyophilized protein or antibody formulation in a diluent such that the protein is dispersed throughout.
  • the reconstituted formulation is suitable for administration (e.g., subcutaneous administration) to a patient to be treated with the protein of interest and, in certain embodiments, may be one which is suitable for parenteral or intravenous administration.
  • An “isotonic” formulation is one which has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm.
  • the term “hypotonic” describes a formulation with an osmotic pressure below that of human blood.
  • the term “hypertonic” is used to describe a formulation with an osmotic pressure above that of human blood. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example.
  • the formulations of the present application can be hypertonic as a result of the addition of salt and/or buffer.
  • a “recombinant AAV vector (rAAV vector) ” refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of AAV origin) that are flanked by at least one, and in embodiments two, AAV inverted terminal repeat sequences (ITRs) .
  • Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins) .
  • a rAAV vector When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection) , then the rAAV vector may be referred to as a “pro-vector” which can be “rescued” by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions.
  • An rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle, particularly an AAV particle.
  • a rAAV vector can be packaged into an AAV virus capsid to generate a “recombinant adeno-associated viral particle (rAAV particle) ” .
  • An “AAV inverted terminal repeat (ITR) ” sequence is an approximately 145-nucleotide sequence that is present at both termini of the native single-stranded AAV genome.
  • the outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome.
  • the outermost 125 nucleotides also contain several shorter regions of self-complementarity (designated A, A’ , B, B’ , C, C’ and D regions) , allowing intrastrand base-pairing to occur within this portion of the ITR.
  • transfected or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells, ” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, and may contain mutations. Mutant progenies that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease) , preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival.
  • the methods of the application contemplate any one or more of these aspects of treatment.
  • phrases “effective amount” and “pharmaceutically effective amount” as used herein refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system.
  • inhibitors refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic.
  • To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and/or amount as compared to that of a reference.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20%or greater (e.g., at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) .
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50%or greater.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
  • delay development of a disease means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.
  • Preventing includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual that may be predisposed to the disease but has not yet been diagnosed with the disease.
  • to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition.
  • an antibody which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody.
  • a “reference” as used herein, refers to any sample, standard, or level that is used for comparison purposes.
  • a reference may be obtained from a healthy and/or non-diseased sample.
  • a reference may be obtained from an untreated sample.
  • a reference is obtained from a non-diseased or non-treated sample of an individual.
  • a reference is obtained from one or more healthy individuals who are not the individual or patient.
  • the terms “patient, ” “subject, ” “individual, ” and the like are used interchangeably herein, and refer to any animal, in some embodiments a mammal, and in some embodiments a human, having a complement system, including a human in need of therapy for, or susceptible to, a condition or its sequelae.
  • the individual may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, mice and humans.
  • the individual is a human.
  • a "human consensus framework” or "acceptor human framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin V L or V H framework sequences.
  • the selection of human immunoglobulin V L or V H sequences is from a subgroup of variable domain sequences.
  • the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) . Examples include for the V L , the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al., supra.
  • the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al.
  • a human consensus framework can be derived from the above in which particular residues, such as when a human framework residue is selected based on its homology to the donor framework by aligning the donor framework sequence with a collection of various human framework sequences.
  • An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • an “affinity-matured” antibody is one with one or more alterations m one or more CDRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration (s) .
  • an affinity-matured antibody has nanomolar or even picomolar affinities for the target antigen.
  • Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio/Technology 10: 779-783 (1992) describes affinity maturation by V H -and V L -domain shuffling. Random mutagenesis of CDR and/or framework residues is described by, for example: Barbas et al. Proc Nat. Acad. Sci.
  • valent denotes the presence of a specified number of binding sites in an antigen binding protein.
  • a natural antibody for example or a full-length antibody has two binding sites and is bivalent.
  • trivalent tetravalent
  • pentavalent hexavalent
  • Antibody effector functions refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fe receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC) ; phagocytosis; down regulation of cell surface receptors (e.g., B cell receptors) ; and B cell activation. "Reduced or minimized" antibody effector function means that which is reduced by at least 50% (alternatively 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) from the wild type or unmodified antibody.
  • effector function is readily determinable and measurable by one of ordinary skill in the art.
  • the antibody effector functions of complement binding, complement dependent cytotoxicity and antibody dependent cytotoxicity are affected.
  • effector function is eliminated through a mutation in the constant region that eliminated glycosylation, e.g., "effectorless mutation. "
  • the effectorless mutation is an N297A or DANA mutation (D265A+N297A) in the C H 2 region. Shields et al., J. Biol. Chem. 276 (9) : 6591-6604 (2001) .
  • effector function can be reduced or eliminated through production techniques, such as expression in host cells that do not glycosylate (e.g., E. coli. ) or in which result in an altered glycosylation pattern that is ineffective or less effective at promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5) : 3466-3473 (2003) .
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g., natural killer (NK) cells, neutrophils and macrophages
  • NK cells natural killer cells
  • monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • ADCC activity of a molecule of interest is assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS USA 95 : 652-656 (1998) .
  • “Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen.
  • C1q the first component of the complement system
  • a CDC assay e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) .
  • Antibody variants with altered Fc region amino acid sequences and increased or decreased C1q binding capability are described in U.S. Pat. No. 6,194,551B1 and WO99/51642. The contents of those patent publications are specifically incorporated herein by reference. See, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000) .
  • Half maximal inhibitory concentration is a measure of the effectiveness of a substance (such as an antibody) in inhibiting a specific biological or biochemical function. It indicates how much of a particular drug or other substance (inhibitor, such as an antibody) is needed to inhibit a given biological process by half. The values are typically expressed as molar concentration. IC 50 is comparable to an "EC 50 " for agonist drug or other substance (such as an antibody) . EC 50 also represents the plasma concentration required for obtaining 50%of a maximum effect in vivo. As used herein, an "IC 50 " is used to indicate the effective concentration of an antibody needed to neutralize 50%of the antigen bioactivity in vitro.
  • IC 50 or EC 50 can be measured by bioassays such as inhibition of ligand binding by FACS analysis (competition binding assay) , cell-based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA) .
  • bioassays such as inhibition of ligand binding by FACS analysis (competition binding assay) , cell-based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA) .
  • a “low-pH dissociation factor” as used herein is defined as the percentage of antibody dissociated at pH 5.8 from the antigen at 25 °C, wherein the antibody is pre-bound to the antigen at pH 7.4.
  • the low-pH dissociation factor may be measured by associating an antibody and an antigen (e.g., the anti-human FD antibody construct and human factor D) at pH 7.4 for 600 seconds, followed by a dissociation period of 600 seconds in a buffer at pH 5.8, and calculation of the percentage of antibody dissociated at pH 5.8 from the antigen.
  • a “neutral-pH dissociation factor” is defined as the percentage of antibody dissociated at pH 7.4 from the antigen at 25°C, wherein the antibody is pre-bound to the antigen at pH 7.4.
  • the neutral-pH dissociation factor may be measured by associating antibody and antigen at pH 7.4 for 600 seconds, followed by a dissociation period of 600 seconds in a buffer at pH 7.4, and calculation of the percentage of antibody dissociated at pH 7.4 from the antigen.
  • the antibody-antigen association and dissociation may be measured in various ways that are with the skill in the art, for instance, using biolayer interferometry.
  • Fc receptor or “FcR” describes a receptor that binds the Fc domain of an antibody.
  • the preferred FcR is a native sequence human FcR.
  • a preferred FcR is one which binds an IgG antibody (agamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor” ) and Fc ⁇ RIIB (an “inhibiting receptor” ) , which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • Fc receptor or “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus.
  • FcRn the neonatal receptor
  • Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18: (12) : 592-8 (1997) ; Ghetie et al., Nature Biotechnology 15 (7) : 637-40 (1997) ; Hinton et al., J. Biol. Chem.
  • Binding to FcRn in vivo and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides having a variant Fc region are administered.
  • WO 2004/42072 (Presta) describes antibody variants which improved or diminished binding to FcRs. See also, e.g., Shields et al., J. Biol. Chem. 9 (2) : 6591-6604 (2001) .
  • references to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X” .
  • reference to “not” a value or parameter generally means and describes “other than” a value or parameter.
  • the method is not used to treat disease of type X means the method is used to treat disease of types other than X.
  • the resent invention in some aspects provides isolated antibody constructs (anti-human FD antibody constructs) comprising an antibody moiety specifically binding to human factor D (anti-human FD antibody moiety, such as any of the anti-human FD antibody moieties described herein) .
  • the anti-human FD antibody construct consists essentially of (or consists of) the anti-human FD antibody moiety.
  • anti-human FD antibody moieties there is also provided anti-human FD antibody moieties.
  • the isolated anti-human FD antibody construct processes any of the properties and activities of the anti-human FD antibody moieties described herein.
  • the isolated anti-human FD antibody construct further comprises a fusion partner (e.g., protein or polypeptide) .
  • the fusion partner is directly fused to the anti-human FD antibody moiety.
  • the fusion partner is fused to the anti-human FD antibody moiety via a linker (e.g., peptide linker) .
  • the fusion partner is an effector protein. Effector proteins can be any protein that produce a specific response to a stimulus, such as altering one or more of the cellular functions in the target cells.
  • the fusion partner is an antibody moiety, such as an antibody moiety specifically binding to a non-FD epitope.
  • the isolated anti-human FD antibody construct is monospecific. In some embodiments, the isolated anti-human FD antibody construct is multispecific (e.g., bispecific) . In some embodiments, the isolated anti-human FD antibody construct is multivalent and monospecific. In some embodiments, the isolated anti-human FD antibody construct is multivalent and multispecific (e.g., bispecific) .
  • the isolated anti-human FD antibody construct further comprises a second antibody moiety (e.g., scFv, Fab, sdAb, or full-length antibody) specifically recognizing a component of the complement pathway.
  • the second antibody moiety specifically recognizes FD.
  • the isolated anti-human FD antibody construct comprises two or more anti-human FD antibody moieties (e.g., scFv) , such as connected to each other in tandem.
  • the second antibody moiety specifically recognizes a component of the complement pathway that is not FD, such as C2 or C5.
  • Anti-human FD antibody moieties are provided.
  • Anti-human FD antibody moiety and “anti-FD antibody moiety” are used interchangeably herein. Referring to “anti-human FD” does not exclude antibody moieties that can bind to a human FD as well as an FD derived from another species. Any of the anti-FD antibody moieties described herein can be used in the anti-human FD antibody constructs described herein.
  • Factor D also known as adipsin, is a 24 kDa serine protease comprising 228 amino acids.
  • the structure of factor D comprises of two antiparallel ⁇ -barrel domains with each barrel containing six ⁇ -strands with the same typology in all enzymes.
  • factor D is predominantly produced by and secreted into the bloodstream by adipocytes.
  • macrophages and monocytes as well as by brain astrocytes to a lesser extent.
  • the levels of factor D in serum can vary, but under normal conditions it is found within the range of 1-2 ⁇ g/mL range. Under healthy conditions, factor D, like other low molecular weight proteins, is filtered through the glomerulus and almost completely reabsorbed within the tubules, where it is then rapidly catabolized intracellularly.
  • Factor D is produced as a proenzyme or zymogen (pro-factor D) that requires subsequent cleavage of a 6-amino acid peptide for maturation. Conversion of pro-factor D into its mature form appears to happen rapidly, either during secretion in the secretory pathway or immediately thereafter. Although there has been some controversy, maturation of pro-factor D into mature factor D is thought to occur predominantly through the action of activated mannose-binding lectin-associated serine protease-3 (MASP-3) , one of the MASPs thought to play a role in the lectin pathway. Despite the need for enzyme-mediated maturation, however, factor D predominantly exists in its mature form in resting blood, likely because MASP-3 has no physiological inhibitors.
  • MASP-3 mannose-binding lectin-associated serine protease-3
  • mature factor D With mature factor D being the predominant form in resting blood, and because it has no known endogenous inhibitors itself, a high level of control is essential to prevent it from inappropriately cleaving endogenous proteins other than its substrate. As such, mature factor D is locked into an inactive state by a self-inhibitory loop. This loop dictates the enzyme’s low reactivity and extreme specificity for its substrate, factor B. Importantly, factor B can only be cleaved by factor D when factor B is bound to C3b or C3 (H2O) . Upon binding to and cleaving factor B, factor D is not permanently incorporated into the complex but is instead recycled in a reversible reaction.
  • the kidney plays an important role in regulating the concentration of factor D via glomerular filtration.
  • the alternative pathway and factor D have been implicated in various other physiological processes.
  • C3b-mediated opsonization is known to be responsible for marking damaged liver cells for removal by phagocytes after acute liver injury. This process enables a scaffold for newly formed cells to develop and helps to prevent persistent inflammation.
  • the alternative pathway and factor D have been shown to be essential in this process. Nonetheless, a fine balance in the level of activation of the alternative pathway is required because an overactive complement cascade is also known to cause extensive hepatic cell death, persistent inflammation, and injury to the liver.
  • factor D has also been implicated in the aging process of the skin.
  • aging it is known that the extracellular matrix of the dermal layer deteriorates as the quantity of senescent cells increases. Expression in human skin samples was higher in older subjects than in younger subjects.
  • Another tissue in which factor D plays a role is adipose tissue.
  • Adipocytes are energy reservoirs that play an important role in energy balance. Not only are they responsible for lipolysis but they are also involved in glucose uptake and triglyceride synthesis.
  • factor D is not the only component of the complement system that is produced in adipose tissue.
  • C3 and factor B are also expressed to some degree in this tissue, where they have been found to activate the proximal part of the alternative pathway (i.e., upstream of C5 cleavage) in the absence of pathogens.
  • factor D has been shown to be important for adipocyte differentiation and lipid accumulation via C3a signaling.
  • no activation of the terminal or lytic part of the pathway is observed, because proteins such as C5 are not expressed in adipose tissue.
  • adipocytes also have endocrine function. In response to certain stimuli, they secrete regulatory molecules that play a role in the metabolic function of other tissues.
  • regulatory molecules include fatty acids and adipokines such as factor D. It has been shown that by controlling the production of C3a via the alternative pathway, factor D indirectly induces insulin secretion from pancreatic beta cells when glucose levels are elevated. Furthermore, factor D/C3a signaling has been found to preserve islet beta cells by blocking cell dedifferentiation and death. It has even been shown that higher levels of circulating factor D are associated with a lower risk of developing diabetes in middle-aged adults.
  • factor D Deficiency in components of the proximal part of the alternative pathway, such as factor D, can lead to an inability to opsonize invading pathogens and to insufficient formation of the MAC. This ultimately limits both phagocytosis and lysis of the invaders.
  • complete deficiency of factor D has been identified as a risk factor for serious bacterial infections.
  • complete factor D deficiency due to a Ser42Stop mutation in both alleles of the gene was observed in a Dutch individual suffering from meningitis.
  • the complete factor D deficiency was linked with a decreased ability to opsonize and phagocytose bacteria and was also observed in other family members.
  • factor D deficiencies are associated with N. gonorrhoeae and with various respiratory infections. Although the underlying factor D mutations are not always the same, they likely result in an unstable protein or an abnormally folded protein that cannot be secreted. Mutations in both alleles are required for complete factor D deficiency, and therefore the mode of inheritance is autosomal recessive.
  • the anti-FD antibody moiety can specifically bind to FD derived from any source, such as any organism that has a complement system, including but not limited to, dogs, cats, pigs, cows, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans.
  • the FD is human FD.
  • the FD is cynomolgus monkey FD (cyno FD) .
  • the factor D is human factor D.
  • binding of the anti-FD antibody moiety to human-factor D is associated with a reduction in the generation of C3bBb in the complement activation pathway in an intact organism.
  • the anti-FD antibody moiety binds to human FD. In some embodiments, the anti-FD antibody moiety binds to human FD only and does not bind to FD derived from another species. In some embodiments, the anti-FD antibody moiety has cross-species reactivity to FD other than human FD. Exemplary non-human FD include, but are not limited to, mouse FD, rat FD, rabbit FD, sheep FD, and cynomolgus monkey FD. In some embodiments, the anti-FD antibody moiety cross-reacts with cynomolgus monkey FD (cyno FD) . In some embodiments, the anti-FD antibody moiety does not cross-react with murine FD.
  • the anti-FD antibody moiety binds to a mature FD molecule. In some embodiments, the anti-FD antibody moiety binds to a pro-FD. In some embodiments, the anti-FD antibody moiety binds to both pro-FD and mature FD.
  • the anti-FD antibody moiety can be any suitable format known in the art.
  • the anti-FD antibody moiety is selected from the group consisting of full-length antibody, Fab, Fab’ , F (ab’ ) 2 , scFv, and a combination thereof.
  • the anti-FD antibody moiety comprises (or consists essentially of, or consists of) a full-length antibody, such as a full-length antibody comprising an Fc fragment derived from IgG4.
  • the anti-FD antibody moiety comprises (or consists essentially of, or consists of) an anti-FD scFv.
  • the anti-FD antibody moiety is murine, chimeric, or humanized antibody.
  • the anti-FD antibody moiety is monospecific. In some embodiments, the anti-FD antibody moiety is multispecific. In some embodiments, the anti-FD antibody moiety is monovalent. In some embodiments, the anti-FD antibody moiety is multivalent.
  • the anti-human FD antibody moiety has one or more mutations (e.g., insertion, deletion, and/or substitution) that reduce off-target binding.
  • the binding of the anti-FD antibody moiety to FD is pH-dependent, wherein the anti-human FD antibody moiety binds more strongly to FD (e.g., human FD) at a neutral pH (e.g., about pH 7.4; such as that found in the blood) than it does at an acidic pH (e.g., about pH 5.8; such as that found in the endosome) .
  • the binding affinity of the pH-dependent anti-FD antibody moiety to FD (e.g., human FD) at about pH 7.4 is at least about 2 times (such as at least about any of 3, 4, 5, 6, 7, 8, 9, 10, or more) of the binding affinity of the pH-dependent anti-FD antibody moiety to FD (e.g., human FD) at about pH 5.8.
  • the anti-FD antibody moiety is affinity-matured.
  • the affinity-matured anti-FD antibody moiety has a binding affinity to FD (e.g., human FD, and/or cyno FD) at least about 2-fold (e.g., at least about any of 3, 4, 5, 10, 50, 100, 1000, or more folds) of that of the parental anti-FD antibody moiety (i.e., not affinity-matured) .
  • the affinity-matured anti-FD antibody moiety has a similar (e.g., within about 2-fold difference) binding affinity to human FD as the parental anti-FD antibody moiety, but has a binding affinity to cynomolgus monkey FD at least about 2-fold (e.g., at least about any of 3, 4, 5, 10, 50, 100, 1000, or more folds) of that of the parental anti-FD antibody moiety.
  • the anti-FD antibody moiety is affinity-matured, and the binding of the anti-FD antibody moiety to FD (e.g., human FD) is pH-dependent.
  • the anti-human FD antibody moiety comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) .
  • an anti-human FD antibody moiety comprising: i) a VH comprising an H-CDR1, an H-CDR2, and an H-CDR3, respectively comprising the amino acid sequence of an H-CDR1 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ) , an H-CDR2 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ) , and an H-CDR3 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ) within a reference VH having the amino acid sequence set forth in SEQ ID NO: 7,
  • the CDR positions are according to Kabat numbering.
  • the affinity of such anti-human FD antibody moiety for FD is comparable (e.g., within about 2-fold difference) to that of a reference antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the affinity of such anti-human FD antibody moiety for FD is at least about 2-fold (e.g., at least about any of 3, 4, 5, 10, 50, 100, 1000, or more folds) of that of a reference antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • an anti-human FD antibody moiety comprising a VH and a VL
  • the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)
  • an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)
  • an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)
  • an anti-human FD antibody moiety comprising a VH and a VL
  • the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)
  • an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions)
  • an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3
  • the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g.,
  • an anti-human FD antibody moiety comprising: (i) a VH comprising an H-CDR1 comprising the amino acid sequence of D-T-Y-V-H (SEQ ID NO: 1) ; an H-CDR2 comprising the amino acid sequence of R-I-D-P-X1-X2-G-X3-T-X4-F-X5-P-R-F-Q-A (SEQ ID NO: 9) , wherein X1 is A or H, X2 is N, S, or Y, X3 is L or H, X4 is T or H, and X5 is D, V, L, or H; and an H-CDR3 comprising the amino acid sequence of A-M-E-Y (SEQ ID NO: 3) ; and (ii) a VL comprising an L-CDR1 comprising the amino acid sequence of S-A-X6-S-D-V-S-X7-M-Y (SEQ ID NO: 3) ; and (ii
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence homology to SEQ ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 8.
  • the VH comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 10 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) in the VH domain
  • the VL comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 10 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) in the VL domain.
  • the one or more amino acid variations are in one or more of the CDRs. In some embodiments, the one or more amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) are in one or more of the framework regions (FRs) . In some embodiments, the two or more amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) are in both CDRs and FRs.
  • the amino acid residue at position 24 at the VH is A or T; ii) the amino acid residue at position 74 of the VH is K or T; iii) the amino acid residue at position 77 of the VH is N or S; iv) the amino acid residue of position 97 at the VH is A or T; v) the amino acid residue at position 98 of the VH is R or Y; and/or vi) the amino acid residue at position 70 of the VL is F or Y; wherein the numbering is according to the Kabat numbering system.
  • the amino acid residue position in VH is relative to a VH comprising the amino acid sequence of SEQ ID NO: 7
  • the amino acid residue position in VL is relative to a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 7, and L- CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 18, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 19.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80%(e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence homology to SEQ ID NO: 18; and a VL comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence homology to SEQ ID NO: 19.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 18, and a VL comprising the
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 23, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 25.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 26, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 27.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 26; and a VL comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 27.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 26, and a VL comprising the amino acid sequence of SEQ ID NO: 27.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 34, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 35.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 34; and a VL comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 35.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 34, and a VL comprising the amino acid sequence of SEQ ID NO: 35.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 216, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 214.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 216, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 216; and a VL comprising the amino acid sequence of SEQ ID NO: 214, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 214.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 216, and a VL comprising the amino acid sequence of SEQ ID NO: 214.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 39, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 41.
  • an anti-human FD antibody moiety that comprises H-CDR1, H- CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 42, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 43.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 42; and a VL comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 43.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 42, and a VL comprising the amino acid sequence of SEQ ID NO: 43.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 44, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 46; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 47, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 49.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 50, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 51.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 50, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 50; and a VL comprising the amino acid sequence of SEQ ID NO: 51, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 51.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 51.
  • the anti-human FD antibody moiety possess pH-dependent binding to human and/or cyno FD.
  • the anti-human FD antibody moiety binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold of that at an acidic pH.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 52, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 54; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 57.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 59.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 58, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 58; and a VL comprising the amino acid sequence of SEQ ID NO: 59, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 59.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 58, and a VL comprising the amino acid sequence of SEQ ID NO: 59.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 63, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 64, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 65.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 66, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 66, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 66; and a VL comprising the amino acid sequence of SEQ ID NO: 67, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 67.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 66, and a VL comprising the amino acid sequence of SEQ ID NO: 67.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 71, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 72, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 73.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 74, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 75.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 74, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 74; and a VL comprising the amino acid sequence of SEQ ID NO: 75, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 75.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 74, and a VL comprising the amino acid sequence of SEQ ID NO: 75.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 76, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 78; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 79, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 81.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 82, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 83.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 82; and a VL comprising the amino acid sequence of SEQ ID NO: 83, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 83.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 82, and a VL comprising the amino acid sequence of SEQ ID NO: 83.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 84, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 85, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 86; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 91.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 90, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 90; and a VL comprising the amino acid sequence of SEQ ID NO: 91, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 91.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 90, and a VL comprising the amino acid sequence of SEQ ID NO: 91.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 92, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 93, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 94; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 98, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 99.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 98; and a VL comprising the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 99.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 98, and a VL comprising the amino acid sequence of SEQ ID NO: 99.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 100, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 101, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 102; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 106, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 107.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 106, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 106; and a VL comprising the amino acid sequence of SEQ ID NO: 107, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 107.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 106, and a VL comprising the amino acid sequence of SEQ ID NO: 107.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 108, an H- CDR2 comprising the amino acid sequence of SEQ ID NO: 109, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 110; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 114, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 115.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 114; and a VL comprising the amino acid sequence of SEQ ID NO: 115, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 115.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 114, and a VL comprising the amino acid sequence of SEQ ID NO: 115.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 116, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 117, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 118; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 122, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 123.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 122, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 122; and a VL comprising the amino acid sequence of SEQ ID NO: 123, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 123.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 122, and a VL comprising the amino acid sequence of SEQ ID NO: 123.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 126.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 168, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 169.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 168, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 168; and a VL comprising the amino acid sequence of SEQ ID NO: 169, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 169.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 168, and a VL comprising the amino acid sequence of SEQ ID NO: 169.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and an VL comprising a L-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 126.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 184, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 185.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 184, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 184; and a VL comprising the amino acid sequence of SEQ ID NO: 185, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 185.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 184, and a VL comprising the amino acid sequence of SEQ ID NO: 185.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 186, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 187.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 186, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 186; and a VL comprising the amino acid sequence of SEQ ID NO: 187, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 187.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 186, and a VL comprising the amino acid sequence of SEQ ID NO: 187.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 126.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 289, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 127.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 289, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 289; and a VL comprising the amino acid sequence of SEQ ID NO: 127, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 127.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 289, and a VL comprising the amino acid sequence of SEQ ID NO: 127.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 190, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 191.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 190, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 190; and a VL comprising the amino acid sequence of SEQ ID NO: 191, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 191.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 190, and a VL comprising the amino acid sequence of SEQ ID NO: 191.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105.
  • an anti-human FD antibody moiety comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 192, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 193.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 192, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 192; and a VL comprising the amino acid sequence of SEQ ID NO: 193, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 193.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 192, and a VL comprising the amino acid sequence of SEQ ID NO: 193.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 194, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 195.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 194, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 194; and a VL comprising the amino acid sequence of SEQ ID NO: 195, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 195.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 194, and a VL comprising the amino acid sequence of SEQ ID NO: 195.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 196, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 197.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 196, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 196; and a VL comprising the amino acid sequence of SEQ ID NO: 197, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 197.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 196, and a VL comprising the amino acid sequence of SEQ ID NO: 197.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 198, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 199.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 198, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 198; and a VL comprising the amino acid sequence of SEQ ID NO: 199, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 199.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 198, and a VL comprising the amino acid sequence of SEQ ID NO: 199.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 57.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 156, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 157.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 156, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 156; and a VL comprising the amino acid sequence of SEQ ID NO: 157, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 157.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 156, and a VL comprising the amino acid sequence of SEQ ID NO: 157.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 158, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 159.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 158, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 158; and a VL comprising the amino acid sequence of SEQ ID NO: 159, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 159.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 158, and a VL comprising the amino acid sequence of SEQ ID NO: 159.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 160, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 161.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 160, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 160; and a VL comprising the amino acid sequence of SEQ ID NO: 161, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 161.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 160, and a VL comprising the amino acid sequence of SEQ ID NO: 161.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 162, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 163.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 162, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 162; and a VL comprising the amino acid sequence of SEQ ID NO: 163, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 163.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 162, and a VL comprising the amino acid sequence of SEQ ID NO: 163.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 164, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 165.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 164; and a VL comprising the amino acid sequence of SEQ ID NO: 165, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 165.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 164, and a VL comprising the amino acid sequence of SEQ ID NO: 165.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 166, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 167.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 166, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 166; and a VL comprising the amino acid sequence of SEQ ID NO: 167, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 167.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 166, and a VL comprising the amino acid sequence of SEQ ID NO: 167.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 57.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 172, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 173.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 172, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 172; and a VL comprising the amino acid sequence of SEQ ID NO: 173, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 173.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 172, and a VL comprising the amino acid sequence of SEQ ID NO: 173.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 174, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 175.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 174, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 174; and a VL comprising the amino acid sequence of SEQ ID NO: 175, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 175.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 174, and a VL comprising the amino acid sequence of SEQ ID NO: 175.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 176, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 177.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 176, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 176; and a VL comprising the amino acid sequence of SEQ ID NO: 177, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 177.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 176, and a VL comprising the amino acid sequence of SEQ ID NO: 177.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 178, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 179.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 178, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 178; and a VL comprising the amino acid sequence of SEQ ID NO: 179, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 179.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 178, and a VL comprising the amino acid sequence of SEQ ID NO: 179.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 180, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 181.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 180, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 180; and a VL comprising the amino acid sequence of SEQ ID NO: 181, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 181.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 180, and a VL comprising the amino acid sequence of SEQ ID NO: 181.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 182, and L- CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 183.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 182, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 182; and a VL comprising the amino acid sequence of SEQ ID NO: 183, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 183.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 182, and a VL comprising the amino acid sequence of SEQ ID NO: 183.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 206, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 207, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 208; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 209, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 210, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 211.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 212, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 213.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 212, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 212; and a VL comprising the amino acid sequence of SEQ ID NO: 213, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 213.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising the amino acid sequence of SEQ ID NO: 213.
  • the anti-human FD antibody moiety possess pH-dependent binding to human and/or cyno FD.
  • the anti-human FD antibody moiety binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold of that at an acidic pH.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 234, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 235, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 236; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 237, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 238, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 239.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 253, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 254.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 253, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 253; and a VL comprising the amino acid sequence of SEQ ID NO: 254, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 254.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 253, and a VL comprising the amino acid sequence of SEQ ID NO: 254.
  • the anti-human FD antibody moiety possess pH-dependent binding to human and/or cyno FD.
  • the anti-human FD antibody moiety binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold of that at an acidic pH.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 240, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 241, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 242; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 243, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 244, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 245.
  • an anti-human FD antibody moiety that comprises H- CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 255, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 256.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 255, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 255; and a VL comprising the amino acid sequence of SEQ ID NO: 256, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 256.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 255, and a VL comprising the amino acid sequence of SEQ ID NO: 256.
  • the anti-human FD antibody moiety possess pH-dependent binding to human and/or cyno FD.
  • the anti-human FD antibody moiety binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold of that at an acidic pH.
  • an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 246, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 247, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 248; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 249, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 250, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 251.
  • an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 257, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 258.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 257, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 257; and a VL comprising the amino acid sequence of SEQ ID NO: 258, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 258.
  • the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 257, and a VL comprising the amino acid sequence of SEQ ID NO: 258.
  • the anti-human FD antibody moiety possess pH-dependent binding to human and/or cyno FD.
  • the anti-human FD antibody moiety binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold of that at an acidic pH.
  • the anti-human FD antibody moiety is a full-length antibody (anti-human FD full-length antibody) .
  • the anti-human FD full-length antibody comprises an Fc region, such as a human Fc region.
  • the Fc region is derived from an IgG molecule, such as any one of the IgG1, IgG2, IgG3, or IgG4 subclass.
  • the anti-human FD full-length antibody comprises a heavy chain constant region derived from IgG4 (e.g., human IgG4) .
  • the Fc region is capable of mediating an antibody effector function, such as ADCC and/or CDC.
  • the Fc region comprises a modification that reduces binding affinity of the Fc region to an Fc receptor (FcR) .
  • the Fc region is derived from an IgG4 Fc (e.g., human IgG4 Fc) .
  • the heavy chain constant region comprises the amino acid sequence of a wild-type human IgG4 heavy chain constant region.
  • the IgG4 heavy chain constant region or Fc region comprises mutations.
  • the heavy chain constant region comprises one or more mutations (e.g., insertion, deletion, and/or substitution) in the hinge region for improved stability.
  • the heavy chain constant region comprises an S228P substitution (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence.
  • the IgG4 heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 132.
  • the heavy chain constant region comprises one or more mutations (e.g., insertion, deletion, and/or substitution) for increased (e.g., increasing at least about any of 20%, 50%, 80%, 90%, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, or more) binding to neonatal FcR (FcRn; e.g., human and/or cyno FcRn) , herein also referred to as “FcRn variants. ” Any FcRn variants that increase the binding of an anti-human FD full-length antibody to an FcRn (e.g., at about pH 5.8 and/or about pH 7.4) can be used herein.
  • FcRn neonatal FcR
  • FcRn variants any FcRn variants that increase the binding of an anti-human FD full-length antibody to an FcRn (e.g., at about pH 5.8 and/or about pH 7.4) can be used herein.
  • the heavy chain constant region comprises M428L/N434A ( “LA” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence.
  • the heavy chain constant region comprises M428L/N434A ( “LA” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132.
  • the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 133.
  • the heavy chain constant region comprises M252Y/V308P/N434Y ( “YPY” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence.
  • the heavy chain constant region comprises M252Y/V308P/N434Y ( “YPY” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 134. In some embodiments, the heavy chain constant region comprises M252Y/N286E/N434Y ( “YEY” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence.
  • the heavy chain constant region comprises M252Y/N286E/N434Y ( “YEY” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the heavy chain constant region comprises L432C/H433S/N434W/Y436L/T437CE ( “N3E” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence.
  • the heavy chain constant region comprises L432C/H433S/N434W/Y436L/T437CE ( “N3E” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132.
  • N3E substitutions relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132.
  • T437CE designates an amino acid substitution of T437C and an insertion of E right after the 437 position (EU numbering) .
  • the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 136.
  • the heavy chain constant region comprises L432C/H433S/N434W/Y436L/T437C ( “N3” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence.
  • the heavy chain constant region comprises L432C/H433S/N434W/Y436L/T437C (“N3” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132.
  • the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 137.
  • the heavy chain constant region comprises M252Y/S254T/T256E ( “YTE” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence. In some embodiments, the heavy chain constant region comprises M252Y/S254T/T256E ( “YTE” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 138.
  • the heavy chain constant region comprises M252Y/S254T/T256E/L432E/H433R/N434F/Y436R/T437Q ( “Y31-YTE” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence.
  • the heavy chain constant region comprises M252Y/S254T/T256E/L432E/H433R/N434F/Y436R/T437Q ( “Y31-YTE” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132.
  • the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 139.
  • any of the mutations e.g., insertion, deletion, and/or substitution
  • “N3E” mutations can be combined with the “YTE” mutations to generate “N3E-YTE” FcRn variant.
  • the S228P mutation (EU numbering) can be combined with any of the FcRn mutations described herein.
  • the heavy chain constant region of an anti-human FD full-length antibody comprises the amino acid sequence of any of SEQ ID NOs: 132-139.
  • the anti-human FD antibody moiety is an anti-human FD full-length antibody that: i) binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold (e.g., at least about any of 2.5, 3, 3.5, 5, 10-fold, or more) of that at an acidic pH; and ii) binds more strongly to FcRn (e.g., human FcRn and/or cyno FcRn) at about pH 5.8 and/or about pH 7.4 than a reference antibody without the FcRn mutation (s) , e.g., the binding affinity to FcRn of the anti-human FD full-length antibody is at least about 2-fold (e.g., at least about any of
  • the anti-human FD antibody moiety is an anti-human FD full-length antibody that: i) binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold (e.g., at least about any of 2.5, 3, 3.5, 5, 10-fold, or more) of that at an acidic pH; and ii) is affinity-matured, e.g., the binding affinity to FD (e.g., human FD and/or cyno FD) is at least about 2-fold (e.g., at least about any of 3, 5, 10, 20-fold, or more) of that of its parental antibody without affinity-maturation.
  • FD e.g., human FD and/or cyno FD
  • a neutral pH
  • the anti-human FD antibody moiety is an anti-human FD full-length antibody that: i) binds more strongly to FcRn (e.g., human FcRn and/or cyno FcRn) at about pH 5.8 and/or about pH 7.4 than a reference antibody without the FcRn mutation (s) , e.g., the binding affinity to FcRn of the anti-human FD full-length antibody is at least about 2-fold (e.g., at least about any of 5, 10, 50, 100, 1000-fold, or more) of that of a reference antibody without the FcRn mutation (s) ; and ii) is affinity-matured, e.g., the binding affinity to FD (e.g., human FD and/or cyno FD) is at least about 2-fold (e.g., at least about any of 2.5, 3, 3.5, 5, 10-fold, or more) of that of its parental antibody without affinity-maturation.
  • the anti-human FD antibody moiety is an scFv (anti-human FD scFv) .
  • the anti-human FD scFv comprises from N’ to C’ : VH-optional linker-VL.
  • the anti-human FD scFv comprises from N’ to C’ : VL-optional linker-VH.
  • Any suitable linker e.g., see “Linker” subsection below
  • an anti-human FD scFv comprising the amino acid sequence of SEQ ID NO: 219 or 220.
  • anti-human FD antibody moieties and anti-human FD antibody constructs that bind to FD (e.g., human FD and/or cyno FD) competitively with any of the anti-human FD antibody moieties or anti-human FD antibody constructs described herein.
  • FD e.g., human FD and/or cyno FD
  • the isolated anti-human FD antibody construct or the anti-human FD antibody moiety comprises an Fc domain. In some embodiments, the anti-human FD antibody moiety is an anti-human FD full-length antibody.
  • the Fc domain is an Fc effector domain, namely, an Fc domain possessing some or all effector functions, including for example complement and ADCC functions.
  • the Fc effector domain is an IgG1 or IgG3 Fc region.
  • one or more amino acid modifications may be introduced into the Fc domain of the antibody moiety, thereby generating an Fc domain variant.
  • the Fc domain variant may comprise a human Fc domain sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc domain) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
  • the Fc domain possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody moiety in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • Fc receptor (FcR) binding assays can be conducted to determine whether the antibody possesses Fc ⁇ R binding (hence likely ADCC activity) , and/or retains FcRn binding ability.
  • FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991) .
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5, 500, 362 (see, e.g., Hellstrom, I. et al. Proc. Nat’ l Acad. Sci. USA 83: 7059-7063 (1986) ) and Hellstrom, I et al., Proc. Nat’ l Acad. Sci. USA 82: 1499-1502 (1985) ; 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987) ) .
  • non-radioactive assays methods may be employed (see, for example, ACTI TM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI) .
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’ l Acad. Sci. USA 95: 652-656 (1998) .
  • C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) ; Cragg, M.S. et al., Blood 101: 1045-1052 (2003) ; and Cragg, M.S. and M.J. Glennie, Blood 103: 2738-2743 (2004) ) .
  • FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’ l. Immunol. 18 (12) : 1759-1769 (2006) ) .
  • Antibodies with reduced effector function include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) .
  • Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581) .
  • the Fc domain of the multispecific construct or the isolated anti-C5 antibody construct does not comprise a mutation that reduces its effector function, such as one or more mutations described herein.
  • the Fc domain of the multispecific construct or the isolated anti-C5 antibody construct comprises one or more of these mutations.
  • the Fc domain is an IgG1 Fc domain. In some embodiments, the IgG1 Fc domain does not comprise L234A mutation and/or a L235A mutation. In some embodiments, the IgG1 Fc domain comprises a L234A mutation and/or a L235A mutation (“LALA” mutation) . In some embodiments, the Fc domain is an IgG3 Fc domain. In some embodiments, the Fc domain is an IgG2 or IgG4 Fc domain. In some embodiments, the Fc domain is a human IgG4 Fc comprising the amino acid sequence of SEQ ID NO: 231.
  • the Fc domain is an IgG4 Fc domain comprising S228P, L234A, and/or L235A mutation. In some embodiments, the Fc domain is an IgG4 Fc fragment comprising an S228P mutation, such as comprising the amino acid sequence of SEQ ID NO: 232. In some embodiments, the Fc domain is an IgG4 Fc fragment comprising S228P/M428L/N434A ( “PLA” ) triple mutations, such as comprising the amino acid sequence of SEQ ID NO: 230.
  • the anti-human FD antibody moiety (or the isolated anti-human FD antibody construct) comprises an Fc domain with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc domain (EU numbering of residues) .
  • alterations are made in the Fc domain that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC) , e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000) .
  • CDC Complement Dependent Cytotoxicity
  • the anti-human FD antibody moiety (or the isolated anti-human FD antibody construct) comprises a variant Fc domain comprising one or more amino acid substitutions which alters half-life and/or changes binding to FcRn.
  • Antibodies with increased half-lives and improved binding to the FcRn which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994) ) , are described in US2005/0014934A1 (Hinton et al. ) .
  • Those antibodies comprise an Fc domain with one or more substitutions therein which alters binding of the Fc domain to FcRn.
  • Fc variants include those with substitutions at one or more of Fc domain residues, e.g., substitution of Fc domain residue 434 (US Patent No. 7,371,826) .
  • Binding affinities of anti-human FD antibody moieties Binding affinities of anti-human FD antibody moieties
  • Binding affinity and specificity of the anti-human FD antibody moieties described herein can be determined experimentally by methods known in the art.
  • the binding of an antibody to a protein antigen can be detected and/or quantified using a variety of techniques such as, but not limited to, Western blot, dot blot, surface plasmon resonance (SPR) method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N. J.
  • SPR surface plasmon resonance
  • BLI Bio-Layer Interferometry
  • RIA e.g., Octet system, ForteBio
  • ECL e.g., IRMA
  • EIA e.g., peptide scans
  • ELISA enzyme-linked immunosorbent assay
  • the anti-human FD antibody moiety specifically binds FD (e.g., human FD and/or cyno FD) with a K D of about 10 -7 M to about 10 -12 M, such as any of about 10 - 7 M to about 10 -10 M, about 10 -8 M to about 10 -11 M, about 10 -9 M to about 10 -11 M, about 10 -8 M to about 10 -10 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, or about 10 -10 M to about 10 -11 M.
  • FD e.g., human FD and/or cyno FD
  • the anti-human FD antibody moiety specifically binds FD (e.g., human FD and/or cyno FD) with a K D of about 10 -8 M to about 10 -12 M, or about 1 ⁇ 10 - 9 M to about 1 ⁇ 10 -12 M.
  • FD e.g., human FD and/or cyno FD
  • the anti-human FD antibody moiety binds more strongly to human FD and/or cyno FD at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) .
  • the anti-human FD antibody moiety specifically binds FD (e.g., human FD and/or cyno FD) with a K D of about 5 ⁇ 10 -10 M to about 10 ⁇ 10 -10 M at about pH 5.8.
  • the anti-human FD antibody moiety specifically binds FD (e.g., human FD and/or cyno FD) with a K D of about 1 ⁇ 10 -10 M to about 5 ⁇ 10 -10 M at about pH 7.4.
  • FD e.g., human FD and/or cyno FD
  • the AP is thought to be constitutively active at a low level due to spontaneous hydrolysis of C3 to form C3 (H2O) .
  • C3 (H2O) behaves like C3b in that it can associate with fB, which make fB susceptible to fD cleavage and activation.
  • the resultant C3 (H2O) Bb then cleaves C3 to produce C3b and C3a to initiate the AP cascade by forming the C3 convertase of the AP, C3bBb. As the initial C3 convertase generates increasing amounts of C3b, an amplification loop is established.
  • the AP amplification loop also participates in the CP and LP once these pathways are activated.
  • the AP consists of two functional entities: an independent complement activation pathway that is unrelated to CP or LP, and an amplification process that does participate and contribute to the full manifestation of CP and LP.
  • the anti-human FD antibody moiety inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the AP. In some embodiments, the anti-human FD antibody moiety inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the AP amplification process or amplification loop. In some embodiments, the anti-human FD antibody moiety does not inhibit (or inhibit at most about any of 10%, 5%, 2%, 1%, or less) the activation of CP or LP.
  • the anti-human FD antibody moiety or use thereof preserves the ability of an individual to combat an infection through the CP and LP.
  • the anti-human FD antibody moiety inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the amplification process or amplification loop.
  • the IC 50 value of the anti-FD antibody moiety in inhibiting the CP and/or LP e.g., inhibiting sheep RBC lysis in 20%human serum
  • the activity of the AP that is inhibited using a method of the invention or an anti-human FD antibody moiety described herein is AP activation induced by one or more of lipopolysaccharide (LPS) , lipooligosaccharide (LOS) , pathogen-associated molecular patterns (PAMPs) , and danger-associated molecular patterns (DAMPs) .
  • LPS lipopolysaccharide
  • LOS lipooligosaccharide
  • PAMPs pathogen-associated molecular patterns
  • DAMPs danger-associated molecular patterns
  • the activity of the AP that is inhibited using a method of invention or an anti-human FD antibody moiety described herein is the generation of C3bBb protein complex.
  • the activity of the AP that is inhibited using a method of the invention or an anti-human FD antibody moiety described herein is FD-dependent.
  • Methods for determining whether a particular antibody described herein inhibits human FD are known in the art. Inhibition of human FD can reduce the APC-mediated cell-lysing ability a subject’s body fluids. Such reductions of the cell-lysing ability present in the body fluid (s) can be measured by methods well known in the art such as, for example, by a conventional hemolytic assay such as the hemolysis assay in chicken erythrocyte hemolysis method as described in, e.g., Hillmen et al. (2004) N Engl. J Med 350 (6) : 552; and Yuan et al. (2017) Haematologica 102 (3) : 466-475.
  • a conventional hemolytic assay such as the hemolysis assay in chicken erythrocyte hemolysis method as described in, e.g., Hillmen et al. (2004) N Engl. J Med 350 (6) : 552; and Yuan et al. (2017) Haematologica 102 (3) : 466-475.
  • the concentration and/or physiologic activity of FD in a body fluid can be measured by methods well known in the art.
  • Methods for measuring FD concentration or activity include, e.g., ELISAs (see, e.g., Corvillo et al. (2021) Int J Mol Sci 22(12) : 6608) .
  • Inhibition of FD can result in the inhibition of alternative pathway activities, such as the production of C3b, as shown in Barratt et al. (2021) Front Immunl. 12: 712572.
  • the ELISA assays can be used to determine the inhibition of LPS-induced C3b deposition by an anti-FD antibody, as described in, e.g., Kimura et al. (2008) Blood.
  • Hemolytic assays can be used to determine the inhibitory activity of an anti-FD antibody on FD-mediated complement activation, and are thus useful for determining potential off-target binding of anti-FD antibodies.
  • These assays include but are not limited to a rabbit red blood cell (RBC) lysis test.
  • RBC red blood cell
  • a rabbit RBC lysis assay may be used to examine AP regulation, or an LPS-based ELISA assay may be used to examine AP complement inhibition.
  • the percentage of lysis is normalized by considering 100%lysis equal to the lysis occurring in the absence of the inhibitor.
  • C3 inhibition can be measured by methods known in the art, including, e.g., flow cytometry and commercial kits (e.g., assays) . See, for example, Mannes et al, (2021) Blood. 137 (4) : 443-455 and “Instructions for Use Complement System Alternative Pathway” Document No. LABEL-DOC-0034, 2.0, December 2018.
  • binding of the anti-human FD antibody moiety to FD is associated with a reduction (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) in the generation of C3bBb in the complement activation pathway in an intact organism (e.g., human) .
  • the anti-human FD antibody moiety inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) LPS-induced C3b and/or C5b-9 deposition.
  • the anti-FD antibody moiety does not inhibit (or inhibit at most about any of 10%, 5%, 2%, 1%, or less) IgM-induced or mannan-induced C3b and/or C5b-9 deposition.
  • Methods of testing the activities (e.g., inhibition of the AP) of anti-human FD antibody moieties are well known in the art, including but not limited to, rabbit RBC lysis assay (e.g., induced by human serum) , or LPS-induced C3b and/or C5b-9 deposition assay. See, e.g., assays in US11434279, the content of which is incorporated herein by reference in its entirety. Also see Example 4 herein.
  • the anti-human FD antibody moiety described herein possess pH-dependent dissociation from factor D (e.g., human FD and/or cyno FD) .
  • factor D e.g., human FD and/or cyno FD
  • Such pH-dependent binding provides for greater persistence of administered antibody or antibody fusion protein molecules, because immune complexes (i.e., the anti-human FD antibody construct bound to factor D) taken up by cells will dissociate in the acidic environment of the endosome and allow the freed antibody or antibody fusion protein to be recycled back out of the cell through the neonatal Fc receptor (FcRn) where it is available to bind to a new factor D molecule.
  • FcRn neonatal Fc receptor
  • the expression “pH-dependent binding” means that the antibody exhibits reduced (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) binding to FD at acidic pH (e.g., about pH 5.8; such as in the endosome) as compared to its binding at neutral pH (e.g., about pH 7.4; such as in the blood) .
  • acidic pH e.g., about pH 5.8; such as in the endosome
  • neutral pH e.g., about pH 7.4; such as in the blood
  • pH-dependency of the anti-human FD antibody moiety described herein can be determined experimentally by methods known in the art, such as in US20220204602, US20220177556, US9,079,949, and US9765135, the contents of each of which are incorporated herein by reference in their entirety. Also see Example 3 herein. pH-dependency for FD binding may be reflected in the differences in binding properties such as binding affinity (e.g., dissociation constant) , kinetic parameters (e.g., association rate and dissociation rate) , and percentage dissociation, at different pH levels. In some embodiments, the pH-dependency of the anti-human FD antibody moiety may be expressed in terms of the ratio of the percentage dissociation.
  • the percentage dissociation may be expressed in terms of the low-pH dissociation factor and the neutral-pH dissociation factor.
  • the pH-dependency of the anti-human FD antibody moiety is expressed in the K off ratio of about pH 5.8 vs.about pH 7.4. In some embodiments, the pH-dependency of the anti-human FD antibody moiety is expressed in the K D ratio of about pH 5.8 vs. about pH 7.4.
  • the pH dependence for FD binding of the anti-human FD antibody moiety can be assessed based on the dissociation of a FD-bound antibody at an acidic pH (e.g., about pH 5.8) or at a neutral pH (e.g., about pH 7.4) .
  • Low-pH dissociation factor namely, the percentage of antibody dissociated at about pH 5.8 from the antigen at about 25°C, wherein the antibody is pre-bound to the antigen at about pH 7.4, can be used to determine the dissociation of an FD-bound antibody at an acidic pH.
  • the low-pH dissociation factor may be measured by associating an antibody and an antigen (e.g., the anti-human FD antibody construct and human/cyno factor D) at pH 7.4 for about 600 seconds, followed by a dissociation period of about 600 seconds in a buffer at about pH 5.8, and calculation of the percentage of antibody dissociated at about pH 5.8 from the antigen.
  • an antigen e.g., the anti-human FD antibody construct and human/cyno factor D
  • the low-pH dissociation factor of the anti-human FD antibody moiety i.e., the %of dissociation of the anti-human FD antibody moiety from the bound FD under an acidic pH (e.g., about pH 5.8) ) is no less than about any of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%.
  • Neutral-pH dissociation factor namely, the percentage of antibody dissociated at about pH 7.4 from the antigen at about 25 °C, wherein the antibody is pre-bound to the antigen at about pH 7.4 and can be used to determine the dissociation of an FD-bound antibody at a neutral pH.
  • the neutral-pH dissociation factor may be measured by associating an antibody and an antigen (e.g., the anti-human FD antibody construct and human/cyno FD) at about pH 7.4 for about 600 seconds, followed by a dissociation period of about 600 seconds in a buffer at about pH 7.4, and calculation of the percentage of antibody dissociated at about pH 7.4 from the antigen.
  • an antigen e.g., the anti-human FD antibody construct and human/cyno FD
  • the neutral-pH dissociation factor of the anti-human FD antibody moiety i.e., the %of dissociation of the anti-human FD antibody moiety from the bound FD under a neutral pH (e.g., about pH 7.4) ) is no more than about any of 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.
  • the ratio of the low-pH dissociation factor over the neutral-pH dissociation factor of the anti-human FD antibody moiety of the present invention is at least about any one of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 20, 50, or more.
  • the percentage of dissociation of the antibody for factor D at pH 5.8 over the percentage of dissociation of the antibody for factor D at pH 7.4 is at least about any of 3, 4, 5, or 6.
  • the anti-human FD antibody moiety binds FD (e.g., human and/or cyno FD) more strongly (such as at least about any of 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, or higher, more strongly) at a neutral pH (such as about pH 7.4) than it does at an acidic pH (such as about pH 5.8) .
  • FD e.g., human and/or cyno FD
  • a neutral pH such as about pH 7.4
  • an acidic pH such as about pH 5.8 .
  • the FcRn variant anti-human FD antibody moiety described herein in some embodiments exhibit prolonged (e.g., increasing at least about any of 10%, 20%, 50%, 70%, 90%, 1.5-fold, 2-fold, 5-fold, 20-fold, or more) serum half-life in vivo, compared to a reference anti-human FD antibody without FcRn binding mutation (s) , such as in mice (including transgenic mice) .
  • the anti-human FD antibody moiety exhibits prolonged serum half-life in other test animals, include but are not limited to, rats, chickens, rabbits, sheep, and cyno monkeys.
  • the anti-human FD antibody moiety exhibits prolonged serum half-life in human.
  • Antibodies with pH-dependent binding to antigens enhance antigen degradation within the cell in endosomes or lysosomes; and more freed or unbound antibodies are rescued via FcRn binding and are ultimately recycled back into the circulation and bind to additional antigen (Igawa et al., Nat Biotechnol. 2010 Nov; 28 (11) : 1203-7) .
  • the pH-dependent antigen binding properties can naturally occur or be created by engineering of the antigen binding interactions.
  • IgG Fc naturally binds to FcRn in a pH-dependent manner.
  • Engineered antibodies with enhanced pH-dependent FcRn binding properties e.g., increased (e.g., increasing at least about any of 2-fold, 5-fold, 20-fold, or more) binding to FcRn at acidic pH (e.g., about pH 5.8) and little (e.g., within about 2-fold difference) or no change binding to FcRn under neutral pH (e.g., about pH 7.4) , that can be rescued and recycled more effectively back to the serum are also known as “recycling antibodies. ” .
  • Engineered antibody Fc that have increased binding affinity to FcRn at both acidic pH (e.g., about pH 5.8) within endosome and at neutral pH (e.g., about pH 7.4) can enhance antigen/antibody complex uptake from the circulation (to reduce serum antigen level) and recycle from endosome back to the circulation to bind more new antigens, thus named as “sweeping” antibody (Igawa et al., PloS One. 2013 May 7; 8 (5) : e63236) .
  • FcRn binding mutation LA has been shown to increase antibody recycling resulting long IgG serum persistence (Yeung et al., J Immunol (2009) 182 (12) : 7663-7671) .
  • FcRn binding mutations Y31-YTE and N3E (Borrok et al., J Biol Chem. 2015 Feb 13; 290 (7) : 4282-90) as well as YEY and YPY (Igawa et al., PloS One. 2013 May 7; 8 (5) : e63236) have been shown to significantly increasing FcRn binding at both pH 7.4, and at pH 5.8.
  • Antibody carrying these Fc mutations observed extended serum persistence and/or reduced serum antigen levels. The contents of each of these references are incorporated herein by reference in their entireties.
  • the anti-human FD antibody moiety has pH-dependent binding to FD (e.g., human and/or cyno FD) and has faster (e.g., at least about any of 50%, 70%, 90%, 1.5-fold, 2-fold, 5-fold, 20-fold, or more faster) dissociation from FD at acidic pH (e.g., about pH 5.8) than that at neutral pH (e.g., about pH 7.4) .
  • FD e.g., human and/or cyno FD
  • faster e.g., at least about any of 50%, 70%, 90%, 1.5-fold, 2-fold, 5-fold, 20-fold, or more faster
  • acidic pH e.g., about pH 5.8
  • neutral pH e.g., about pH 7.4
  • the anti-human FD antibody moiety (e.g., a full-length anti-human FD antibody) has improved (e.g., increasing at least about any of 10%, 20%, 50%, 70%, 90%, 1.5-fold, 2-fold, 5-fold, 20-fold, 100-fold, 1000-fold, or more) binding to FcRn, such as under about pH 7.4 and/or about pH 5.8, e.g., compared to a reference anti-human FD antibody without FcRn binding mutation (s) .
  • the anti-human FD antibody moiety is a recycling antibody moiety.
  • the anti-human FD antibody moiety is a sweeping antibody moiety.
  • the anti-human FD antibody moiety is both a recycling antibody moiety and a pH-dependent FD binding antibody moiety. In some embodiments, the anti-human FD antibody moiety is both a sweeping antibody moiety and a pH-dependent FD binding antibody moiety. In some embodiments, the binding affinity of an anti-human FD full-length antibody to FcRn at about pH 7.4 is about 10 -11 M to about 10 -5 M, such as about 10 -10 M to about 10 -6 M, or about 10 -9 M to about 10 -8 M.
  • the binding affinity of an anti-human FD full-length antibody to FcRn at about pH 5.8 is about 10 -11 M to about 10 -7 M, such as about 10 -11 M to about 10 -8 M, or about 10 -10 M to about 10 -8 M.
  • the isolated anti-human FD antibody constructs described herein further comprise a fusion partner, or a second antibody moiety that specifically recognizes a component of the complement pathway (hereinafter also referred to as “complement protein” ) .
  • complement protein a component of the complement pathway
  • an isolated anti-human FD antibody construct comprising: an anti-human FD antibody moiety (e.g., any of the anti-human FD antibody moieties described herein) and a second antibody moiety specifically recognizing a component of the complement pathway (e.g., FD, or non-FD complement protein) .
  • Any complement protein can be used herein.
  • Exemplary fusion partners include, but are not limited to, anti-FD antibody moieties (e.g., any of the anti-human FD antibody moieties described herein) , anti-C2 antibody moieties, or anti-C5 antibody moieties.
  • the isolated anti-human FD antibody construct is monospecific.
  • the isolated anti-human FD antibody construct is multivalent.
  • the isolated anti-human FD antibody construct is multispecific ( “multispecific construct” ) .
  • the second antibody moiety is selected from the group consisting of: a full-length antibody, Fab, Fab’ , F (ab) 2 , F (ab’ ) 2 , scFv, sdAb, and a combination thereof. In some embodiments, the second antibody moiety is an scFv.
  • the complement system plays an important role in the pathology of many autoimmune, inflammatory, and ischemic diseases. Inappropriate complement activation and its deposition on host cells can lead to complement-mediated lysis and/or injury of cells and target tissues, as well as tissue destruction due to the generation of powerful mediators of inflammation.
  • the complement system also known as complement cascade, is a part of the immune system that enhances (complements) the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promote inflammation, and attack the pathogen’s cell membrane. It is part of the innate immune system, which is not adaptable and does not change during an individual’s lifetime.
  • the complement system can, however, be recruited and brought into action by antibodies generated by the adaptive immune system.
  • the alternative complement pathway AP
  • the classical pathway CP
  • the lectin pathway LP
  • the CP is initiated by antigen-antibody complexes
  • the LP is activated by binding of lectins to sugar molecules on microbial surfaces
  • the AP is constitutively active at a low level but can be quickly amplified on bacterial, viral, and parasitic cell surfaces due to the lack of regulatory proteins.
  • Host cells are usually protected from AP complement activation by regulatory proteins. But in some situations, such as when the regulatory proteins are defective or missing, the AP can also be activated uncontrollably on host cells, leading to complement-mediated disease or disorder.
  • the CP consists of components C1, C2, and C4, and converges with the AP at the C3 activation step.
  • the LP consists of mannose-binding lectins (MBLs) and MBL-associated serine proteases (MASPs) and shares with the CP the components C4 and C2.
  • MBLs mannose-binding lectins
  • MASPs MBL-associated serine proteases
  • the AP consists of components C3 and several factors, such as factor B, factor D, properdin, and the fluid phase regulator factor H.
  • Complement activation consists of three stages: (a) recognition, (b) enzymatic activation, and (c) membrane attack leading to cell death. The first phase of CP complement activation begins with C1.
  • C1 is made up of three distinct proteins: a recognition subunit, C1q, and the serine protease subcomponents, C1r and C1s, which are bound together in a calcium-dependent tetrameric complex, C1r2 s2.
  • An intact C1 complex is necessary for physiological activation of C1 to result. Activation occurs when the intact C1 complex binds to immunoglobulin complexed with antigen. This binding activates C1s which then cleaves both the C4 and C2 proteins to generate C4a and C4b, as well as C2a and C2b.
  • the C4b and C2a fragments combine to form the C3 convertase, C4b2a, which in turn cleaves C3 to form C3a and C3b.
  • Activation of the LP is initiated by MBL binding to certain sugars on the target surface and this triggers the activation of MBL-associated serine proteases (MASPs) which then cleave C4 and C2 in a manner analogous to the activity of C1s of the CP, resulting in the generation of the C3 convertase, C4b2a.
  • MASPs MBL-associated serine proteases
  • the CP and LP are activated by different mechanisms, but they share the same components C4 and C2 and both pathways lead to the generation of the same C3 convertase, C4b2a.
  • C3b and C3a are central events of the complement pathway for two reasons. It initiates the AP amplification loop because surface deposited C3b is a central intermediate of the AP C3 convertase C3bBb. Both C3a and C3b are biologically important. C3a is proinflammatory and together with C5a are referred to as anaphylatoxins. C3b and its further cleavage products also bind to complement receptors present on neutrophils, eosinophils, monocytes and macrophages, thereby facilitating phagocytosis and clearance of C3b-opsonized particles.
  • C3b can associate with C4b2a or C3bBb to form the C5 convertase of the CP and LP, and AP, respectively, to activate the terminal complement sequence, leading to the production of C5a, a potent proinflammatory mediator, and the assembly of the lytic membrane attack complex (MAC) , C5-C9.
  • MAC lytic membrane attack complex
  • Defective complement action is a cause of several human glomerular diseases including atypical hemolytic uremic syndrome (aHUS) , anti-neutrophil cytoplasmic antibody mediated vasculitis (ANCA) , C3 glomerulopathy, IgA nephropathy, immune complex membranoproliferative glomerulonephritis, renal ischemic reperfusion injury, lupus nephritis, membranous nephropathy, and chronic transplant mediated glomerulopathy.
  • Aberrant complement component activation has been proposed as markers in various types of cancers and their clinical outcomes. Lung cancer patients show significantly higher plasma levels of complement proteins and activation fragments than do control donors, and elevated complement levels are correlated with lung tumor size.
  • Complement-related proteins are also elevated in biological fluids from patients with other types of tumors. See, for example, Pio et al. Semin Immunol. 2013 Feb; 25 (1) : 54-64. Inhibition of the complement cascade has been proposed for glomerular diseases and cancer treatment.
  • the second antibody moiety specifically recognizes FD.
  • the isolated anti-human FD antibody construct comprises two or more anti-human FD antibody moieties (e.g., scFv) , such as two or more any of the anti-human FD antibody moieties described herein.
  • the two or more anti-human FD antibody moieties are fused to each other in tandem.
  • the two or more anti-human FD antibody moieties are on different polypeptide chains within the isolated anti-human FD antibody construct.
  • the two or more anti-human FD antibody moieties bind to the same FD epitope.
  • the two or more anti-human FD antibody moieties bind to at least two different FD epitopes. In some embodiments, each of the two or more anti-human FD antibody moieties binds to a different FD epitope.
  • the second antibody moiety specifically recognizes complement component 2 ( “C2” ) . In some embodiments, the second antibody moiety specifically recognizes complement component 5 ( “C5” ) .
  • the isolated anti-human FD antibody construct comprises an anti-human FD antibody moiety (e.g., any of the anti-human FD antibody moieties described herein) and one or more antibody moieties (e.g., scFvs) specifically recognizing a complement protein that is not FD, such as fused to each other or with the anti-human FD antibody moiety in tandem, or on different polypeptide chains.
  • the isolated anti-human FD antibody construct comprises (or consists essentially of, or consists of) one polypeptide chain. In some embodiments, the isolated anti-human FD antibody construct comprises (or consists essentially of, or consists of) two or more (e.g., 2) polypeptide chains, such as a heterodimer or a homodimer formed in the presence of an Fc domain.
  • the isolated anti-human FD antibody construct comprises an anti-human FD scFv ( “scFv1” ; e.g., any of the anti-human FD scFvs described herein) fused to a second scFv (scFv2) specifically recognizing a component of the complement pathway.
  • the scFv2 is an anti-C2 scFv.
  • the scFv2 is an anti-C5 scFv.
  • the scFv (e.g., scFv1 or scFv2) comprises from N-terminus to C-terminus: VH-optional linker-VL.
  • the scFv (e.g., scFv1 or scFv2) comprises from N-terminus to C-terminus: VL-optional linker-VH.
  • the isolated anti-human FD antibody construct comprises one or more linkers between the anti-human FD antibody moiety (e.g., anti-FD scFv) and the second antibody moiety (e.g., scFv) specifically recognizing the complement protein.
  • the length, the degree of flexibility, and/or other properties of the linker (s) include but not are limited to influencing the affinity, specificity, or avidity, for one or more particular antigens or epitopes. For example, longer linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another.
  • a linker (such as peptide linker) comprises flexible residues (such as glycine (G) and serine (S) ) so that anti-human FD antibody moiety (e.g., scFv) and the second antibody moiety specifically recognizing the complement protein are free to move relative to each other.
  • the linker is a peptide linker.
  • the linker is a non-cleavable linker.
  • the linker is a cleavable linker.
  • linker considerations include the effect on physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as planned degradation) , rigidity, flexibility, immunogenicity, modulation of antibody binding, the ability to be incorporated into a micelle or liposome, and the like.
  • the peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence.
  • a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO1996/034103.
  • the peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, 200, or more, amino acids long. In some embodiments, the peptide linker is no more than about any of 200, 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or fewer, amino acids long.
  • the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.
  • the peptide linker does not comprise any polymerization activity.
  • the characteristics of a peptide linker, which comprise the absence of the promotion of secondary structures, are known in the art and described, e.g., in Dall’A cqua et al. (Biochem. (1998) 37, 9266-9273) , Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9 (1) , 73-80) .
  • the peptide linker does not promote the formation of any secondary structures.
  • the linkage of the domains to each other can be provided by, e.g., genetic engineering.
  • the peptide linker can be a stable linker, which is not cleavable by proteases, especially by Matrix metalloproteinases (MMPs) .
  • MMPs Matrix metalloproteinases
  • the peptide linker is a flexible linker.
  • exemplary flexible linkers include glycine polymers (G) n, where n is an integer of at least one, and glycine-serine polymers (including, for example, (GS) n, where n is an integer of at least one) , glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.
  • the linker is a GS linker.
  • the isolated anti-human FD antibody construct (e.g., multispecific construct) comprises two polypeptide chains each comprising a subunit of the Fc domain, and the two polypeptide chains dimerize through the Fc domain.
  • the isolated anti-human FD antibody construct comprising an Fc domain has one or more of the following properties: i) has at least about 20% (e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, or more) longer half-life in vivo; and ii) has at least about 20%(e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, or more) stronger activity in inhibiting one or more of CP, LP, AP, and TP, compared to a same isolated anti-human FD antibody construct (e.g., multispecific construct) without the Fc domain (e.g., different antibody moieties are connected via a G4S linker) .
  • i has at least about 20% (e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold,
  • the anti-human FD antibody moiety is fused to the second antibody moiety via an Fc domain, such as an Fc domain comprising the amino acid sequence of SEQ ID NO: 230.
  • an Fc domain such as an Fc domain comprising the amino acid sequence of SEQ ID NO: 230.
  • Any of the Fc domains described under the “Fc domain” subsection above can be used herein in the linker.
  • the Fc domain is derived from human IgG4 Fc.
  • the Fc domain is flanked by GGGGS (SEQ ID NO: 221) on each end of its sequence.
  • the Fc domain is flanked by a linker (e.g., SEQ ID NO: 227) on one or each end of its sequence, then situated in between the anti-human FD antibody moiety (e.g., anti-FD scFv1) and the second antibody moiety (e.g, . scFv2) .
  • a linker e.g., SEQ ID NO: 227
  • the Fc domain is flanked by GGGGSGGGGS (SEQ ID NO: 227) on each end of its sequence (hereinafter also referred to as “flanked Fc domain linker” ) , then situated in between the anti-human FD antibody moiety (e.g., anti-FD scFv1) and the second antibody moiety (e.g., scFv2) .
  • the Fc domain comprises the amino acid sequence of SEQ ID NO: 230.
  • the flanked Fc domain linker comprises the amino acid sequence of SEQ ID NO: 233.
  • the anti-human FD antibody moiety e.g., scFv
  • the anti-human FD antibody moiety is fused to the fusion partner or the second antibody moiety (e.g., scFv) directly.
  • the anti-human FD antibody moiety e.g., scFv
  • the anti-human FD antibody moiety is fused to the fusion partner or the second antibody moiety (e.g., scFv) via a linker (e.g., peptide linker) .
  • the linker within an scFv (e.g., anti-human FD scFv and/or the scFv against the complement protein) , and/or the linker between two or more antibody moieties within the anti-human FD antibody construct, is independently is selected from the group consisting of SEQ ID NOs: 221-229, such as any of SEQ ID NOs: 221 and 226-229.
  • the isolated anti-human FD antibody construct comprises from N terminus to C terminus: an anti-human FD scFv (e.g., any of the anti-human FD scFvs described herein) , an optional linker, and an anti-C2 scFv or an anti-C5 scFv.
  • the isolated anti-human FD antibody construct comprises from N terminus to C terminus: an anti-C2 scFv or anti-C5 scFv, an optional linker, and an anti-human FD scFv (e.g., any of the anti-human FD scFvs described herein) .
  • the isolated anti-human FD antibody construct comprises from N terminus to C terminus: an anti-human FD scFv (e.g., any of the anti-human FD scFvs described herein) , a first optional linker, an Fc domain, a second optional linker, and an anti-C2 scFv or an anti-C5 scFv.
  • an anti-human FD scFv e.g., any of the anti-human FD scFvs described herein
  • the isolated anti-human FD antibody construct comprises from N terminus to C terminus: an anti-C2 scFv or an anti-C5 scFv, a first optional linker, an Fc domain, a second optional linker, and an anti-human FD scFv (e.g., any of the anti-human FD scFvs described herein) .
  • an anti-human FD scFv e.g., any of the anti-human FD scFvs described herein
  • the isolated anti-human FD antibody construct comprises an Fc domain as whole or part of a linker
  • the two polypeptide chains each comprising an Fc subunit dimerize through the Fc domain.
  • the complement factor the second antibody moiety binds to is complement factor 2 (C2) , such that the anti-human FD antibody construct is an anti-human FD/anti-C2 multispecific construct.
  • C2 complement factor 2
  • Any anti-C2 antibody or antigen binding fragment thereof described in WO2023143583 (the content of which is incorporated herein by reference in its entirety) can be used in the anti-C2 antibody moieties described herein.
  • the anti-C2 antibody moiety is an anti-C2 scFv.
  • Human C2 is an 83 kDa protein (100 kDa glycosylated) that is produced as an inactive, heavily glycosylated zymogen consisting of five domains: three N-terminal complement-control-protein (CCP) domains, a von Willebrand factor A-type (VWA) domain, and a C-terminal trypsin-like serine proteinase (SP) domain.
  • CCP complement-control-protein
  • VWA von Willebrand factor A-type
  • SP C-terminal trypsin-like serine proteinase
  • the last two domains, VWA and SP form the C2a fragment (residues 224-732, 70 kDa) that is produced in the proteolytic activation cascade of the classical and lectin pathways.
  • the larger fragment C2a is 57.4 kDa (70 kDa glycosylated) and provides the catalytic center to the convertase complexes of the classical and lectin-binding pathways of complement activation.
  • C2 bound to C4b is cleaved by classical (C1s) or lectin (MASP2) proteases to produce C4bC2a, a very short-lived C3 convertase that in turn cleaves C3 to C3a and C3b, leading ultimately to formation of Membrane Attack Complex (MAC) and lysis of bacteria and damaged cells.
  • C2 has the same serine protease domain as C4bC2a but in an inactive zymogen-like conformation, requiring cofactor-induced conformational change for activity.
  • the anti-C2 antibody moiety can specifically bind to C2 derived from any source, such as any organism that has a complement system, including but not limited to, dogs, cats, pigs, cows, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans.
  • the C2 is murine C2.
  • the C2 is human C2.
  • the C2 is cynomolgus monkey C2.
  • the anti-C2 antibody moiety binds to C2. In some embodiments, the anti-C2 antibody moiety binds to C2a. In some embodiments, the anti-C2 antibody moiety has cross-species reactivity to C2 other than human C2, and/or to C2a other than human C2a. Exemplary non-human C2 and C2a include, but are not limited to, mouse C2 and C2a, rat C2 and C2a, rabbit C2 and C2a, sheep C2 and C2a, cynomolgus monkey C2 and C2a. In some embodiments, the anti-C2 antibody moiety cross-reacts with cynomolgus C2 and/or C2a.
  • the anti-C2 antibody moiety can be any suitable format known in the art.
  • the anti-C2 antibody moiety is selected from the group consisting of full-length antibody, Fab, Fab’ , F (ab’ ) 2 , scFv, sdAb, and a combination thereof.
  • the anti-C2 antibody moiety comprises an sdAb that binds to C2.
  • the anti-C2 antibody moiety is a full-length antibody, such as a full-length antibody comprising an Fc fragment derived from IgG4 (e.g., an IgG4 Fc comprising a PLA mutation, such as SEQ ID NO: 230) .
  • the anti-C2 antibody moiety comprises (or consists essentially of, or consists of) an anti-C2 scFv.
  • the anti-C2 antibody moiety is murine, chimeric, humanized, or human antibody.
  • the anti-C2 antibody moiety is monospecific.
  • the anti-C2 antibody moiety is multispecific.
  • the anti-C2 antibody moiety is monovalent. In some embodiments, the anti-C2 antibody moiety is multivalent.
  • the binding of the anti-C2 antibody moiety to C2 is pH-dependent, and wherein the anti-C2 antibody moiety binds more strongly to C2 at a neutral pH (e.g., about pH 7.4; such as that found in the blood) than it does at an acidic pH (e.g., about pH 5.8; such as that found in the endosome) .
  • a neutral pH e.g., about pH 7.4; such as that found in the blood
  • an acidic pH e.g., about pH 5.8; such as that found in the endosome
  • the binding affinity of the pH-dependent anti-C2 antibody moiety to C2 (e.g., human C2) at about pH 7.4 is at least about 3 times (such as at least about any of 4, 5, 6, 7, 8, 9, 10, or more) times higher than the binding affinity of the pH-dependent anti-C2 antibody moiety to C2 (e.g., human C2) at about pH 5.8.
  • the anti-C2 antibody moiety comprises: (i) a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 259, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 260, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 261; and (ii) a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 262, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 263, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 264.
  • the anti-C2 antibody moiety comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 265, or a variant thereof that has at least about 80% (e.g., at least about any of 85%, 90%, 95%, 97%, 99%, or more) amino acid homology to SEQ ID NO: 265; and a VL domain comprising the amino acid sequence of SEQ ID NO: 266, or a variant thereof that has at least about 80% (e.g., at least about any of 85%, 90%, 95%, 97%, 99%, or more) amino acid homology to SEQ ID NO: 266.
  • the anti-C2 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 265, and a VL comprising the amino acid sequence of SEQ ID NO: 266.
  • the anti-C2 antibody moiety is an scFv, such as an anti-C2 scFv comprising the amino acid sequence of SEQ ID NO: 267 or 268.
  • the anti-C2 antibody moiety comprises: (i) a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 269, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 270, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 271; and (ii) a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 272, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 273, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 274.
  • the anti-C2 antibody moiety comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 275, or a variant thereof that has at least about 80% (e.g., at least about any of 85%, 90%, 95%, 97%, 99%, or more) amino acid homology to SEQ ID NO: 275; and a VL domain comprising the amino acid sequence of SEQ ID NO: 276, or a variant thereof that has at least about 80% (e.g., at least about any of 85%, 90%, 95%, 97%, 99%, or more) amino acid homology to SEQ ID NO: 276.
  • the anti-C2 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 275, and a VL comprising the amino acid sequence of SEQ ID NO: 276.
  • the anti-C2 antibody moiety is an scFv, such as an anti-C2 scFv comprising the amino acid sequence of SEQ ID NO: 277 or 278.
  • the anti-C2 antibody moiety specifically binds C2 (e.g., human C2) with a K D of about 10 -7 M to about 10 -12 M, such as any of about 10 -7 M to about 10 -10 M, about 10 -8 M to about 10 -11 M, about 10 -9 M to about 10 -11 M, about 10 -8 M to about 10 -10 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, or about 10 -10 M to about 10 -11 M.
  • the anti-C2 antibody moiety specifically binds C2 (e.g., human C2) with a K D of about 1 ⁇ 10 -10 M to about 5 ⁇ 10 -10 M.
  • the anti-C2 antibody moieties described herein inhibit (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) the CP and/or LP. In some embodiments, the anti-C2 antibody moieties described herein inhibit no more than about 70% (e.g., no more than about any of 60%, 50%, 40%, 30%, 20%, 10%, or less) of the CP and/or LP. In some embodiments, the anti-C2 antibody moiety does not inhibit (or inhibits at most about any of 10%, 5%, 2%, 1%, or less) the activation of AP.
  • the anti-C2 antibody moiety inhibits about 50%to about 80%of the CP and/or LP. In some embodiments, the IC 50 value of the anti-C2 antibody moiety in inhibiting the AP (e.g., inhibiting rabbit RBC lysis in 20%human serum) is undetectable. In some embodiments, the anti-C2 antibody moiety inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) IgM-induced and/or mannan-induced C3b and/or C5b-9 deposition. In some embodiments, the anti-C2 antibody moiety does not inhibit (or inhibit at most about any of 10%, 5%, 2%, 1%, or less) LPS-induced C3b and/or C5b-9 deposition.
  • the anti-human FD/anti-C2 multispecific construct i) inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the CP and/or LP, such as about 50%to about 80%of the CP and/or LP, and ii) inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the AP.
  • the anti-human FD/anti-C2 multispecific construct inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) all of CP, LP, AP, and TP. In some embodiments, the anti-human FD/anti-C2 multispecific construct inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) IgM-, mannan-, and LPS-induced C3b and C5b-9 deposition.
  • the anti-human FD/anti-C2 multispecific construct inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) sheep RBC lysis and rabbit RBC lysis (e.g., induced by human serum) .
  • the complement factor the second antibody moiety binds to is complement factor 5 (C5) , such that the anti-human FD antibody construct is an anti-human FD/anti-C5 multispecific construct.
  • C5 complement factor 5
  • Any anti-C5 antibody or antigen binding fragment thereof described in US20220204602, US20220177556, US11578137, WO2022134047, and PCT/US2023/063305 can be used in the anti-C5 antibody moieties described herein, the content of each of which is incorporated herein by reference in their entirety.
  • the anti-C5 antibody moiety is an anti-C5 scFv.
  • the C5 gene encodes a preproprotein is proteolytically processed to generate multiple protein products, including the C5 alpha chain, C5 beta chain, C5a anaphylatoxin and C5b.
  • Human C5 is a 188 kDa protein that is comprised of the C5 alpha and beta chains, which are linked by a disulfide bridge. Cleavage of the alpha chain by a convertase enzyme results in the formation of the C5a anaphylatoxin, which possesses potent spasmogenic and chemotactic activity, and the C5b macromolecular cleavage product.
  • the C5b macromolecular cleavage product can form a complex with the complement component C6, which then becomes the membrane attack complex (MAC) . Mutations in this gene cause complement component 5 deficiency, a disease characterized by recurrent bacterial infections.
  • MAC membrane attack complex
  • the anti-C5 antibody moiety can specifically bind to C5 derived from any source, such as any organism that has a complement system, including but not limited to, dogs, cats, pigs, cows, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans.
  • the C5 is murine C5.
  • the C5 is human C5.
  • the anti-C5 antibody moiety binds to C5. In some embodiments, the anti-C5 antibody moiety binds to C5b. In some embodiments, the anti-C5 antibody moiety has cross-species reactivity to C5 other than human C5, and/or to C5b other than human C5b. Exemplary non-human C5 and C5b include, but are not limited to, mouse C5 and C5b, rat C5 and C5b, rabbit C5 and C5b, sheep C5 and C5b, cynomolgus monkey C5 and C5b. In some embodiments, the anti-C5 antibody moiety cross-reacts with cynomolgus C5 and/or C5b. In some embodiments, the anti-C5 antibody moiety does not bind to C5a.
  • the anti-C5 antibody moiety can be any suitable format known in the art.
  • the anti-C5 antibody moiety is selected from the group consisting of full-length antibody, Fab, Fab’ , F (ab’ ) 2 , scFv, sdAb, and a combination thereof.
  • the anti-C5 antibody moiety comprises an sdAb that binds to C5.
  • the anti-C5 antibody moiety is a full-length antibody, such as a full-length antibody comprising an Fc fragment derived from IgG4 (e.g., an IgG4 Fc comprising a PLA mutation, such as SEQ ID NO: 230) .
  • the anti-C5 antibody moiety comprises (or consists essentially of, or consists of) an anti-C5 scFv.
  • the anti-C5 antibody moiety is murine, chimeric, humanized, or human antibody.
  • the anti-C5 antibody moiety is monospecific.
  • the anti-C5 antibody moiety is multispecific.
  • the anti-C5 antibody moiety is monovalent. In some embodiments, the anti-C5 antibody moiety is multivalent.
  • the binding of the anti-C5 antibody moiety to C5 is pH-dependent, and wherein the anti-C5 antibody moiety binds more strongly to C5 at a neutral pH (e.g., about pH 7.4; such as that found in the blood) than it does at an acidic pH (e.g., about pH 5.8; such as that found in the endosome) .
  • a neutral pH e.g., about pH 7.4; such as that found in the blood
  • an acidic pH e.g., about pH 5.8; such as that found in the endosome
  • the binding affinity of the pH-dependent anti-C5 antibody moiety to C5 (e.g., human C5) at about pH 7.4 is at least about 3 times (such as at least about any of 4, 5, 6, 7, 8, 9, 10, or more) times higher than the binding affinity of the pH-dependent anti-C5 antibody moiety to C5 (e.g., human C5) at about pH 5.8.
  • the anti-C5 antibody moiety binds to an epitope in the ⁇ -chain of C5, and/or an epitope in the ⁇ -chain of C5.
  • the anti-C5 antibody moiety comprises (i) a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 279, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 280, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 281; and (ii) a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 282, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 283, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 284.
  • the anti-C5 antibody moiety comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 285, or a variant thereof that has at least about 80% (e.g., at least about any of 85%, 90%, 95%, 97%, 99%, or more) amino acid homology to SEQ ID NO: 285; and a VL domain comprising the amino acid sequence of SEQ ID NO: 286, or a variant thereof that has at least about 80% (e.g., at least about any of 85%, 90%, 95%, 97%, 99%, or more) amino acid homology to SEQ ID NO: 286.
  • the anti-C5 antibody moiety comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 285, and a VL domain comprising the amino acid sequence of SEQ ID NO: 286. In some embodiments, the anti-C5 antibody moiety is an scFv comprising the amino acid sequence of SEQ ID NO: 287 or 288.
  • the anti-C5 antibody moiety specifically binds C5 (e.g., human C5) with a K D of about 10 -7 M to about 10 -12 M, such as any of about 10 -7 M to about 10 -10 M, about 10 -8 M to about 10 -11 M, about 10 -9 M to about 10 -11 M, about 10 -8 M to about 10 -10 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, or about 10 -10 M to about 10 -11 M.
  • the anti-C5 antibody moiety specifically binds C5 (e.g., human C5) with a K D of about 1 ⁇ 10 -9 M to about 5 ⁇ 10 -9 M.
  • the anti-human FD/anti-C5 multispecific construct i) inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the TP, and ii) inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the AP.
  • the anti-human FD/anti-C5 multispecific construct inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) IgM-, mannan-, and LPS-induced C5b-9 deposition.
  • the anti-human FD/anti-C5 multispecific construct inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) LPS-induced C3b deposition. In some embodiments, the anti-human FD/anti-C5 multispecific construct does not inhibit (or inhibits at most about any of 10%, 5%, 2%, 1%, or less) IgM-induced or mannan-induced C3b deposition.
  • the anti-FD/anti-C5 multispecific construct inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) sheep RBC lysis and rabbit RBC lysis (e.g., induced by human serum) .
  • the activity of the complement pathway that is inhibited by an anti-human FD/anti-C5 multispecific construct is complement pathway activation induced by one or more of LPS, LOS, PAMPs, and DAMPs.
  • the activity of complement signaling that is inhibited by the anti-human FD/anti-C5 multispecific construct is the generation of C5a protein, the generation of C5b protein, and/or the formation of MAC.
  • the activity of the complement pathway that is inhibited by anti-human FD/anti-C5 multispecific construct is C5-dependent.
  • Methods of testing the activities of the multispecific construct described herein, such as inhibition of one or more of AP, CP, LP, and TP are well known in the art, including but not limited to, rabbit RBC lysis assay (e.g., induced by human serum) , sheep RBC lysis assay (e.g., induced by human serum) , IgM-induced C3b and/or C5b-9 deposition assay, mannan-induced C3b and/or C5b-9 deposition assay, or LPS-induced C3b and/or C5b-9 deposition assay.
  • rabbit RBC lysis assay e.g., induced by human serum
  • sheep RBC lysis assay e.g., induced by human serum
  • IgM-induced C3b and/or C5b-9 deposition assay e.g., mannan-induced C3b and/or C5b-9 deposition assay
  • one or more of the antibody moieties of the isolated anti-human FD antibody constructs (e.g., multispecific construct) or the anti-human FD antibody moieties of the present application is a chimeric antibody.
  • Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984) ) .
  • a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from mouse) and a human constant region.
  • a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • a chimeric antibody is a humanized antibody.
  • a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • HVRs e.g., CDRs, (or portions thereof) are derived from a non-human antibody
  • FRs or portions thereof
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived) , e.g., to restore or improve antibody specificity or affinity.
  • a non-human antibody e.g., the antibody from which the HVR residues are derived
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 15 1 : 2296 (1993) ) ; Framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 8 9 : 4285 (1992) ; and Presta et al. J. Immunol., 151: 2623 (1993) ) ; human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front.
  • one or more of the antibody moieties of the isolated anti-human FD antibody constructs (e.g., multispecific construct) of the present application is a human antibody (known as human domain antibody, or human dAb) .
  • Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) , Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008) , and Chen, Mol. Immunol. 47 (4) : 912-21 (2010) . Transgenic mice or rats capable of producing fully human single-domain antibodies (or dAb) are known in the art. See, e.g., US20090307787A1, U.S. Pat. No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.
  • Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge.
  • Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes.
  • the endogenous immunoglobulin loci have generally been inactivated.
  • Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.
  • Human antibodies can also be made by hybridoma-based methods.
  • Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (See, e.g., Kozbor J. Immunol., 133: 3001 (1984) ; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987) ; and Boerner et al., J. Immunol., 147: 86 (1991) ) .
  • Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl.
  • Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain.
  • antibody variants comprising one or more amino acid substitutions are included in the isolated anti-human FD antibody constructs (e.g., multispecific construct) or anti-human FD antibody moieties described herein.
  • Sites of interest for substitutional mutagenesis include the HVRs (or CDRs) and FRs.
  • Conservative substitutions are shown in Table B under the heading of “Preferred substitutions. ” More substantial changes are provided in Table B under the heading of “exemplary substitutions, ” and as further described below in reference to amino acid side chain classes.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
  • Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody) .
  • a parent antibody e.g., a humanized or human antibody
  • the resulting variant (s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity) .
  • Alterations may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR “hotspots, ” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008) ) , and/or SDRs (a-CDRs) , with the resulting variant VH or VL being tested for binding affinity.
  • HVR “hotspots, ” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008) )
  • SDRs a-CDRs
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis) .
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of HVR “hotspots” or CDRs.
  • each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • a residue or group of target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
  • a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
  • Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino-and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N-or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • one or more antibody moieties of the isolated anti-human FD antibody constructs is altered to increase or decrease the extent to which the construct is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the C H 2 domain of the Fc domain. See, e.g., Wright et al. TIBTECH 15: 26-32 (1997) .
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc) , galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in the antibody moiety may be made in order to create antibody variants with certain improved properties.
  • the antibody moiety (or the isolated anti-human FD antibody construct) has a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc domain.
  • the amount of fucose in such antibody may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc domain (EU numbering of Fc domain residues) ; however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L. ) ; US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd) .
  • Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004) ; Yamane-Ohnuki et al. Biotech.
  • Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986) ; US Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially at Example 11) , and knockout cell lines, such as alpha-1, 6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) ; Kanda, Y. et al., Biotechnol. Bioeng., 94 (4) : 680-688 (2006) ; and WO2003/085107) .
  • the antibody moiety (or the isolated anti-human FD antibody construct) has bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc domain of the antibody is bisected by GlcNAc.
  • Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al. ) ; US Patent No. 6, 602, 684 (Umana et al. ) ; and US 2005/0123546 (Umana et al. ) .
  • Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc domain are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.) ; WO 1998/58964 (Raju, S. ) ; and WO 1999/22764 (Raju, S. ) .
  • cysteine engineered antibody moieties e.g., “thioMAbs, ” in which one or more residues of one or more of the antibody moieties in an isolated anti-human FD antibody construct herein are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the antibody.
  • reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein.
  • any one or more of the following residues may be substituted with cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc domain.
  • Cysteine engineered antibody moieties may be generated as described, e.g., in U.S. Patent No. 7, 521, 541.
  • nucleic acid molecules encoding any of the isolated anti-human FD antibody constructs are also contemplated.
  • a nucleic acid e.g., isolated nucleic acid
  • mRNAs comprising such nucleic acids.
  • the mRNA is formulated in liposomes or lipid nanoparticles.
  • vectors comprising any of the nucleic acids (e.g., isolated nucleic acid) described herein, such as viral vectors (e.g., AAV vector or lentiviral vector) .
  • isolated host cell comprising or expressing (e.g., one or more polypeptide components of) any of the isolated anti-human FD antibody constructs (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) described herein. Also provided are isolated host cells comprising a nucleic acid encoding (e.g., one or more polypeptide components of) the isolated anti-human FD antibody construct described herein. Also provided are isolated host cells comprising a vector comprising a nucleic acid encoding (e.g., one or more polypeptide components of) the isolated anti-human FD antibody construct described herein.
  • the present application also includes variants to these nucleic acid sequences.
  • the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding (e.g., one or more polypeptide components of) the isolated anti-human FD antibody constructs (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) or various antibody moieties described herein under at least moderately stringent hybridization conditions.
  • the isolated anti-human FD antibody constructs e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs
  • various antibody moieties described herein under at least moderately stringent hybridization conditions.
  • the present application also provides vectors in which a nucleic acid of the present application is inserted.
  • the nucleic acid can be clone into a number of types of vectors.
  • the nucleic acid can be clone into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) , and in other virology and molecular biology manuals.
  • Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193) .
  • the nucleic acid encoding the isolated anti-human FD antibody construct comprises sequences or features in addition to the coding sequence encoding the isolated anti-human FD antibody construct.
  • the nucleic acid can be an mRNA (or a DNA sequence encoding the mRNA) comprising optional features that improve targeting of the isolated anti-human FD antibody construct encoded by the mRNA and to increase stability of the mRNA.
  • the mRNA encoding the anti-human FD antibody moiety or the isolated anti-human FD antibody construct comprises a nucleic acid sequence encoding a signal peptide.
  • Signal peptides are short peptides that may be present at the N-terminus or the C-terminus of a newly synthesized protein, that may function to properly translocate the protein.
  • the signal peptide assists with translocating the anti-human FD antibody moiety or the isolated anti-human FD antibody construct encoded by the coding sequence of the mRNA.
  • the signal peptide translocates the anti-human FD antibody moiety or the isolated anti-human FD antibody construct to the cellular membrane.
  • the mRNA comprises one or more untranslated regions (UTRs) .
  • the UTR of the mRNA may be involved in various regulatory aspects of gene expression. It should be understood that the UTRs (e.g., the 5’ UTRs and/or the 3’ UTRs) provided herein are examples, and that the mRNA may comprise any UTR from any gene. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide synthetic (e.g., artificial UTRs) which are not variants of wild type genes. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location.
  • UTR may be inverted, shortened, lengthened, made chimeric with one or more other 5’ UTRs or 3’ UTRs.
  • the term “altered” as it relates to a UTR sequence means that the UTR has been changed in some way in relation to a reference sequence.
  • a 3’ or 5’ UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3’ or 5’ ) comprise a variant UTR.
  • a double, triple or quadruple UTR such as a 5’ or 3’ UTR may be used.
  • a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. It is also within the scope of the present invention to have patterned UTRs.
  • patterned UTRs are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.
  • the UTRs provided herein may also include translation enhancer elements (TEE) .
  • TEE translation enhancer elements
  • the mRNA comprises a 5’ UTR.
  • the 5’ UTRs provided herein may be recognized by the ribosome, thereby allowing the ribosome to bind and initiate translation of the mRNA (e.g., translation of the coding sequence and/or nucleic acid encoding a signal peptide of the mRNA) .
  • the 5’ UTR is upstream from the coding sequence of the mRNA.
  • the mRNA comprises a 3’ UTR.
  • the 3’ UTRs provided herein may be involved in translation termination (e.g., translation of the coding sequence and/or nucleic acid encoding a signal peptide of the mRNA) , and can also be important for post-transcriptional modifications.
  • the 3’ UTR is downstream from the coding sequence of the mRNA.
  • the 3’ UTR immediately follows the translation stop codon of the coding sequence of the mRNA.
  • the mRNA comprises one or more stop codons before the 3’ UTR.
  • the mRNA comprises a 5’ UTR and a 3’ UTR, such as any of the 5’ UTRs and 3’ UTRs provided herein.
  • the 5’ UTR and the 3’ UTR are derived from the same species.
  • the 5’ UTR and the 3’ UTR are not derived from the same species.
  • the 5’ UTR is synthetic, and the 3’ UTR is not synthetic.
  • the 5’ UTR is not synthetic, and the 3’ UTR is synthetic.
  • the mRNA comprises one or more additional features, such as but not limited to a poly (A) sequence, one or more chemical modifications, a 5’ cap, or a combination thereof.
  • the mRNA comprises a poly (A) sequence (e.g., a polyadenylation sequence) .
  • Poly (A) sequences consist of multiple adenosine monophosphates in succession.
  • the poly (A) sequence is crucial for translation of the mRNA.
  • the poly (A) sequence is downstream of the coding sequence of the mRNA.
  • the poly (A) sequence is downstream of a 3’ UTR of the mRNA.
  • the poly (A) sequence has a length of about 50 nucleotides or longer, such as about 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, or longer. In some embodiments, the poly (A) sequence has a length of about 150 nucleotides or shorter, such as about 100 nucleotides, 90 nucleotides, 80 nucleotides, 70 nucleotides, 50 nucleotides, or shorter. In some embodiments, the poly (A) sequence has a length of about 105 nucleotides.
  • the present application also contemplates vectors comprising a nucleic acid encoding any of the isolated anti-human FD antibody constructs described herein (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) .
  • vector refers to DNA or RNA molecules that comprise a coding nucleotide sequence that can be transcribed into RNAs or expressed into proteins.
  • the vector contains one or more regulatory elements operably linked to the nucleotide sequence encoding the isolated anti-human FD antibody construct.
  • the coding nucleotide sequence in the construct can be transcribed or expressed.
  • the vector comprises a promoter that is operably linked to the coding nucleotide sequence, such that the promoter controls the transcription or expression of the coding nucleotide sequence.
  • a promoter may be positioned 5’ (upstream) of a coding nucleotide sequence under its control. The distance between the promoter and the coding sequence may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.
  • the vector comprises a 5’ UTR and/or a 3’ UTR that regulates the transcription or expression of the coding nucleotide sequence.
  • the promoter is a U6 promoter.
  • the promoter is a Poly II promoter as discussed in the sections described above.
  • Suitable vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular) ; nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) .
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the transcription or expression of coding nucleotide sequences to which they are operatively linked. Such vectors are referred to herein as “expression vectors” .
  • the expression vector is selected from the group consisting of a viral vector, a bacterial vector, and a mammalian cell vector.
  • Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses (AAV) , herpes viruses, and lentiviruses.
  • AAV adeno-associated viruses
  • a murine stem cell virus (MSCV) vector is used to express a desired nucleic acid. MSCV vectors have been demonstrated to efficiently express desired nucleic acids in cells.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193) .
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for transcription or expression of the nucleic acid in a host cell.
  • the recombinant expression vector includes one or more regulatory elements, which may be selected on the basis of the host cells to be used for transcription or expression, which is operatively linked to the nucleic acid sequence to be transcribed or expressed.
  • “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element (s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell) .
  • the vector is a recombinant adeno-associated virus (rAAV) vector.
  • the rAAV vector is a vector derived from an AAV serotype, including without limitation, AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV capsid serotype or the like.
  • the nucleic acid in the AAV comprises an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV capsid serotype or the like.
  • the nucleic acid in the AAV further encodes an isolated anti-human FD antibody construct (e.g., anti-human FD antibody moiety, anti-human FD/anti-C2 multispecific construct, or anti-human FD/anti-C5 multispecific construct) as described herein.
  • the vector is encapsidated in a rAAV particle.
  • the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV2 V708K, AAV2-HBKO, AAVDJ8, AAVPHP. B, AAVPHP. eB, AAVBR1, AAVHSC15, AAVHSC17, goat AAV, AAV1/AAV2 chimeric, bovine AAV, mouse AAV, or raAV2/HboV1 serotype capsid.
  • the vector is a lentiviral vector.
  • nucleic acids described herein or the vectors can be delivered directly into a host cell or an individual (e.g., human) for the purpose of gene therapy.
  • Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, electroporation, nanoparticles, exosomes, microvesicles, or gene-gun, naked DNA and artificial virions.
  • RNA or DNA viral based systems for the delivery of nucleic acids has high efficiency in targeting a virus to specific cells and trafficking the viral payload to the cellular nuclei.
  • a method of i) expressing any of the isolated anti-human FD antibody constructs e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs
  • a complement-mediated disease in an individual e.g., human
  • a viral vector such as an AAV or a lentiviral vector
  • a nucleic acid encoding any of the isolated anti-human FD antibody constructs described herein
  • the vector is an rAAV vector.
  • the construct is flanked by one or more AAV inverted terminal repeat (ITR) sequences.
  • the construct is flanked by two AAV ITRs.
  • the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs.
  • the AAV ITRs are AAV2 ITRs.
  • the vector further comprises a stuffer nucleic acid.
  • the stuffer nucleic acid is located upstream or downstream of the nucleic acid encoding the isolated anti-human FD antibody construct.
  • the vector is a self-complementary rAAV vector.
  • the vector comprises a first nucleic acid sequence encoding the anti-human FD antibody construct and a second nucleic acid sequence encoding a complement of the anti-human FD antibody construct, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length.
  • the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal resolution sequence.
  • the vector is encapsidated in a rAAV particle.
  • the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV2 V708K, AAV2-HBKO, AAVDJ8, AAVPHP.
  • B AAVPHP.
  • the vector e.g., viral vector
  • the vector allows delivery of the nucleic acid to the eye.
  • Suitable viral vectors for eye delivery include, but are not limited to, AAV2, AAV4, AAV5, and AAV8.
  • the delivery method includes, but is not limited to nanoparticles, nanospheres, microparticles, microspheres, liposomes, nonbiodegradable implantable devices, biodegradable implantable devices, microcatheters, microneedles, micropumps, or port drug delivery system.
  • kits for inhibiting complement activation or reducing the activity of a complement system and/or treating diseases or conditions (such as complement-mediated diseases or disorders) in an individual (e.g., human) .
  • the methods comprise administering an effective amount of an anti-human FD antibody construct described herein (or pharmaceutical composition thereof) , a nucleic acid encoding the anti-human FD antibody construct, or a vector (e.g., an AAV vector or lentiviral vector) comprising a nucleic acid encoding the anti-human FD antibody construct, to an individual (e.g., human) .
  • the individual is a mammal (e.g., human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. ) .
  • the individual is a human.
  • the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc.
  • the complement-mediated disease or disorder is an AP-mediated disease or AP-mediated disorder.
  • AP-mediated pathologies and conditions that can be treated with the anti-human FD antibody constructs (or pharmaceutical compositions thereof) and methods of the invention include, but are not limited to, macular degeneration (MD) , age-related macular degeneration (AMD) , ischemia reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, post-surgery systemic inflammatory syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD) , paroxysmal nocturnal hemoglobinuria (PNH) syndrome, autoimmune hemolytic anemia (AIHA) , Gaucher disease, myasthenia gravis, neuromyelitis optica (NMO) , multiple sclerosis, delayed graft function, antibody-mediated rejection, atypical hemolytic uremic syndrome (aHUS) , central retinal vein occlusion (CRVO) , central retinal artery occlusion (CRAO) , epidermolysis bullosa,
  • a method of treating a complement-mediated disease in an individual comprising administering to the individual an effective amount of any of anti-human FD antibody constructs (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) described herein (or pharmaceutical composition thereof) .
  • anti-human FD antibody constructs e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs
  • a method of treating a complement-mediated disease in an individual comprising administering to the individual an effective amount of a vector (e.g., viral vector, such as AAV vector or lentiviral vector) encoding any of the anti-human FD antibody constructs (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) described herein (or pharmaceutical composition thereof) .
  • a vector e.g., viral vector, such as AAV vector or lentiviral vector
  • anti-human FD antibody constructs e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs
  • the anti-human FD antibody construct is a multispecific construct (e.g., an anti-human FD/anti-C2 multispecific construct, or an anti-human FD/anti-C5 multispecific construct) , or pharmaceutical compositions thereof) .
  • a method of treating a complement-mediated disease in an individual comprising administering to the individual an effective amount of a vector (e.g., viral vector, such as AAV vector or lentiviral vector) encoding any of the multispecific anti-human FD antibody constructs described herein or pharmaceutical compositions thereof.
  • the complement-mediated disease is AP-and/or FD-mediated disease.
  • the complement-mediated disease is CP-and/or LP-and/or C2-mediated disease (e.g., anti-human FD/anti-C2 multispecific constructs can be used) .
  • the complement-mediated disease is TP-and/or C5-mediated disease (e.g., anti-human FD/anti-C5 multispecific constructs can be used) .
  • the complement-mediated disease is an eye disease.
  • the eye disease is selected from the group selecting of: macular degeneration, AMD, glaucoma, diabetic retinopathy, autoimmune uveitis, dry eye disorder, neuromyelitis optica (NMO) , central retinal vein occlusion (CRVO) , and subcorneal pustular dermatosis, and any combinations thereof.
  • the anti-human FD antibody construct (or pharmaceutical composition thereof) or the vector (e.g., viral vector) encoding the anti-human FD antibody construct (or pharmaceutical composition thereof) , is directly administered to the eye.
  • anti-human FD antibody constructs provided herein can be used in combination with other treatment modalities, such as, for example anti-inflammatory therapies, and the like.
  • anti-inflammatory therapies that can be used in combination with the anti-human FD antibody constructs or methods of the invention include, for example, therapies that employ steroidal drugs, as well as therapies that employ non-steroidal drugs.
  • a method of inhibiting complement activation or reducing the activity of a complement system comprising administering (such as systemically administering, for example by subcutaneous or intravenous administration) to the individual an effective amount of: i) an anti-human FD antibody construct, such as any of the anti-human FD antibody constructs described herein (e.g., anti-human FD antibody moiety, anti-human FD/anti-C2 multispecific construct, or anti-human FD/anti-C5 multispecific construct) , or a pharmaceutical composition thereof; or ii) a vector (e.g., an AAV vector or lentiviral vector) comprising a nucleic acid encoding an anti-human FD antibody construct, or a pharmaceutical composition thereof.
  • a complement system e.g., activity of AP
  • administering such as systemically administering, for example by subcutaneous or intravenous administration
  • administering such as systemically administering, for example by subcutaneous or intravenous administration
  • administering such as systemically administering, for example
  • the anti-human FD antibody construct, the vector, or pharmaceutical composition thereof is administered by subcutaneous administration.
  • the method inhibits complement activation (e.g., AP activation) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.
  • the method reduces the activity of a complement system (e.g., activity of AP) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.
  • Dosages and desired drug concentrations of pharmaceutical compositions of the present application may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics, ” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46.
  • dosages which may be administered in a method of the invention to a subject range in amount from 0.5 ⁇ g to about 50 mg per kilogram of body weight of the subject. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of subject and type of disease state being treated, the age of the subject and the route of administration.
  • the anti-human FD antibody construct or a nucleic acid (e.g., viral vector) encoding thereof (or pharmaceutical composition thereof) is administered for a single time. In some embodiments, the anti-human FD antibody construct or a nucleic acid (e.g., viral vector) encoding thereof (or pharmaceutical composition thereof) is administered for multiple times (such as any of 2, 3, 4, 5, 6, or more times) . In some embodiments, the anti-human FD antibody construct or a nucleic acid (e.g., viral vector) encoding thereof (or pharmaceutical composition thereof) is administered once per week, once 2 weeks, once per month, once per 2 months, once per 3 months, once per 6 months, once per year, or the like.
  • the anti-human FD antibody construct or a nucleic acid (e.g., viral vector) encoding thereof (or pharmaceutical composition thereof) is administered at a dose of between about 0.1 mg/dose and about 20 mg/dose. In some embodiments, the anti-human FD antibody construct or a nucleic acid (e.g., viral vector) encoding thereof (or pharmaceutical composition thereof) is administered to the individual (such as to the eye of the individual) once.
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, include but not limited to, the type and severity of the disease being treated, the type and age of the subject, etc.
  • the anti-human FD antibody construct is formulated in a reconstituted and liquid formulation.
  • Parenteral administration of the anti-human FD antibody construct or a nucleic acid (e.g., viral vector) encoding thereof (or pharmaceutical composition thereof) includes any route of administration characterized by physical breaching of a tissue of an individual and administration of the pharmaceutical composition through the breach in the tissue.
  • Parental administration can be local, regional or systemic.
  • Parenteral administration thus includes, but is not limited to, administration by injection of the composition, by application of the composition through a surgical incision or through a tissue-penetrating non-surgical wound, and the like.
  • Parenteral administration is contemplated to include, but is not limited to, intravenous, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, or intrasternal injection.
  • the vector e.g., plasmid or viral vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the delivery is via intravenous, transdermal, mucosal, or other delivery methods. Such delivery may be either via a single dose, or multiple doses.
  • the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choice, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc..
  • the anti-human FD antibody construct or nucleic acid (e.g., viral vector) encoding thereof of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a unit dose is discrete amount of the anti-human FD antibody construct or the nucleic acid (e.g., viral vector) encoding thereof comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to an individual or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions of the invention will vary, depending upon the identity, size, and condition of the individual treated and further depending upon the route by which the composition is to be administered.
  • the anti-human FD antibody constructs described herein can be made by introducing a nucleic acid into a host cell, allowing expression of the protein, and purifying the protein from the host cell extract or supernatant.
  • Methods of making proteins comprising antibody moieties and constructing nucleic acids or vectors encoding thereof, as well as purifying proteins comprising antibody moieties are known in the art. E.g., see US20220204602.
  • Proteins comprising antibody moieties can be obtained using methods known in the art, such as by immunizing a non-human mammal and obtaining hybridomas therefrom, or by cloning a library of antibodies using molecular biology techniques known in the art and subsequence selection or by using phage display. Nucleic acid constructs encoding any one of the antibodies or antigen-binding fragments described herein, vectors, and host cells for preparation are also provided.
  • polypeptide portion of the constructs or targeting moieties described herein may be prepared by any of the known protein expression and purification methods in the art.
  • DNA sequence encoding the polypeptide portion of the constructs or targeting moieties can be fully synthesized. After obtaining such sequence, it is cloned into a suitable expression vector, then transfected into a suitable host cell. The transfected host cells are cultured, and the supernatant is harvested and purified to obtain the polypeptide portion of the constructs or targeting moieties described herein.
  • polypeptide portion of the constructs or targeting moieties described herein can also be obtained by conventional immunization methods, such as by immunizing a mammal (e.g., mouse, llama) , and obtaining the constructs or targeting moieties from the serum; or from a hybridoma, such as by fusing B cells from lymph nodes and/or spleen of immunized animal with myeloma cells. Purification can be followed.
  • immunization methods such as by immunizing a mammal (e.g., mouse, llama) , and obtaining the constructs or targeting moieties from the serum; or from a hybridoma, such as by fusing B cells from lymph nodes and/or spleen of immunized animal with myeloma cells. Purification can be followed.
  • Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975) , or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567) .
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) .
  • the hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E.
  • antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990) . Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries.
  • the invention is a cell or cell line (such as host cells) that produces at least one of the anti-human FD antibody constructs described herein.
  • the cell or cell line is a genetically modified cell.
  • the cell or cell line is a hybridoma.
  • Hybrid cells are generally produced from mass fusions between murine splenocytes, which are highly enriched for B-lymphocytes, and myeloma “fusion partner cells” (Alberts et al., Molecular Biology of the Cell (Garland Publishing, Inc. 1994) ; Harlow et al., Antibodies. A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988) .
  • the cells in the fusion are subsequently distributed into pools that can be analyzed for the production of antibodies with the desired specificity. Pools that test positive can be further subdivided until single cell clones are identified that produce antibodies of the desired specificity. Antibodies produced by such clones are referred to as monoclonal antibodies.
  • Nucleic acids encoding the anti-human FD antibody constructs described herein, or the heavy chain or light chain or fragments thereof, can be obtained and used in accordance with recombinant nucleic acid techniques for the production of the specific immunoglobulin, immunoglobulin chain, or a fragment or variant thereof, in a variety of host cells or in an in vitro translation system.
  • the antibody-encoding nucleic acids, or fragments thereof can be placed into suitable prokaryotic or eukaryotic vectors, e.g., expression vectors, and introduced into a suitable host cell by an appropriate method, e.g., transformation, transfection, electroporation, infection, such that the nucleic acid is operably linked to one or more expression control elements, e.g., in the vector or integrated into the host cell genome.
  • suitable prokaryotic or eukaryotic vectors e.g., expression vectors
  • suitable host cell e.g., transformation, transfection, electroporation, infection
  • the invention provides isolated nucleic acid molecules comprising a nucleic acid sequence encoding a heavy chain and/or a light chain, as well as fragments thereof.
  • a nucleic acid molecule comprising sequences encoding both the light and heavy chain, or fragments thereof, can be engineered to contain a synthetic signal sequence for secretion of the antibody, or fragment, when produced in a cell.
  • the nucleic acid molecule can contain specific DNA links which allow for the insertion of other antibody sequences and maintain the translational reading frame so to not alter the amino acids normally found in antibody sequences.
  • the antibody-encoding nucleic acids, or fragments thereof, can be subjected to various recombinant nucleic acid techniques known to those skilled in the art such as site-directed mutagenesis.
  • the expression vectors may contain a variety of elements for controlling expression, including without limitation, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be selected as appropriate by a person of ordinary skill in the art.
  • a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression may be employed.
  • Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2012) .
  • the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and fragments thereof.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) , human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney virus promoter, the avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the muscle creatine promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • Moloney virus promoter the avian
  • inducible promoters are also contemplated as part of the invention.
  • the use of an inducible promoter in the invention provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • tissue-specific promoter or cell-type specific promoter which is a promoter that is active only in a desired tissue or cell.
  • Tissue-specific promoters are well known in the art and include, but are not limited to, the HER-2 promoter and the PSA associated promoter sequences.
  • Selectable markers may be selected to allow selection of the host cells inserted with the vector from those not, for example, the selectable markers may be genes that confer antibiotic resistance.
  • the two or more polypeptides of a construct or a targeting moiety are encoded by a single vector. In some embodiments, the two or more polypeptides of a construct or a targeting moiety are encoded by two or more vectors.
  • Host cells containing the vector may be useful in expression or cloning of the isolated nucleic acids. Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. The expression of antibodies and antigen-binding fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pluckthun, A. BioTechnology 9: 545-551 (1991) .
  • eukaryotic cells in culture are also available to those skilled in the art as an option for production of antibodies or antigen-binding fragments thereof, see recent reviews, for example Ref, M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560.
  • Higher eukaryotic cells in particular, those derived from multicellular organisms can be used for expression of glycosylated polypeptides. Suitable higher eukaryotic cells include, without limitation, invertebrate cells and insect cells, and vertebrate cells.
  • the host cell is E. coli.
  • the host cell is a Chinese hamster ovary (CHO) cell or an HEK293 cell.
  • the vector can be introduced to the host cell using any suitable methods known in the art, including, but not limited to, DEAE-dextran mediated delivery, calcium phosphate precipitate method, cationic lipids mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of a vector of interest are well known in the art.
  • the host cells comprise two or more vectors each encoding a polypeptide of any of the protein constructs or targeting moieties described herein.
  • the host cells comprise a single vector comprising isolated nucleic acids encoding two or more polypeptides of any of the protein constructs or targeting moieties described herein.
  • the present application provides methods of making the polypeptide portion of any of the isolated anti-human FD antibody constructs described herein, comprising i) culturing a host cell comprising any of the isolated nucleic acids described herein or any of the vectors described herein, or any of the isolated host cells described herein, under a condition suitable for the expression of the polypeptide portion of any of the anti-human FD antibody constructs described herein, and ii) obtaining the expressed polypeptide portion of any of the anti-human FD antibody constructs described herein from said host cell (e.g., from the cell culture) .
  • the isolated host cells are cultured under conditions that allow expression of the isolated nucleic acids inserted in the vectors.
  • Suitable conditions for expression of polynucleotides may include, without limitation, suitable medium, suitable density of host cells in the culture medium, presence of necessary nutrients, presence of supplemental factors, suitable temperatures and humidity, and absence of microorganism contaminants.
  • suitable medium suitable density of host cells in the culture medium
  • suitable temperatures and humidity suitable temperatures and humidity, and absence of microorganism contaminants.
  • a person with ordinary skill in the art can select the suitable conditions as appropriate for the purpose of the expression.
  • the methods of making further comprises purifying any of the obtained polypeptide portion of any of the anti-human FD antibody constructs described herein.
  • the polypeptides expressed by the host cell can assemble together (e.g., form a polypeptide complex such as a dimer) and produce any of the polypeptide portion of any of the anti-human FD antibody constructs described herein.
  • the polypeptide complex may be formed inside the host cell.
  • the polypeptide complex may be formed inside the host cell with the aid of relevant enzymes and/or cofactors.
  • the polypeptide complex may be secreted out of the cell.
  • individual polypeptides may be secreted out of the host cell then form a polypeptide complex outside of the host cell.
  • the two or more polypeptides of any of the anti-human FD antibody constructs described herein may be separately expressed and allowed to form (e.g., dimerize) a protein complex under suitable conditions.
  • the first polypeptide and the second polypeptide may be combined in a suitable buffer and allow the first protein monomer and the second protein monomer to dimerize through appropriate interactions such as hydrophobic interactions.
  • the first polypeptide and the second polypeptide may be combined in a suitable buffer containing an enzyme and/or a cofactor which can promote the dimerization of the first polypeptide and the second polypeptide.
  • the first polypeptide and the second polypeptide may be combined in a suitable vehicle and allow them to react with each other in the presence of a suitable reagent and/or catalyst.
  • the expressed polypeptide (s) and/or the polypeptide complex can be collected using any suitable methods.
  • the polypeptide (s) and/or the polypeptide complex can be expressed intracellularly, in the periplasmic space or be secreted outside of the cell into the medium. If the polypeptide and/or the polypeptide complex are expressed intracellularly, the host cells containing the polypeptide and/or the polypeptide complex may be lysed and polypeptide and/or the polypeptide complex may be isolated from the lysate by removing the unwanted debris by centrifugation or ultrafiltration. If the polypeptide and/or the polypeptide complex is secreted into periplasmic space of E.
  • the cell paste may be thawed in the presence of agents such as sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) for about 30 min, and cell debris can be removed by centrifugation (Carter et al., BioTechnology 10: 163-167 (1992) ) .
  • agents such as sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) for about 30 min, and cell debris can be removed by centrifugation (Carter et al., BioTechnology 10: 163-167 (1992) ) .
  • the supernatant of the cell culture may be collected and concentrated using a commercially available protein concentration filter, for example, an Amincon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor and/or an antibiotic may be included in the collection and concentration steps to inhibit protein degradation and/or growth of contaminated micro
  • the expressed polypeptide (s) and/or the polypeptide complex can be further purified by a suitable method, such as without limitation, affinity chromatography, hydroxylapatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, ion exchange fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column) , chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation (see, for review, Bonner, P. L., Protein purification, published by Taylor &Francis. 2007; Janson, J. C., et al., Protein purification: principles, high resolution methods and applications, published by Wiley-VCH, 1998) .
  • a suitable method such as without limitation, affinity chromatography, hydroxylapatite chromatography
  • polypeptides and/or polypeptide complexes can be purified by affinity chromatography.
  • protein A chromatography or protein A/G (fusion protein of protein A and protein G) chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising a component derived from antibody CH2 domain and/or CH3 domain (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983) ) ; Zettlit, K. A., Antibody Engineering, Part V, 531-535, 2010) .
  • protein G chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising IgG ⁇ 3 heavy chain (Guss et al., EMBO J. 5: 1567 1575 (1986) ) .
  • protein L chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising ⁇ light chain (Sudhir, P., Antigen engineering protocols, Chapter 26, published by Humana Press, 1995; Nilson, B. H. K. et al., J. Biol. Chem., 267, 2234-2239 (1992) ) .
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the bakerbond ABX resin J.T. Baker, Phillipsburg, N.J.
  • MabSelect SuRe is used for purification.
  • the method further comprises analyzing the purified protein product, such as by SDS-PAGE and/or SEC-HPLC.
  • the antibody moieties described herein can be obtained, for example, by molecular cloning the antibody sequences and identifying antibodies suitable for making the anti-human FD antibody constructs (e.g., anti-human FD antibody moiety, or multispecific construct) .
  • Binding affinity and specificity of the antibody or antibody moieties described herein can be determined experimentally by methods known in the art.
  • the binding of an antibody to a protein antigen can be detected and/or quantified using a variety of techniques such as, but not limited to, Western blot, dot blot, surface plasmon resonance (SPR) method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J. ) , Bio-Layer Interferometry (BLI) (e.g.
  • Binding of an anti-FD antibody moiety to FD, or the binding of the second antibody moiety to the complement protein can be measured by using a variety of techniques such as, but not limited to, Western blot, dot blot, SPR method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J. ) , BLI (e.g. Octet system, ForteBio) , RIA, ECL, IRMA, EIA, peptide scans, and ELISA. See, e.g., Benny K. C.
  • the activity of the anti-C2 antibody can also be carried out by biological assays. Methods for determining whether a particular antibody described herein inhibits C2 cleavage are known in the art. Inhibition of human C2 can reduce the cell-lysing ability of complement in a subject’s body fluids. Such reductions of the cell-lysing ability of complement present in the body fluid (s) can be measured by methods well known in the art such as, for example, by a conventional hemolytic assay such as the hemolysis assay in chicken erythrocyte hemolysis method as described in, e.g., Hillmen et al. (2004) N Engl. J Med 350 (6) : 552.
  • sheep erythrocytes coated with hemolysin or chicken erythrocytes sensitized with anti-chicken erythrocyte antibody are used as target cells.
  • the percentage of lysis is normalized by considering 100%lysis equal to the lysis occurring in the absence of the inhibitor.
  • IgM-mediated C3b formation assays can be used to determine the inhibitory activity of the anti-C2 antibodies on classical pathway induced C3b formation. Plate-bound and immobilized IgM can resemble immune complex and activate the classical pathway complement. In this assay, human IgM is coated onto ELISA plate, and after washing with PBS buffer, normal human serum (1%or 50%) in GVB++ buffer is added to the plate and incubated at room temperature for 60 min. After washing, the amount of C3b generated and bound to the plate is detected by anti-C3b/iC3b antibodies.
  • MBL mannan-binding lectin
  • MASPs mannan-binding lectin associated serine proteases
  • Methods for determining whether a candidate compound inhibits the cleavage of human C2 into forms C2a and C2b are known in the art and described in, e.g., Thomas et al. (1996) Mol Immunol 33 (17-18) : 1389-401; and Evans et al. (1995) Mol Immunol 32 (16) : 1183-95.
  • concentration and/or physiologic activity of C2a and C2b in a body fluid can be measured by methods well known in the art.
  • Methods for measuring C2a concentration or activity include, e.g., biolayer interferometry, RIAs, or ELISAs (see, e.g., Wurzner et al. (1991) Complement Inflamm 8: 328-340) .
  • candidate agents capable of inhibiting human complement component C2 can be screened.
  • the activity of the anti-C5 antibody can also be carried out by biological assays.
  • Methods for determining the inhibitory activity of anti-C5 antibodies on the classical pathway are known in the art and described in, e.g., Zelek et al, (2020) Immunology. 161 (2) : 103-113.
  • Inhibition of C5 prevents the generation of C5a and C5b, which subsequently inhibits leukocyte and platelet activation.
  • anti-C5 complement inhibition during extracorporeal circulation can be measured by methods known in the art. See, for example, Rinder et al. (1995) J Clin Invest. 96 (3) : 1564-1572.
  • Generation of C5a can be measured by methods known in the art. See, for example, Volokina et al. (2015) Blood. 126 (2) : 278-279.
  • compositions comprising i) any one of the anti-human FD antibody constructs (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) , or any of the isolated nucleic acids encoding the anti-human FD antibody constructs, or any of the vectors (e.g., a viral vector such as an AAV) comprising such isolated nucleic acids, or any mRNA comprising or encoded by such isolated nucleic acids, and ii) an optional pharmaceutically acceptable carrier.
  • the anti-human FD antibody constructs e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs
  • isolated nucleic acids encoding the anti-human FD antibody constructs or any of the vectors (e.g., a viral vector such as an AAV) comprising such isolated nu
  • Suitable formulations of the anti-human FD antibody construct described herein, a nucleic acid encoding the anti-human FD antibody construct, or a vector (e.g., viral vector) comprising a nucleic acid encoding the anti-human FD antibody construct, can be obtained by mixing the anti-human FD antibody construct (or the nucleic acid encoding the anti-human FD antibody construct, or the vector comprising a nucleic acid encoding the anti-human FD antibody construct) having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ) .
  • the formulations can be lyophilized formulations or aqueous solutions. In some embodiments, the formulation is suitable for application to the eye.
  • the pharmaceutical composition further comprises additional ingredients.
  • Additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • Additional ingredients which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA) , which is incorporated herein by reference. Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall (e.g., surfactant, such as polysorbate (e.g., polysorbate 80) or poloxamer) .
  • surfactant such as polysorbate (e.g., polysorbate 80) or poloxamer
  • acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine,
  • the pharmaceutical compositions In order for the pharmaceutical compositions to be used for in vivo administration, they must be sterile.
  • the pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes.
  • the pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the formulations of the pharmaceutical compositions may be prepared by any method known or hereafter developed in the art of pharmacology. Preparations include but are not limited to, bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-or multi-dose unit.
  • the pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1, 3-butane diol, for example.
  • a non-toxic parenterally-acceptable diluent or solvent such as water or 1, 3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides.
  • Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems.
  • Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for parenteral, intramuscular, intradermal, subcutaneous, or intravenous routes of administration.
  • Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
  • the pharmaceutical composition is for eye (e.g., intraocular) administration.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a unit dose is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to an individual or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • kits comprising any one of the anti-human FD antibody constructs (or the nucleic acid encoding the anti-human FD antibody construct, or the vector (e.g., viral vector) comprising a nucleic acid encoding the anti-human FD antibody construct) described herein.
  • kits of the present application are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags) , and the like. Kits may optionally provide additional components such as buffers and interpretative information.
  • the present application thus also provides articles of manufacture.
  • the article of manufacture can comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include vials (such as sealed vials) , bottles, jars, flexible packaging, and the like.
  • the container holds a composition, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) .
  • a mouse anti-human FD IgG4 monoclonal antibody comprising the VH amino acid sequence of SEQ ID NO: 140 and VL amino acid sequence of SEQ ID NO: 141 was used to generate humanized anti-human FD antibodies and variants thereof.
  • CDR-grafting was used to generate humanized anti-human FD antibodies.
  • the conserved N-linked glycosylation sequence motif in H-CDR2 was removed by site-specific mutation.
  • the humanized 118A1 with (indicated with “p2” ) or without the H-CDR2 mutation were expressed and purified from CHO cells. The binding affinities of the recombinant, humanized anti-FD antibodies were determined using SPR.
  • humanized anti-FD antibodies showed the same or less than 2-fold reduction of binding K D compared to the murine “parental” anti-FD antibody 118A1 (achimeric form of 118A1 clone with human Fc served as reference control) .
  • the binding kinetics and affinity of the humanized anti-FD antibodies to human FD were determined by SPR (Table 1) . Also, removing conserved N-linked glycosylation sequence motif in H-CDR2 did not affect much of the binding affinity of the humanized antibodies (e.g., compare z16 and z16-p2, compare z17 and z17-p2) .
  • Clone z16-p2 was selected for further sequence engineering for affinity maturation.
  • each amino acid of the six CDRs of the anti-FD scFv clone comprising VH and VL sequences of z16-p2 was individually mutated to all 20 amino acids using site-directed mutagenesis.
  • DNA primers containing an NNS codon encoding 20 amino acids were used to introduce mutations at each targeted CDR position.
  • the degenerate primers were used in site-directed mutagenesis reactions. Briefly, each degenerate primer was phosphorylated.
  • the PCR products were purified, then electroporated into BL21 for colony formation and production of scFv fragments.
  • One clone (1L8-B6 ⁇ 3L10E9 (mAb-18) ) has two combined CDR mutations included SEQ ID NOs: 124 and 126) .
  • SEQ ID NOs: 124 and 126) As shown in FIG. 1, FIG. 2A, and FIG. 2B, compared to the parental humanized z16-p2 clone, several clones with single amino acid substitutions significantly improved binding to both human FD and cynomolgus FD.
  • z16-p2 served as reference anti-human FD control and an irrelevant scFv clone served as negative control.
  • a histidine substitution was introduced to each position of the six CDRs of clone mAb-42 (IgG4 format) by mutagenesis, a method known as histidine scanning.
  • HEK293 cells were transiently transfected with the expression plasmids encoding a mutated mAb-42 heavy chain and a wild-type light chain, and cultivated with serum-free medium in shaking flasks for 5-7 days.
  • the light chain Ab variants were obtained in a similar way by co-transfecting HEK293 cells with expression plasmids encoding a mutated light chain and a wild-type heavy chain.
  • the expression supernatant was collected by centrifugation for binding characterization. IgG from the expression supernatant was used to measure FD binding under either pH 7.4 or pH 5.8 using Gator TM (Probe Life, Inc. ) .
  • the pH-differential binding variants with multiple histidine mutations were further created by co-transfecting HEK293 cells with expression plasmids encoding heavy or light chain carrying binding beneficial, single histidine mutation.
  • Variants with multiple binding beneficial histidine mutations within an antibody chain were created by mutagenesis by introducing the beneficial mutations into a same antibody chain.
  • the pH-dependent binding variants were purified from the expression supernatant by Protein-Aaffinity chromatography for affinity determinations.
  • Purified antibodies were dialyzed into 20 mM histidine buffer with 150 mM NaCl and quantitated by Nanodrop (Thermo Fisher Scientific, Inc., USA) before affinity measurement using Gator TM (Probe Life, Inc. ) .
  • the binding sensorgrams of exemplary pH-binding engineered anti-FD variants to human and cyno FD are shown in FIG. 4.
  • the binding affinity and pH-dependent binding activities of representative anti-FD variants are shown in Table 4.
  • mAb-42 served as control.
  • pH-binding engineered anti-FD variants maintained similar binding dissociation constant at pH 7.4 to human and/or cyno FD, but had reduced binding dissociation constant at pH 5.8, compared to parental mAb-42.
  • Sequences of 5 of the pH-binding engineered variants are shown in Table 5.
  • Rabbit red blood cells were prepared by centrifuging buffer diluted with rabbit blood at 1500 rpm for 5 min at 4°C. After washing, the cells were resuspended in AP buffer, and the cell density was adjusted to 4 ⁇ 10 8 cells/ml. Serially diluted anti-FD antibodies were added to the assay wells containing Normal Human Serum (NHS) to a final concentration of 50%or 30%. Distilled water or EDTA was used as the positive control (PC) or negative control (NC) , respectively. Rabbit RBC was added at last to the antibody and NHS mixture (1 ⁇ 10 7 cells per well) and incubated at 37°C for 40 min.
  • NHS Normal Human Serum
  • the lysis reaction was stopped by addition of ice-cold 40 mM EDTA in PBS. The reaction mixture was centrifuged for 5 min at 1500 rpm. The supernatant was collected and transferred to a flat-bottom 96-well plate. Color of the supernatant was measured at OD 405 nm on a Microplate reader.
  • LPS-C3b ELISA was used to assess the inhibition of the alternative pathway by anti-FD antibodies.
  • NHS samples final concentration 10%
  • AP complement activation was stopped by adding 100 ⁇ l/well pre-chilled 40 mM EDTA.
  • Anti-FD affinity-matured IgG4 variants were evaluated for inhibition of NHS-induced rabbit RBC lysis (FIG. 5) and LPS-induced C3b deposition (FIG. 6) . As shown in FIG. 5, all the representative affinity-matured anti-FD IgG4 variants completely inhibited 50%NHS-induced rabbit RBC lysis, and exhibited stronger inhibition than the parental z16-p2 clone.
  • mAb-42 was the most potent mAb with an IC50 value of 1.49 ⁇ g/ml (Table 6) . The same constructs were assessed for inhibition of the alternative pathway using an LPS-induced C3b deposition assay. All constructs inhibited the alternative pathway (FIG. 6) , and the affinity-matured variants showed stronger AP inhibition than the parental z16-p2 clone.
  • the corresponding IC50 values for both lysis and deposition assay are listed in Table 6.
  • Anti-FD pH-binding engineered variants were also evaluated for inhibition of NHS-induced rabbit RBC lysis (FIG. 7) and LPS-induced C3b deposition (FIG. 8) . As shown in FIG. 7, all pH-binding engineered variants completely inhibited 30%NHS-induced rabbit RBC lysis (FIG. 7) .
  • the IC50 values of inhibition of rRBC lysis by mAb-42-2536 and mAb-42-2821 in 30%NHS were 0.71 ⁇ g/ml and 0.74 ⁇ g/ml, respectively, which were comparable to the parental mAb-42 clone. All pH-dependent constructs also inhibited the alternative pathway with similar potency as the parental mAb-42 clone (FIG.
  • Example 5 Combined pH-dependent target binding and optimized FcRn binding at pH 7.4 for effective target removal from circulation
  • a panel of FcRn binding mutations that increase FcRn binding at pH 5.8 and at pH 7.4 were engineered into an exemplary mAb-42 pH-dependent anti-FD variant from Example 3 (herein also referred to as “mAb-42pH” ) , to reduce total FD concentration in serum.
  • the affinity-matured mAb-42 clone was engineered with pH-dependent FD binding mutations (mAb42-pH) and/or with various FcRn binding mutations that show increased FcRn binding affinity at both pH 5.8 and 7.4 (FIG. 9) .
  • All antibodies were in an IgG4 backbone with an S228P mutation (herein referred to as “IgG4P” , SEQ ID NO: 132) in the hinge region for improved stability.
  • the variants containing different FcRn binding mutations were tested for antigen sweeping activity.
  • “LA” variant SEQ ID NO: 133) : S228P, M428L+N434A.
  • YPY S228P, M252Y+V308P+N434Y.
  • YEY S228P, M252Y+N286E+N434Y.
  • N3E S228P, L432C+H433S+N434W+Y436L+T437CE.
  • N3 S228P, L432C+H433S+N434W+Y436L+T437C.
  • YTE SEQ ID NO: 138
  • M252Y+S254T+T256E S228P
  • M252Y+S254T+T256E+L432E+H433R+N434F+Y436R+T437Q S228P, M252Y+S254T+T256E+L432E+H433R+N434F+Y436R+T437Q.
  • FcRn binding affinities of mAb-42pH variants containing various FcRn binding mutations were determined using BLI (Gator TM , Probe Life Inc., USA) . Briefly, biotinylated FcRn (AcroBio) was captured on streptavidin (SA) -coupled sensor probes and equilibrated in kinetic (K) buffer (phosphate-buffered saline containing 0.02%bovine serum albumin and 0.002%Tween 20, pH 7.4) . Antibodies were captured onto the SA-sensors by dipping them into 200 ⁇ L of transfection supernatant for 600 seconds at pH 7.4.
  • K kinetic buffer
  • the biosensors were then incubated with human FD prepared in the K buffer at pH 7.4 (40 nM) for 600 seconds followed by a 600-second dissociation period in K Buffer, pH 7.4 or pH 5.8.
  • the data were processed and analyzed by the Gator evaluation software.
  • the binding kinetics of the FcRn binding variants are shown in FIG. 10 and the binding affinities of the FcRn binding variants under pH 7.4 and pH 5.8 are summarized in Table 8.
  • mAb-42pH with S228P mutation in IgG4 served as control ( “mAb42pH-IgG4P” ) .
  • mAb42pH-IgG4P showed expected FcRn binding affinity under pH 5.8 but no detectable binding ( “N/A” ) under pH 7.4.
  • a humanized FD/FcRn transgenic mouse model was generated.
  • Mature human FD (hFD) cDNA was cloned into the pCAGGS vector at an EcoRI restriction site by the infusion cloning method (kit from Takara) .
  • the entire expression cassette with CMV IE enhancer to rabbit beta globin polyA was ligated into an AAV vector with TBG promoter at a BstXI restriction site. Positive clones were screened by Sma I digestion.
  • Super-coiled endotoxin-free plasmid was prepared by the EndoFree plasmid kit (Cat. No. 12362, Qiagen) , and was used for AAV8 virus production.
  • the packaging, purification, and titer determination of AAV8 virus was accomplished by using standard procedures.
  • Scid/FcRn humanized mice (Strain number: 018441, Jackson Laboratory, Bar Harbor, Maine, USA) were injected by retro-orbital intravenous (I.V.) route with an AAV8 virus containing human mature FD (3 ⁇ 10 11 gene copies/mouse) . After two weeks, blood samples were collected and processed for human FD protein detection.
  • 96-well plates were coated with 50 ⁇ l of a capture anti-human FD mAb (generated in-house) at a final concentration of 2 ⁇ g/ml in PBS at 37°C for 1 hr. Following three washes with washing buffer, the plates were incubated with 200 ⁇ l of blocking buffer at room temperature (RT) for 1 hr. After washing, the plates were incubated with 50 ⁇ l of diluted mouse plasma samples or standards in the blocking solution at RT for 1 hr. Plasma dilution started at 1/25, followed by a two-fold serial dilution. Recombinant hFD standard dilution started at 125 ng/ml and was serially diluted by 2-fold.
  • a capture anti-human FD mAb generated in-house
  • the plates were incubated with 50 ⁇ l of biotin-conjugated detection anti-human FD mAb (generated in-house) at a final concentration of 2 ⁇ g/ml in blocking solution at RT for 1 hr. After washing, the plates were developed with 100 ⁇ l of HRP substrate (Cat. No. 34029, Thermo Scientific) for 3 min. The reaction was stopped with 50 ⁇ l of 2N H 2 SO 4 and the plate was read at 450 nm in a micro plate reader.
  • HRP substrate Cat. No. 34029, Thermo Scientific
  • mAb-42 (not antigen pH-dependent engineering) constructed in IgG4P-LA backbone (SEQ ID NO: 133; mAb42-IgG4P-LA, or “42WT” ) and its pH-dependent FD-binding and/or FcRn binding variants were administered to hFD/FcRn-Scid mice via retro-orbital I.V. infusion at 40 mg/kg dosage.
  • the plates were incubated with 50 ⁇ l of anti-human IgG4 HRP (1: 2000 dilution, Invitrogen, A10654) in blocking solution at RT for 1 hr. After washing, the plates were developed with 100 ⁇ l of HRP substrate (Thermo Scientific, 34029) for 3 min. The reaction was stopped with 50 ⁇ l of 2N H 2 SO 4 and the plate was read at 450 nm in a microplate reader. To measure free anti-hFD mAbs, 96-well plates were coated with 50 ⁇ l of recombinant hFD at a final concentration of 2 ⁇ g/ml in bicarbonate buffer at 37°C for 1 hr.
  • the plates were incubated with 200 ⁇ l of blocking buffer at RT for 1 hr. After washing, the plates were incubated with 50 ⁇ l of diluted plasma samples or standards in the blocking solution at RT for 1 hr. Two-fold serial dilutions starting at 1/200-1/500 dilution of mouse plasma were made. Two-fold dilutions were also made for antibody standards starting at 500ng/ml-1000 ng/ml. After washing, the plates were incubated with 50 ⁇ l of anti-human IgG4 HRP (1: 2000 dilution, Invitrogen, A10654) in blocking solution at RT for 1 hr.
  • the plates were developed with 100 ⁇ l of HRP substrate (Thermo Scientific, 34029) for 3 min. The reaction was stopped with 50 ⁇ l of 2N H 2 SO 4 and the plate was read at 450 nm in a microplate reader.
  • mAb42-IgG4P-LA 42WT (no anti-FD pH-dependent binding engineering)
  • mAb42pH-IgG4P-LA 42Mut
  • mAb42pH-IgG4P-YEY mAb42pH-IgG4P-YPY
  • mAb42pH-IgG4P-N3E N3E
  • mAb42pH-IgG4P-Y31-YTE YTE
  • MAbs containing mutations that increased FcRn binding at both pH 7.4 and 5.8 include mAb42pH-IgG4P-Y31-YTE (lines with asterisk, FIG. 11A) , mAb42pH-IgG4P-YEY (lines with triangles, FIG. 11A) , and mAb42pH-IgG4P-YPY (lines with filled circles, FIG. 11A) .
  • FcRn binding sweeping mutation N3E showed a similar PK profile as 42Mut and 42WT, YTE, YEY, and YPY showed faster clearance in mice than the other FcRn binding mutant mAbs (FIG. 11A) .
  • Human IgG4 levels of mice injected with YPY variant were consistently lower than that of mice injected with other mAbs, even starting from 2 hrs post-injection. When the mice were measured for total plasma hFD levels, plasma hFD level increased following mAb administration, as expected (FIG. 11B) . As shown in FIG.
  • mAbs containing both pH-dependent hFD-binding mutations in the Fab domain and FcRn-binding mutations in the Fc domain accumulated overall less total plasma hFD by day 7, compared to mAb42-IgG4P-LA (42WT) , which does not contain pH-dependent hFD-binding mutations in the Fab domain.
  • mAb42-IgG4P-LA (42WT) and mAb42pH-IgG4P-LA (42Mut) This suggests that pH-dependent hFD binding contributed to plasma hFD reduction.
  • FcRn binding sweeping mutations YPY produced the strongest hFD reduction effect, or antigen-sweeping effect, compared with other Fc mutations; particularly, the YPY variant completely ablated hFD accumulation by day 15 (FIG. 11B) .
  • the ratios of total plasma IgG4 mAb and total hFD in the hFD Scid/FcRn mice were compared for each mAb, higher IgG4/hFD ratios were observed for FcRn binding sweeping mutations YEY and N3E from day 5 onwards (FIG. 11C) .
  • the YEY and N3E mutations showed higher binding affinity to FcRn at both pH 5.8 and pH 7.4.
  • IgG4/hFD ratios may have been due to higher binding affinity to FcRn at pH 5.8 which improved antibody recycling (or high total IgG4) (see FIG. 11A) and higher binding affinity to FcRn at pH 7.4 which promoted antigen sweeping activity (or low hFD level) (see FIG. 11B) .
  • mice 40 mg/kg of either mAb42-IgG4P-LA (42WT) or mAb42pH-IgG4P-N3E (N3E) was injected into hFD/FcRn-Scid mice.
  • mice injected with 42WT showed a sudden drop of free IgG4 concentration between day 5 and day 7 and remained low thereafter, while mice injected with N3E showed a slow, gradual decrease in free plasma IgG4 levels over time (FIG. 12B) .
  • the pH-dependent FD and FcRn binding properties of N3E may account for the enhanced release and recycling of free mAb from the mAb/hFD complex. Free IgG4 can continue to bind, neutralize, and sweep circulating hFD into endosomes.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

This invention relates to anti-human factor D (FD) antibody constructs. Also provided are methods of treating a complement-mediated disease using the anti-human FD antibody constructs, pharmaceutical compositions thereof, and methods of making thereof.

Description

ANTI-HUMAN FACTOR D ANTIBODY CONSTRUCTS AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
This patent application claims priority benefits of International Patent Application No. PCT/CN2023/116638 filed on September 3, 2023, and International Patent Application No. PCT/CN2023/116639 filed on September 3, 2023, the contents of each of which are incorporated herein by reference in their entirety.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
The contents of the electronic sequence listing (792252001141seqlist. xml; Size: 370, 943 bytes; and Date of Creation: August 9, 2024) are herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
This invention relates to anti-human factor D (FD) antibody constructs and uses thereof.
BACKGROUND OF THE INVENTION
The complement system is part of innate immunity that plays a key role in host defense. Complement also plays a pathogenic role in human inflammatory diseases. Activation of the complement system occurs via three different pathways, the classical pathway (CP) , the lectin pathway (LP) and the alternative pathway (AP) . The CP is initiated by antigen-antibody binding. The LP is triggered when mannose-binding lectins (MBL) interact with surface sugar molecules on microorganisms. Activation of both pathways leads to the assembly of the CP C3 convertase C4b2a, although direct cleavage of C3 by MBL-associated serine proteases can also occur. The AP is a self-amplification loop driven by the AP C3 convertase, C3bBb. AP activation can occur secondary to CP or LP activation, or is initiated independently. In the latter case, a low level spontaneous C3 “tick-over” generates the initial C3bBb, which rapidly propagates the AP in the absence of adequate regulation. Thus, it is generally assumed that AP activation on non-self surfaces with no or insufficient negative regulation is considered a default process, whereas autologous cells typically avoid this outcome with the help of multiple membrane-bound and fluid phase complement inhibitory proteins. Under certain conditions, altered, damaged or stressed autologous cells and tissues can also activate AP and cause inflammatory injury.
Factor D (FD) is an essential enzyme for AP complement activation. It cleaves factor B after the latter is bound to C3b to produce an active C3 convertase, C3bBb. Factor D is a serine protease of approximately 24 kDa in size and it circulates in the blood as a constitutively active enzyme after being generated from pro-factor D by enzymatic action of mannose binding lectin-associated serine protease-3 (MASP-3) . Compared with other complement proteins in blood, the concentration of factor D in blood is rather low (approximately 2 μg/ml) . While the latter fact may suggest that therapeutic inhibition of factor D activity in blood is possible and might be easily achieved, previous studies have shown that factor D has a fast turnover, and accordingly the consensus in the complement research field is that it would not be feasible to block factor D systemically.
Small molecule antagonists of FD have been tested in clinical trials and showed good AP inhibition and efficacy. However, the treatment regimen requires high and frequent doses, resulting in reported liver toxicities. Anti-FD biologics for treatment of macular diseases have been evaluated in clinical trials. The anti-FD Fab fragment was administered locally into the vitreous of eyes to maintain good pharmacokinetics of the drug. The clinical trial showed good efficacy in phase II trials but failed to reach primary end points in phase III trials.
All references cited herein, including patent applications, patent publications, and Genbank Accession numbers are herein incorporated by reference, as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety.
BRIEF SUMMARY OF THE INVENTION
The present application provides anti-human FD antibody constructs, including anti-human FD antibody constructs having pH-dependent binding to factor D and/or affinity maturation.
In some aspects, provided herein is an isolated antibody construct (anti-human FD antibody construct) comprising an antibody moiety specifically bindings to human factor D (anti-human FD antibody moiety) , wherein the anti-human FD antibody moiety comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL) , wherein: (i) the VH comprises a heavy chain CDR1 ( “H-CDR1” ) comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to 3 amino acid variations; a heavy chain CDR2 ( “H-CDR2” ) comprising the amino acid sequence of SEQ ID  NO: 2, or a variant thereof comprising up to 3 amino acid variations; and a heavy chain CDR3 (“H-CDR3” ) comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to 3 amino acid variations; and (ii) the VL comprises a light chain CDR1 ( “L-CDR1” ) comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to 3 amino acid variations; a light chain CDR2 ( “L-CDR2” ) comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to 3 amino acid variations; and a light chain CDR3 ( “L-CDR3” ) comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to 3 amino acid variations. In some embodiments, (i) the VH comprises an H-CDR1 comprising the amino acid sequence of D-T-Y-V-H (SEQ ID NO: 1) ; an H-CDR2 comprising the amino acid sequence of R-I-D-P-X1-X2-G-X3-T-X4-F-X5-P-R-F-Q-A (SEQ ID NO: 9) , wherein X1 is A or H, X2 is N, S, or Y, X3 is L or H, X4 is T or H, and X5 is D, V, L, or H; and an H-CDR3 comprising the amino acid sequence of A-M-E-Y (SEQ ID NO: 3); and (ii) the VL comprises an L-CDR1 comprising the amino acid sequence of S-A-X6-S-D-V-S-X7-M-Y (SEQ ID NO: 10) , wherein X6 is R, N, or S, and X7 is Y, D, or V; an L-CDR2 comprising the amino acid sequence of X8-T-S-N-L-A-S (SEQ ID NO: 252) , wherein X8 is D or H;and an L-CDR3 comprising the amino acid sequence of Q-Q-W-S-S-X9-P-P-W-X10 (SEQ ID NO: 11) , wherein X9 is Y or H, X10 is T, R, or H.
In some embodiments according to any of the isolated anti-human FD antibody constructs described above, (1) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (2) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 23, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 25; (3) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and the VL comprises an L- CDR1 comprising the amino acid sequence of SEQ ID NO: 39, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 41; (4) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 44, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 46; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 47, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 49; (5) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 52, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 54; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 57; (6) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 63, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 64, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 65; (7) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 71, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 72, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 73; (8) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 76, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 78; the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 79, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 81; (9) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 84, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 85, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 86; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid  sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89; (10) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 92, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 93, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 94; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97; (11) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 100, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 101, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 102; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105; (12) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 108, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 109, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 110; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113; (13) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 116, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 117, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 118; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121; (14) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 126; (15) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and an L-CDR3 comprising the amino acid sequence of SEQ ID  NO: 126; (16) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (17) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 126; (18) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89; (19) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105; (20) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97; (21) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113; (22) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2  comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121; (23) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 57; (24) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89; (25) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105; (26) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97; (27) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113; (28) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino  acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121; (29) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 57; (30) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89; (31) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105; (32) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97; (33) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113; (34) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid  sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121; (35) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 206, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 207, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 208; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 209, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 210, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 211; (36) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 234, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 235, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 236; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 237, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 238, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 239; (37) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 240, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 241, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 242; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 243, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 244, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 245; or (38) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 246, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 247, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 248; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 249, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 250, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 251.
In some embodiments according to any of the isolated anti-human FD antibody constructs described above, the anti-human FD antibody moiety comprises: a VH comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 80%amino acid sequence homology to SEQ ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 80%amino acid sequence homology to SEQ ID NO: 8. In some embodiments, i) the amino acid residue at position 24 of the VH is A or T; ii) the amino acid residue at position 54 of the VH is A or H; iii) the amino acid residue at position 55 of the VH is N, S, or Y; iv) the amino acid residue at position 57 of the VH is H or L; v) the amino acid residue at position 61 of the VH is D, H, L, or V; vi) the amino acid residue at  position 74 of the VH is K or T; vii) the amino acid residue at position 26 of the VL is N, R, or S; viii) the amino acid residue at position 31 of the VL is D, V, or Y; ix) the amino acid residue at position 49 of the VL is D or H; x) the amino acid residue at position 70 of the VL is F or Y; xi) the amino acid residue at position 93 of the VL is H or Y; and/or xii) the amino acid residue at position 97 of the VL is T, H, or R; wherein the numbering is according to the Kabat numbering system.
In some embodiments according to any of the isolated anti-human FD antibody constructs described above, (1) the VH comprises the amino acid sequence of SEQ ID NO: 7, and the VL comprises the amino acid sequence of SEQ ID NO: 8; (2) the VH comprises the amino acid sequence of SEQ ID NO: 18, and the VL comprises the amino acid sequence of SEQ ID NO: 19; (3) the VH comprises the amino acid sequence of SEQ ID NO: 26, and the VL comprises the amino acid sequence of SEQ ID NO: 27; (4) the VH comprises the amino acid sequence of SEQ ID NO: 34, and the VL comprises the amino acid sequence of SEQ ID NO: 35; (5) the VH comprises the amino acid sequence of SEQ ID NO: 42, and the VL comprises the amino acid sequence of SEQ ID NO: 43; (6) the VH comprises the amino acid sequence of SEQ ID NO: 50, and the VL comprises the amino acid sequence of SEQ ID NO: 51; (7) the VH comprises the amino acid sequence of SEQ ID NO: 58, and the VL comprises the amino acid sequence of SEQ ID NO: 59; (8) the VH comprises the amino acid sequence of SEQ ID NO: 66, and the VL comprises the amino acid sequence of SEQ ID NO: 67; (9) the VH comprises the amino acid sequence of SEQ ID NO: 74, and the VL comprises the amino acid sequence of SEQ ID NO: 75; (10) the VH comprises the amino acid sequence of SEQ ID NO: 82, and the VL comprises the amino acid sequence of SEQ ID NO: 83; (11) the VH comprises the amino acid sequence of SEQ ID NO: 90, and the VL comprises the amino acid sequence of SEQ ID NO: 91; (12) the VH comprises the amino acid sequence of SEQ ID NO: 98, and the VL comprises the amino acid sequence of SEQ ID NO: 99; (13) the VH comprises the amino acid sequence of SEQ ID NO: 106, and the VL comprises the amino acid sequence of SEQ ID NO: 107; (14) the VH comprises the amino acid sequence of SEQ ID NO: 114, and the VL comprises the amino acid sequence of SEQ ID NO: 115; (15) the VH comprises the amino acid sequence of SEQ ID NO: 122, and the VL comprises the amino acid sequence of SEQ ID NO: 123; (16) the VH comprises the amino acid sequence of SEQ ID NO: 156, and the VL comprises the amino acid sequence of SEQ ID NO: 157; (17) the VH comprises the amino acid sequence of SEQ ID NO:  158, and the VL comprises the amino acid sequence of SEQ ID NO: 159; (18) the VH comprises the amino acid sequence of SEQ ID NO: 160, and the VL comprises the amino acid sequence of SEQ ID NO: 161; (19) the VH comprises the amino acid sequence of SEQ ID NO: 162, and the VL comprises the amino acid sequence of SEQ ID NO: 163; (20) the VH comprises the amino acid sequence of SEQ ID NO: 164, and the VL comprises the amino acid sequence of SEQ ID NO: 165; (21) the VH comprises the amino acid sequence of SEQ ID NO: 166, and the VL comprises the amino acid sequence of SEQ ID NO: 167; (22) the VH comprises the amino acid sequence of SEQ ID NO: 168, and the VL comprises the amino acid sequence of SEQ ID NO: 169; (23) the VH comprises the amino acid sequence of SEQ ID NO: 172, and the VL comprises the amino acid sequence of SEQ ID NO: 173; (24) the VH comprises the amino acid sequence of SEQ ID NO: 174, and the VL comprises the amino acid sequence of SEQ ID NO: 175; (25) the VH comprises the amino acid sequence of SEQ ID NO: 176, and the VL comprises the amino acid sequence of SEQ ID NO: 177; (26) the VH comprises the amino acid sequence of SEQ ID NO: 178, and the VL comprises the amino acid sequence of SEQ ID NO: 179; (27) the VH comprises the amino acid sequence of SEQ ID NO: 180, and the VL comprises the amino acid sequence of SEQ ID NO: 181; (28) the VH comprises the amino acid sequence of SEQ ID NO: 182, and the VL comprises the amino acid sequence of SEQ ID NO: 183; (29) the VH comprises the amino acid sequence of SEQ ID NO: 184, and the VL comprises the amino acid sequence of SEQ ID NO: 185; (30) the VH comprises the amino acid sequence of SEQ ID NO: 186, and the VL comprises the amino acid sequence of SEQ ID NO: 187; (31) the VH comprises the amino acid sequence of SEQ ID NO: 190, and the VL comprises the amino acid sequence of SEQ ID NO: 191; (32) the VH comprises the amino acid sequence of SEQ ID NO: 192, and the VL comprises the amino acid sequence of SEQ ID NO: 193; (33) the VH comprises the amino acid sequence of SEQ ID NO: 194, and the VL comprises the amino acid sequence of SEQ ID NO: 195; (34) the VH comprises the amino acid sequence of SEQ ID NO: 196, and the VL comprises the amino acid sequence of SEQ ID NO: 197; (35) the VH comprises the amino acid sequence of SEQ ID NO: 198, and the VL comprises the amino acid sequence of SEQ ID NO: 199; (36) the VH comprises the amino acid sequence of SEQ ID NO: 212, and the VL comprises the amino acid sequence of SEQ ID NO: 213; (37) the VH comprises the amino acid sequence of SEQ ID NO: 253, and the VL comprises the amino acid sequence of SEQ ID NO: 254; (38) the VH comprises the amino acid sequence of SEQ ID NO: 255, and the VL comprises the amino acid  sequence of SEQ ID NO: 256; (39) the VH comprises the amino acid sequence of SEQ ID NO: 257, and the VL comprises the amino acid sequence of SEQ ID NO: 258; (40) the VH comprises the amino acid sequence of SEQ ID NO: 289, and the VL comprises the amino acid sequence of SEQ ID NO: 127; or (41) the VH comprises the amino acid sequence of SEQ ID NO: 216, and the VL comprises the amino acid sequence of SEQ ID NO: 214.
In some embodiments according to any of the isolated anti-human FD antibody constructs described above, the anti-human FD antibody moiety cross-reacts with a cynomolgus monkey factor D (cyno FD) .
In some embodiments according to any of the isolated anti-human FD antibody constructs described above, the isolated anti-human FD antibody moiety is pH-dependent, wherein the anti-human FD antibody moiety binds more strongly to human FD at a neutral pH than it does at an acidic pH.
In some embodiments according to any of the isolated anti-human FD antibody constructs described above, the isolated anti-human FD antibody moiety is selected from the group consisting of: a full-length antibody, Fab, Fab’ , F (ab) 2, F (ab’ ) 2, scFv, and a combination thereof. In some embodiments, the anti-human FD antibody moiety is a full-length antibody (anti-human FD full-length antibody) . In some embodiments, the anti-human FD full-length antibody comprises a heavy chain constant region derived from IgG4 (e.g., human IgG4) . In some embodiments, the heavy chain constant region comprises the amino acid sequence of any of SEQ ID NOs: 132-139. In some embodiments, the anti-human FD antibody moiety is an scFv (anti-human FD scFv) . In some embodiments, the anti-human FD scFv comprises the amino acid sequence of SEQ ID NO: 219 or 220.
In some embodiments according to any of the isolated anti-human FD antibody constructs described above, the isolated anti-human FD antibody construct further comprises a second antibody moiety specifically recognizing a component of the complement pathway. In some embodiments, the second antibody moiety is selected from the group consisting of: a full-length antibody, Fab, Fab’ , F (ab) 2, F (ab’ ) 2, scFv, sdAb, and a combination thereof. In some embodiments, the component of the complement pathway is complement component 2 (C2) . In some embodiments, the component of the complement pathway is complement component 5 (C5) . In some embodiments, the anti-human FD antibody moiety is an scFv (scFv1) , and the second antibody moiety is an scFv (scFv2) . In some embodiments, the isolated anti-human FD  antibody construct comprises from N-terminus to C-terminus: (i) scFv1-optional linker-scFv2; (ii) scFv2-optional linker-scFv1; (iii) scFv1-optional linker-Fc domain-optional linker-scFv2; or (iv) scFv2-optional linker-Fc domain-optional linker-scFv1. In some embodiments, i) the linker comprises the amino acid sequence of any of SEQ ID NOs: 221-229; and/or ii) the Fc domain comprises the amino acid sequence of SEQ ID NO: 230.
Also provided are isolated nucleic acids encoding any one of the isolated anti-human FD antibody constructs described above. Also provided are vectors comprising any of the isolated nucleic acids described herein. In some embodiments, the vector is a viral vector, such as an adeno-associated virus (AAV) vector or a lentiviral vector. Also provided are host cells comprising any of the isolated nucleic acids described herein, or any of the vectors described herein.
In another aspect, there is provided a method for making an isolated anti-human FD antibody construct (e.g., any one of the anti-human FD antibody constructs described herein) , comprising i) culturing a host cell comprising any of the isolated nucleic acids described herein or any of the vectors described herein, or any of the host cells described herein, under a condition suitable for the expression of the isolated anti-human FD antibody construct; and ii) obtaining the expressed isolated anti-human FD antibody construct from said host cell.
In another aspect, there is provided a pharmaceutical composition comprising any one of the isolated anti-human FD antibody constructs described above, any of the isolated nucleic acids described above, or any of the vectors described above, and a pharmaceutically acceptable carrier.
In another aspect, there is provided a method for treating a complement-mediated disease in an individual, comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein. In some embodiments, the complement-mediated disease is selected from the group consisting of: macular degeneration (MD) , age-related macular degeneration (AMD) , ischemia reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, post-surgery systemic inflammatory syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD) , paroxysmal nocturnal hemoglobinuria (PNH) syndrome, autoimmune hemolytic anemia (AIHA) , Gaucher disease, myasthenia gravis, neuromyelitis optica (NMO) , multiple sclerosis, delayed graft function, antibody-mediated rejection, atypical hemolytic uremic syndrome (aHUS) , central retinal vein occlusion (CRVO) ,  central retinal artery occlusion (CRAO) , epidermolysis bullosa, sepsis, septic shock, organ transplantation, inflammation, C3 glomerulopathy (C3G) , membranous nephropathy, IgA nephropathy (IgAN) , glomerulonephritis, thrombotic microangiopathies secondary to systemic lupus erythematosus (SLE-TMA) , anti-neutrophil cytoplasmic antibody (ANCA) -mediated vasculitis, Shiga toxin induced HUS, antiphospholipid antibody-induced pregnancy loss, graft versus host disease (GvHD) , bullous pemphigoid, hidradenitis suppurativa, dermatitis herpetiformis, sweets syndrome, pyoderma gangrenosum, palmo-plantar pustulosis &pustular psoriasis, rheumatoid neutrophilic dermatoses, subcorneal pustular dermatosis, bowel-associated dermatosis-arthritis syndrome, neutrophilic eccrine hidradenitis, linear IgA disease, and any combinations thereof.
In another aspect, there is provided a method for reducing the activity of a complement system in an individual, comprising administering to the individual an effective amount of any of the pharmaceutical compositions described above.
These and other aspects and advantages of the present invention will become apparent from the subsequent detailed description and the appended claims. It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts binding activities of representative anti-FD scFv variants to human FD. z16-p2 was the parental humanized anti-FD scFv. Clone z16-p2 served as reference anti-human FD control. An irrelevant antibody clone served as negative control.
FIGs. 2A and 2B depict binding activities of representative anti-FD scFv variants to cynomolgus monkey FD. z16-p2 was the parental humanized anti-FD scFv. Clone z16-p2served as reference anti-human FD control. An irrelevant antibody clone served as negative control.
FIG. 3A depicts binding activities of representative anti-FD IgG4 monoclonal antibody (mAb) variants to human FD. FIG. 3B depicts binding activities of representative IgG4 mAb variants to cynomolgus FD. Parental z16-p2 in IgG4 format served as control.
FIG. 4 depicts Bio-Layer Interferometry (BLI) sensorgrams of pH-binding-engineered anti-FD variants to human and cynomolgus monkey FD.
FIG. 5 depicts inhibition of 50%NHS (normal human serum) -induced rabbit RBC lysis by affinity-matured anti-FD mAb variants. Parental humanized z16-p2 clone served as control. Distilled water served as positive control. EDTA served as negative control.
FIG. 6 depicts inhibition of LPS-induced C3b deposition (in 10%NHS) by affinity-matured anti-FD mAb variants. Parental humanized z16-p2 clone served as control.
FIG. 7 depicts inhibition of 30%NHS-induced rabbit RBC lysis by pH-binding-engineered anti-FD mAbs. Parental affinity-matured mAb-42 served as control.
FIG. 8 depicts inhibition of LPS-induced C3b deposition (in 10%NHS) by pH-binding-engineered anti-FD mAbs. Parental affinity-matured mAb-42 served as control.
FIG. 9 depicts FcRn binding optimization mutations for enhancement of IgG recycling and reduction of antigen serum levels.
FIG. 10 depicts optimization of FcRn binding for antigen sweeping using BLI. The tests were conducted in duplicates.
FIG. 11A depicts a graph of total plasma human IgG4 levels across time in hFD Scid/FcRn mice injected with 40 mg/kg of mAb42-IgG4P (WT) or mAb42pH FcRn binding variants (Mut, YEY, YPY, N3E, and YTE) . FIG. 11B depicts a graph of total plasma hFD levels across time in hFD Scid/FcRn mice injected with 40 mg/kg of mAb42-IgG4P (WT) or mAb42pH FcRn binding variants (Mut, YEY, YPY, N3E, and YTE) . FIG. 11C depicts a graph of the ratio of total plasma IgG4 mAb to total plasma hFD across time in hFD Scid/FcRn mice injected with 40 mg/kg of mAb42-IgG4P (WT) or mAb42pH FcRn binding variants (Mut, YEY, YPY, N3E, and YTE) . The tests were conducted in duplicates.
FIG. 12A depicts a graph of the total plasma IgG4 levels of mAb42-IgG4P-LA (42WT) and mAb42pH-IgG4P-N3E (N3E) in hFD Scid/FcRn mice injected with 40 mg/kg of either mAb. FIG. 12B depicts a graph of the total free plasma IgG4 levels of 42WT and N3E in hFD Scid/FcRn mice injected with 40 mg/kg of either mAb. FIG. 12C depicts a graph of the total hFD levels across time in the hFD Scid/FcRn mice injected with 40 mg/kg of either 42WT or N3E mAb. FIG. 12D depicts a graph of the ratio of total plasma IgG4 mAb to total plasma hFD across time in hFD Scid/FcRn mice injected with 40 mg/kg of either 42WT or N3E mAb. The tests were conducted in duplicates.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides various anti-human FD antibody constructs comprising antibody moieties specifically binding to human factor D (anti-human FD antibody moiety) . The anti-human FD antibody constructs described herein have one or more superior properties: i) have reduced immunogenicity (e.g., compared to parental mouse Ab) ; ii) have reduced N-linked glycosylation potential; iii) have improved cross-reactivity to cynomolgus monkey FD (e.g., affinity-matured variants) , enabling the extrapolation of monkey study results to human; iv) retain similar binding affinity or have improved binding affinity to human FD (e.g., affinity-matured variant, humanized variant, or pH-dependent FD binding variant) ; v) some variants have stronger binding to FD under neutral pH (e.g., about pH 7.4; such as in plasma) but less binding to FD under acidic pH (e.g., about pH 5.8; such as in endosomes) , enabling the anti-FD antibodies to dissociate from FD in the acidic environment of endosomes and allowing the freed anti-FD antibodies to recycle back out of the cells to bind other free FD in the plasma; vi) some variants have optimized FcRn binding at pH 5.8 and/or pH 7.4, which improve antibody recycling with extended serum half-lives (high FcRn binding at pH 5.8) , and/or improve antigen-sweeping activity (to reduce free FD; high FcRn binding at pH 7.4) ; vii) exhibit strong inhibition of Normal Human Serum (NHS) -induced rabbit RBC lysis and LPS-induced C3b deposition, reflecting the strong potency of the antibodies in the inhibition of alternative pathway; and viii) demonstrate superior in vivo pharmacokinetics (longer and/or higher antibody level) and antigen-sweeping activity (lower FD accumulation in plasma) .
I. Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.
The term “antibody” herein is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) , full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. The term “antibody moiety” refers to a full-length antibody or an antigen-binding fragment thereof.
An “antibody” may refer an immunoglobulin molecule or a fragment thereof which is able to specifically bind to a specific epitope of an antigen (including the basic 4-chain antibody unit) . Antibodies can be intact immunoglobulins derived from natural sources, or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ( “intrabodies” ) , antigen-binding fragments (such as Fv, Fab, Fab’ , F (ab) 2 and F (ab’ ) 2) , as well as single chain antibodies (scFv) , heavy chain antibodies, such as camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Bird et al., 1988, Science 242: 423-426) .
A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL” , respectively. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3) . CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991) . The three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as lgG1 (γ1 heavy chain) , lgG2 (γ2 heavy chain) , lgG3 (γ3 heavy chain) , lgG4 (γ4 heavy chain) , lgA1 (α1 heavy chain) , or lgA2 (α2 heavy chain) .
The term “antigen-binding fragment” as used herein refers to an antibody fragment including, for example, a diabody, a Fab, a Fab’ , a F (ab’ ) 2, an Fv fragment, a disulfide stabilized  Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv’ ) , a disulfide stabilized diabody (ds diabody) , a single-chain Fv (scFv) , an scFv dimer (bivalent diabody) , a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a single domain antibody (sdAb) (e.g., a camelized single domain antibody) , a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
“Fv” is the minimum antibody fragment, which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy-and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
“Single-chain Fv, ” also abbreviated as “sFv” or “scFv, ” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994) .
The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or  more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1) . Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds) , Appleton &Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH) , immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated α, δ, ε, γ and μ, respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.
The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL” , respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites. Heavy-chain only antibodies from the Camelidae species have a single heavy chain variable region, which is referred to as “VHH” . VHH is thus a special type of VH.
The term “variable” refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability  is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR) . The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991) ) . The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes) , each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256: 495-97 (1975) ; Hongo et al., Hybridoma, 14 (3) : 253-260 (1995) , Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) ; Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) ) , recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567) , phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991) ; Marks et al., J. Mol. Biol. 222: 581-597 (1992) ;  Sidhu et al., J. Mol. Biol. 338 (2) : 299-310 (2004) ; Lee et al., J. Mol. Biol. 340 (5) : 1073-1093 (2004) ; Fellouse, Proc. Natl. Acad. Sci. USA 101 (34) : 12467-12472 (2004) ; and Lee et al., J. Immunol. Methods 284 (1-2) : 119-132 (2004) , and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993) ; Jakobovits et al., Nature 362: 255-258 (1993) ; Bruggemann et al., Year in Immunol. 7: 33 (1993) ; U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992) ; Lonberg et al., Nature 368: 856-859 (1994) ; Morrison, Nature 368: 812-813 (1994) ; Fishwild et al., Nature Biotechnol. 14: 845-851 (1996) ; Neuberger, Nature Biotechnol. 14: 826 (1996) ; and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995) .
The terms “full-length antibody, ” “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically full-length 4-chain antibodies include those with heavy and light chains including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.
The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10) residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in greater detail in, for example, EP 404, 097; WO 93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) .
The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is (are) identical with or homologous to corresponding sequences in antibodies derived from another  species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984) ) . Chimeric antibodies of interest herein include PRIMATTZFACTORantibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest. As used herein, “humanized antibody” is used a subset of “chimeric antibodies. ”
As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977) ; Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991) ; Chothia et al., J. Mol. Biol. 196: 901-917 (1987) ; Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997) ; MacCallum et al., J. Mol. Biol. 262: 732-745 (1996) ; Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008) ; Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003) ; and Honegger and Plückthun, J. Mol. Biol., 309: 657-670 (2001) , where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table A as a comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008) ; Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010) ; and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015) . The contents of the references cited in this paragraph are incorporated herein by reference in their entireties for use in the present application and for possible inclusion in one or more claims herein.
Table A: CDR Definitions
1Residue numbering follows the nomenclature of Kabat et al., supra
2Residue numbering follows the nomenclature of Chothia et al., supra
3Residue numbering follows the nomenclature of MacCallum et al., supra
4Residue numbering follows the nomenclature of Lefranc et al., supra
5Residue numbering follows the nomenclature of Honegger and Plückthun, supra
The expression “variable-domain residue-numbering as in Kabat” or “amino-acid-position numbering as in Kabat, ” and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or hypervariable region (HVR) of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of 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.
Unless indicated otherwise herein, the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra with minor modification. The “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody.
“Framework” or “FR” residues are those variable-domain residues other than the CDR residues as herein defined.
As used herein, an “immunoassay” refers to any binding assay that uses an antibody capable of binding specifically to a target molecule to detect and quantify the target molecule.
“Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate  having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin. A suitable human acceptor antibody may be one selected from a conventional database, e.g., the KABAT database, Los Alamos database, the AbM, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody. A human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs. A suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody. The prior art describes several ways of producing such humanized antibodies (see, for example, EP-A-0239400 and EP-A-054951) . For further details, see, e.g., Jones et al., Nature 321: 522-525 (1986) ; Riechmann et al., Nature 332: 323-329 (1988) ; and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992) . See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma &Immunol. 1: 105-115 (1998) ; Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995) ; Hurle and Gross, Curr. Op. Biotech. 5: 428-433 (1994) ; and U.S. Pat. Nos. 6,982,321 and 7,087,409.
A “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991) ; Marks et al., J. Mol. Biol., 222: 581 (1991) . Also available for the preparation of human  monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) ; Boerner et al., J. Immunol., 147 (1) : 86-95 (1991) . See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001) . Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology) . See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
The term “donor antibody” refers to an antibody (monoclonal, and/or recombinant) which contributes the amino acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner, so as to provide the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralizing activity characteristic of the donor antibody.
The term “acceptor antibody” refers to an antibody (monoclonal and/or recombinant) heterologous to the donor antibody, which contributes all (or any portion, but in some embodiments all) of the amino acid sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the first immunoglobulin partner. In certain embodiments a human antibody is the acceptor antibody.
The term “attach, ” “attached, ” “fuse, ” or “fused” as used herein, refers to connecting or uniting by a bond, link, force or tie in order to keep two or more components together, which encompasses either direct or indirect attachment such that, for example, where a first polypeptide is directly bound to a second polypeptide or material, and, for example, where one or more intermediate compounds (e.g., amino acids, peptides, polypeptides, etc. ) are disposed between the first polypeptide and the second polypeptide or material.
“Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art,  for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) , or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, %amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32 (5) : 1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5 (1) : 113, 2004) .
“Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60%homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50%homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CH1, CH2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.
The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ( “κ” ) and lambda ( “λ” ) , based on the amino acid sequences of their constant domains.
The “CH1 domain” (also referred to as “C1” of “H1” domain) usually extends from about amino acid 118 to about amino acid 215 (EU numbering system) .
“Hinge region” is generally defined as a region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985) ) . Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-Sbonds in the same positions.
The “CH2 domain” of a human IgG Fc domain (also referred to as “C2” domain) usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22: 161-206 (1985) .
The “CH3 domain” (also referred to as “C3” domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc domain (i.e., from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG) .
The term “Fc domain” or “fragment crystallizable region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc domains and variant Fc domains. Although the boundaries of the Fc domain of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc domain is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc domain may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc domains for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B) , IgG3 and IgG4.
The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds. Two antibodies or antibody moieties may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.
As used herein, a first antibody or fragment thereof “competes” for binding to a target antigen with a second antibody or fragment thereof when the first antibody or fragment thereof inhibits the target antigen binding of the second antibody of fragment thereof by at least about 50%(such as at least about any one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%or 99%) in the presence of an equimolar concentration of the first antibody or fragment thereof, or  vice versa. A high throughput process for “binning” antibodies based upon their cross-competition is described in PCT Publication No. WO 03/48731.
As use herein, the terms “specifically binds, ” “specifically recognizing, ” and “is specific for” refer to measurable and reproducible interactions, such as binding between a target and an antibody or antibody moiety, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody or antibody moiety that specifically recognizes a target (which can be an epitope) is an antibody or antibody moiety that binds this target with greater affinity, avidity, more readily, and/or with greater duration than its bindings to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10%of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA) . In some embodiments, an antibody that specifically binds a target has a dissociation constant (KD) of ≤10-5 M, ≤10-6 M, ≤10-7 M, ≤10-8 M, ≤10-9 M, ≤10-10 M, ≤10-11 M, or ≤10-12 M. In some embodiments, an antibody specifically binds an epitope on a protein that is conserved among the protein from different species. In some embodiments, specific binding can include, but does not require exclusive binding. Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORETM -tests and peptide scans.
The term “specificity” refers to selective recognition of an antigen binding protein or antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term “multispecific” as used herein denotes that an antigen binding protein or an antibody has two or more antigen-binding sites of which at least two bind a different antigen or a different epitope of the same antigen. “Bispecific” as used herein denotes that an antigen binding protein or an antibody has two different antigen-binding specificities. The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind the same epitope of the same antigen.
“Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen) . Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (e.g.,  antibody and antigen) . The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd) . Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present application. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.
An “on-rate, ” “rate of association, ” “association rate, ” or “kon” as used herein can also be determined as described above using methods such as biolayer interferometry and surface plasmon resonance (SPR) .
An “isolated” antibody is one that has been identified, separated and/or recovered from a component of its production environment (e.g., natural or recombinant) . Preferably, the isolated polypeptide is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95%by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99%by weight; (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.
An “isolated” nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is in a form other than in  the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies described herein existing naturally in cells. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in its normal context in a living subject is not “isolated, ” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is “isolated. ” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
The term “hybridoma, ” as used herein refers to a cell resulting from the fusion of a B-lymphocyte and a fusion partner such as a myeloma cell. A hybridoma can be cloned and maintained indefinitely in cell culture and is able to produce monoclonal antibodies. A hybridoma can also be considered to be a hybrid cell.
The terms “nucleic acid molecule” , “nucleic acid” and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or unnatural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide. An “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally  occurring state, i.e., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
“Complementary” as used herein to refer to a nucleic acid, refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ( “base pairing” ) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and or at least about 75%, or at least about 90%, or at least about 95%of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In some embodiments, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
The term “vector, ” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. A vector may be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector  may be either a self-replicating extrachromosomal vector or a vector which integrates into a host genome. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors. ”
“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting there from. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
The terms “polypeptide” and “peptide” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or unnatural amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, a “polypeptide” includes modifications, such as deletions, additions, and substitutions (generally conservative in nature) , to the native sequence, as long as the polypeptide maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
As used herein, “conjugated” refers to covalent attachment of one molecule to a second molecule.
“Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so  that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis. In various embodiments, the variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%identical to the reference sequence.
The term “regulating” as used herein can mean any method of altering the level or activity of a substrate. Non-limiting examples of regulating with regard to a protein include affecting expression (including transcription and/or translation) , affecting folding, affecting degradation or protein turnover, and affecting localization of a protein. Non-limiting examples of regulating with regard to an enzyme further include affecting the enzymatic activity. “Regulator” refers to a molecule whose activity includes affecting the level or activity of a substrate. A regulator can be direct or indirect. A regulator can function to activate or inhibit or otherwise modulate its substrate.
The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
A “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.
The “diluent” of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, such as a formulation reconstituted after lyophilization. Exemplary diluents include sterile water,  bacteriostatic water for injection (BWFI) , a pH buffered solution (e.g., phosphate-buffered saline) , sterile saline solution, Ringer’s solution or dextrose solution. In an alternative embodiment, diluents can include aqueous solutions of salts and/or buffers.
A “preservative” is a compound which can be added to the formulations herein to reduce bacterial activity. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (amixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds) , and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferred preservative herein is benzyl alcohol.
The terms “pharmaceutical formulation” and “pharmaceutical composition” refer to a preparation which is in such form as to permit the biological activity of the active ingredient (s) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations may be sterile.
A “sterile” formulation is aseptic or essentially free from living microorganisms and their spores.
A “stable” formulation is one in which the protein therein essentially retains its physical and chemical stability and integrity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993) . Stability can be measured at a selected temperature for a selected time period. For rapid screening, the formulation may be kept at 40℃. for 2 weeks to 1 month, at which time stability is measured. Where the formulation is to be stored at 2-8℃., generally the formulation should be stable at 30℃. or 40℃. for at least 1 month and/or stable at 2-8℃. for at least 2 years. Where the formulation is to be stored at 30℃., generally the formulation should be stable for at least 2 years at 30℃. and/or stable at 40℃. for at least 6 months. For example, the extent of aggregation during storage can be used as an indicator of protein stability. Thus, a “stable” formulation may be one wherein less than about 10%and preferably less than about 5%of the protein are present as an aggregate in the formulation. In  other embodiments, any increase in aggregate formation during storage of the formulation can be determined.
A “reconstituted” formulation is one which has been prepared by dissolving a lyophilized protein or antibody formulation in a diluent such that the protein is dispersed throughout. The reconstituted formulation is suitable for administration (e.g., subcutaneous administration) to a patient to be treated with the protein of interest and, in certain embodiments, may be one which is suitable for parenteral or intravenous administration.
An “isotonic” formulation is one which has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 250 to 350 mOsm. The term “hypotonic” describes a formulation with an osmotic pressure below that of human blood. Correspondingly, the term “hypertonic” is used to describe a formulation with an osmotic pressure above that of human blood. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer, for example. The formulations of the present application can be hypertonic as a result of the addition of salt and/or buffer.
A “recombinant AAV vector (rAAV vector) ” refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequence not of AAV origin) that are flanked by at least one, and in embodiments two, AAV inverted terminal repeat sequences (ITRs) . Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins) . When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection) , then the rAAV vector may be referred to as a “pro-vector” which can be “rescued” by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. An rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle, particularly an AAV particle. A rAAV vector can be packaged into an AAV virus capsid to generate a “recombinant adeno-associated viral particle (rAAV particle) ” .
An “AAV inverted terminal repeat (ITR) ” sequence, a term well-understood in the art, is an approximately 145-nucleotide sequence that is present at both termini of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two  alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter regions of self-complementarity (designated A, A’ , B, B’ , C, C’ and D regions) , allowing intrastrand base-pairing to occur within this portion of the ITR.
The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.
The terms “host cell, ” “host cell line, ” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells, ” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, and may contain mutations. Mutant progenies that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease) , preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival. The methods of the application contemplate any one or more of these aspects of treatment.
The terms “effective amount” and “pharmaceutically effective amount” as used herein refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,  95%, 99%, or 100%) and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system.
The terms “inhibition” or “inhibit” refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and/or amount as compared to that of a reference. In certain embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20%or greater (e.g., at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) . In another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50%or greater. In yet another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.
“Preventing” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual that may be predisposed to the disease but has not yet been diagnosed with the disease.
As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, an antibody which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody.
A “reference” as used herein, refers to any sample, standard, or level that is used for comparison purposes. A reference may be obtained from a healthy and/or non-diseased sample. In some examples, a reference may be obtained from an untreated sample. In some examples, a reference is obtained from a non-diseased or non-treated sample of an individual. In some examples, a reference is obtained from one or more healthy individuals who are not the individual or patient.
As used herein, the terms “patient, ” “subject, ” “individual, ” and the like are used interchangeably herein, and refer to any animal, in some embodiments a mammal, and in some embodiments a human, having a complement system, including a human in need of therapy for, or susceptible to, a condition or its sequelae. The individual may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, mice and humans. In some embodiments, the individual is a human.
A "human consensus framework" or "acceptor human framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) . Examples include for the VL, the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al. Alternatively, a human consensus framework can be derived from the above in which particular residues, such as when a human framework residue is selected based on its homology to the donor framework by aligning the donor framework sequence with a collection of various human framework sequences. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
An “affinity-matured” antibody is one with one or more alterations m one or more CDRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration (s) . In some embodiments, an affinity-matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio/Technology 10: 779-783 (1992) describes affinity maturation by VH-and VL-domain shuffling. Random mutagenesis of CDR and/or framework residues is described by, for example:  Barbas et al. Proc Nat. Acad. Sci. USA 91: 3809-3813 (1994) ; Schier et al. Gene 169: 147-155 (1995) ; Yelton et al. J. Immunol. 155: 1994-2004 (1995) ; Jackson et al., J. Immunol. 154(7) : 3310-9 (1995) ; and Hawkins et al, J. Mal. Biol. 226: 889-896 (1992) .
The term “valent” as used herein denotes the presence of a specified number of binding sites in an antigen binding protein. A natural antibody for example or a full-length antibody has two binding sites and is bivalent. As such, the terms "trivalent" , "tetravalent" , "pentavalent" and "hexavalent" denote the presence of two binding site, three binding sites, four binding sites, five binding sites, and six binding sites, respectively, in an antigen binding protein.
“Antibody effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fe receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC) ; phagocytosis; down regulation of cell surface receptors (e.g., B cell receptors) ; and B cell activation. "Reduced or minimized" antibody effector function means that which is reduced by at least 50% (alternatively 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) from the wild type or unmodified antibody. The determination of antibody effector function is readily determinable and measurable by one of ordinary skill in the art. In a preferred embodiment, the antibody effector functions of complement binding, complement dependent cytotoxicity and antibody dependent cytotoxicity are affected. In some embodiments, effector function is eliminated through a mutation in the constant region that eliminated glycosylation, e.g., "effectorless mutation. " In one aspect, the effectorless mutation is an N297A or DANA mutation (D265A+N297A) in the CH2 region. Shields et al., J. Biol. Chem. 276 (9) : 6591-6604 (2001) . Alternatively, additional mutations resulting in reduced or eliminated effector function include: K322A and L234A/L235A (LALA) . Alternatively, effector function can be reduced or eliminated through production techniques, such as expression in host cells that do not glycosylate (e.g., E. coli. ) or in which result in an altered glycosylation pattern that is ineffective or less effective at promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5) : 3466-3473 (2003) .
“Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted lg bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages) enable these cytotoxic effector  cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are required for killing of the target cell by this mechanism. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. Fe expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991) . To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS USA 95 : 652-656 (1998) .
“Effector cells” are leukocytes which express one or more FcRs and perform effector functions. In one aspect, the effector cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC) , natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils. The effector cells may be isolated from a native source, e.g., blood. Effector cells generally are lymphocytes associated with the effector phase, and function to produce cytokines (helper T cells) , killing cells in infected with pathogens (cytotoxic T cells) or secreting antibodies (differentiated B cells) .
“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) , may be performed. Antibody variants with altered Fc region amino acid sequences and increased or decreased C1q binding capability are described in U.S. Pat. No. 6,194,551B1 and WO99/51642. The contents of those patent publications are specifically incorporated herein by reference. See, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000) .
Half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a substance (such as an antibody) in inhibiting a specific biological or biochemical function. It indicates how much of a particular drug or other substance (inhibitor, such as an antibody) is needed to inhibit a given biological process by half. The values are typically expressed as molar  concentration. IC50 is comparable to an "EC50" for agonist drug or other substance (such as an antibody) . EC50 also represents the plasma concentration required for obtaining 50%of a maximum effect in vivo. As used herein, an "IC50" is used to indicate the effective concentration of an antibody needed to neutralize 50%of the antigen bioactivity in vitro. IC50 or EC50 can be measured by bioassays such as inhibition of ligand binding by FACS analysis (competition binding assay) , cell-based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA) .
A “low-pH dissociation factor” as used herein is defined as the percentage of antibody dissociated at pH 5.8 from the antigen at 25 ℃, wherein the antibody is pre-bound to the antigen at pH 7.4. The low-pH dissociation factor may be measured by associating an antibody and an antigen (e.g., the anti-human FD antibody construct and human factor D) at pH 7.4 for 600 seconds, followed by a dissociation period of 600 seconds in a buffer at pH 5.8, and calculation of the percentage of antibody dissociated at pH 5.8 from the antigen. A “neutral-pH dissociation factor” is defined as the percentage of antibody dissociated at pH 7.4 from the antigen at 25℃, wherein the antibody is pre-bound to the antigen at pH 7.4. The neutral-pH dissociation factor may be measured by associating antibody and antigen at pH 7.4 for 600 seconds, followed by a dissociation period of 600 seconds in a buffer at pH 7.4, and calculation of the percentage of antibody dissociated at pH 7.4 from the antigen. The antibody-antigen association and dissociation may be measured in various ways that are with the skill in the art, for instance, using biolayer interferometry.
“Fc receptor” or “FcR” describes a receptor that binds the Fc domain of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (agamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcγRII receptors include FcγRIIA (an “activating receptor” ) and FcγRIIB (an “inhibiting receptor” ) , which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M.Annu. Rev. Immunol. 15: 203-234 (1997) . FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991) ; Capel et al., Immunomethods 4: 25-34 (1994) ; and de Haas et  al., J. Lab. Clin. Med. 126: 330-41 (1995) . Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein.
The term “Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus. Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994) . Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18: (12) : 592-8 (1997) ; Ghetie et al., Nature Biotechnology 15 (7) : 637-40 (1997) ; Hinton et al., J. Biol. Chem. 279 (8) : 6213-6 (2004) ; WO 2004/92219 (Hinton et al. ) . Binding to FcRn in vivo and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides having a variant Fc region are administered. WO 2004/42072 (Presta) describes antibody variants which improved or diminished binding to FcRs. See also, e.g., Shields et al., J. Biol. Chem. 9 (2) : 6591-6604 (2001) .
It is understood that embodiments described herein include “consisting of” and/or “consisting essentially of” embodiments.
Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X” .
As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat disease of type X means the method is used to treat disease of types other than X.
The term “about X-Y” used herein has the same meaning as “about X to about Y. ”
As used herein and in the appended claims, the singular forms "a, " "or, " and "the" include plural referents unless the context clearly dictates otherwise.
II. Anti-factor D antibody constructs
The resent invention in some aspects provides isolated antibody constructs (anti-human FD antibody constructs) comprising an antibody moiety specifically binding to human factor D (anti-human FD antibody moiety, such as any of the anti-human FD antibody moieties described herein) . In some embodiments, the anti-human FD antibody construct consists essentially of (or consists of) the anti-human FD antibody moiety. Hence in some embodiments, there is also provided anti-human FD antibody moieties. In some embodiments, the isolated anti-human FD  antibody construct processes any of the properties and activities of the anti-human FD antibody moieties described herein.
In some embodiments, the isolated anti-human FD antibody construct further comprises a fusion partner (e.g., protein or polypeptide) . In some embodiments, the fusion partner is directly fused to the anti-human FD antibody moiety. In some embodiments, the fusion partner is fused to the anti-human FD antibody moiety via a linker (e.g., peptide linker) . In some embodiments, the fusion partner is an effector protein. Effector proteins can be any protein that produce a specific response to a stimulus, such as altering one or more of the cellular functions in the target cells. In some embodiments, the fusion partner is an antibody moiety, such as an antibody moiety specifically binding to a non-FD epitope.
In some embodiments, the isolated anti-human FD antibody construct is monospecific. In some embodiments, the isolated anti-human FD antibody construct is multispecific (e.g., bispecific) . In some embodiments, the isolated anti-human FD antibody construct is multivalent and monospecific. In some embodiments, the isolated anti-human FD antibody construct is multivalent and multispecific (e.g., bispecific) .
In some embodiments, the isolated anti-human FD antibody construct further comprises a second antibody moiety (e.g., scFv, Fab, sdAb, or full-length antibody) specifically recognizing a component of the complement pathway. In some embodiments, the second antibody moiety specifically recognizes FD. Hence in some embodiments, the isolated anti-human FD antibody construct comprises two or more anti-human FD antibody moieties (e.g., scFv) , such as connected to each other in tandem. In some embodiments, the second antibody moiety specifically recognizes a component of the complement pathway that is not FD, such as C2 or C5.
Anti-human FD antibody moieties
“Anti-human FD antibody moiety” and “anti-FD antibody moiety” are used interchangeably herein. Referring to “anti-human FD” does not exclude antibody moieties that can bind to a human FD as well as an FD derived from another species. Any of the anti-FD antibody moieties described herein can be used in the anti-human FD antibody constructs described herein.
Factor D, also known as adipsin, is a 24 kDa serine protease comprising 228 amino acids. The structure of factor D comprises of two antiparallel β-barrel domains with each barrel containing six β-strands with the same typology in all enzymes. Unlike most proteins of the  complement system, which are synthesized by the liver and immune cells, factor D is predominantly produced by and secreted into the bloodstream by adipocytes. However, it is also synthesized by macrophages and monocytes, as well as by brain astrocytes to a lesser extent. The levels of factor D in serum can vary, but under normal conditions it is found within the range of 1-2 μg/mL range. Under healthy conditions, factor D, like other low molecular weight proteins, is filtered through the glomerulus and almost completely reabsorbed within the tubules, where it is then rapidly catabolized intracellularly.
Factor D is produced as a proenzyme or zymogen (pro-factor D) that requires subsequent cleavage of a 6-amino acid peptide for maturation. Conversion of pro-factor D into its mature form appears to happen rapidly, either during secretion in the secretory pathway or immediately thereafter. Although there has been some controversy, maturation of pro-factor D into mature factor D is thought to occur predominantly through the action of activated mannose-binding lectin-associated serine protease-3 (MASP-3) , one of the MASPs thought to play a role in the lectin pathway. Despite the need for enzyme-mediated maturation, however, factor D predominantly exists in its mature form in resting blood, likely because MASP-3 has no physiological inhibitors.
With mature factor D being the predominant form in resting blood, and because it has no known endogenous inhibitors itself, a high level of control is essential to prevent it from inappropriately cleaving endogenous proteins other than its substrate. As such, mature factor D is locked into an inactive state by a self-inhibitory loop. This loop dictates the enzyme’s low reactivity and extreme specificity for its substrate, factor B. Importantly, factor B can only be cleaved by factor D when factor B is bound to C3b or C3 (H2O) . Upon binding to and cleaving factor B, factor D is not permanently incorporated into the complex but is instead recycled in a reversible reaction. Accordingly, the kidney plays an important role in regulating the concentration of factor D via glomerular filtration. In addition to host defense against pathogens, the alternative pathway and factor D have been implicated in various other physiological processes. For example, C3b-mediated opsonization is known to be responsible for marking damaged liver cells for removal by phagocytes after acute liver injury. This process enables a scaffold for newly formed cells to develop and helps to prevent persistent inflammation. The alternative pathway and factor D have been shown to be essential in this process. Nonetheless, a fine balance in the level of activation of the alternative pathway is required because an  overactive complement cascade is also known to cause extensive hepatic cell death, persistent inflammation, and injury to the liver.
More recently, factor D has also been implicated in the aging process of the skin. During aging, it is known that the extracellular matrix of the dermal layer deteriorates as the quantity of senescent cells increases. Expression in human skin samples was higher in older subjects than in younger subjects. Another tissue in which factor D plays a role is adipose tissue. Adipocytes are energy reservoirs that play an important role in energy balance. Not only are they responsible for lipolysis but they are also involved in glucose uptake and triglyceride synthesis. Interestingly, factor D is not the only component of the complement system that is produced in adipose tissue. Other factors, including C3 and factor B, are also expressed to some degree in this tissue, where they have been found to activate the proximal part of the alternative pathway (i.e., upstream of C5 cleavage) in the absence of pathogens. In fact, factor D has been shown to be important for adipocyte differentiation and lipid accumulation via C3a signaling. In contrast, no activation of the terminal or lytic part of the pathway is observed, because proteins such as C5 are not expressed in adipose tissue. As part of their role in energy homeostasis, adipocytes also have endocrine function. In response to certain stimuli, they secrete regulatory molecules that play a role in the metabolic function of other tissues. These regulatory molecules include fatty acids and adipokines such as factor D. It has been shown that by controlling the production of C3a via the alternative pathway, factor D indirectly induces insulin secretion from pancreatic beta cells when glucose levels are elevated. Furthermore, factor D/C3a signaling has been found to preserve islet beta cells by blocking cell dedifferentiation and death. It has even been shown that higher levels of circulating factor D are associated with a lower risk of developing diabetes in middle-aged adults.
Deficiency in components of the proximal part of the alternative pathway, such as factor D, can lead to an inability to opsonize invading pathogens and to insufficient formation of the MAC. This ultimately limits both phagocytosis and lysis of the invaders. As such, it is not surprising that complete deficiency of factor D has been identified as a risk factor for serious bacterial infections. For example, complete factor D deficiency due to a Ser42Stop mutation in both alleles of the gene was observed in a Dutch individual suffering from meningitis. The complete factor D deficiency was linked with a decreased ability to opsonize and phagocytose bacteria and was also observed in other family members. Other case studies have also found  factor D deficiencies to be associated with N. gonorrhoeae and with various respiratory infections. Although the underlying factor D mutations are not always the same, they likely result in an unstable protein or an abnormally folded protein that cannot be secreted. Mutations in both alleles are required for complete factor D deficiency, and therefore the mode of inheritance is autosomal recessive.
The anti-FD antibody moiety can specifically bind to FD derived from any source, such as any organism that has a complement system, including but not limited to, dogs, cats, pigs, cows, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans. In some embodiments, the FD is human FD. In some embodiments, the FD is cynomolgus monkey FD (cyno FD) .
In some embodiments, the factor D is human factor D. In some embodiments, binding of the anti-FD antibody moiety to human-factor D is associated with a reduction in the generation of C3bBb in the complement activation pathway in an intact organism.
In some embodiments, the anti-FD antibody moiety binds to human FD. In some embodiments, the anti-FD antibody moiety binds to human FD only and does not bind to FD derived from another species. In some embodiments, the anti-FD antibody moiety has cross-species reactivity to FD other than human FD. Exemplary non-human FD include, but are not limited to, mouse FD, rat FD, rabbit FD, sheep FD, and cynomolgus monkey FD. In some embodiments, the anti-FD antibody moiety cross-reacts with cynomolgus monkey FD (cyno FD) . In some embodiments, the anti-FD antibody moiety does not cross-react with murine FD.
In some embodiments, the anti-FD antibody moiety binds to a mature FD molecule. In some embodiments, the anti-FD antibody moiety binds to a pro-FD. In some embodiments, the anti-FD antibody moiety binds to both pro-FD and mature FD.
The anti-FD antibody moiety can be any suitable format known in the art. In some embodiments, the anti-FD antibody moiety is selected from the group consisting of full-length antibody, Fab, Fab’ , F (ab’ ) 2, scFv, and a combination thereof. In some embodiments, the anti-FD antibody moiety comprises (or consists essentially of, or consists of) a full-length antibody, such as a full-length antibody comprising an Fc fragment derived from IgG4. In some embodiments, the anti-FD antibody moiety comprises (or consists essentially of, or consists of) an anti-FD scFv. In some embodiments, the anti-FD antibody moiety is murine, chimeric, or humanized antibody. In some embodiments, the anti-FD antibody moiety is monospecific. In some embodiments, the  anti-FD antibody moiety is multispecific. In some embodiments, the anti-FD antibody moiety is monovalent. In some embodiments, the anti-FD antibody moiety is multivalent.
In some embodiments, the anti-human FD antibody moiety has one or more mutations (e.g., insertion, deletion, and/or substitution) that reduce off-target binding. In some embodiments, the binding of the anti-FD antibody moiety to FD (e.g., human FD) is pH-dependent, wherein the anti-human FD antibody moiety binds more strongly to FD (e.g., human FD) at a neutral pH (e.g., about pH 7.4; such as that found in the blood) than it does at an acidic pH (e.g., about pH 5.8; such as that found in the endosome) . In some embodiments, the binding affinity of the pH-dependent anti-FD antibody moiety to FD (e.g., human FD) at about pH 7.4 is at least about 2 times (such as at least about any of 3, 4, 5, 6, 7, 8, 9, 10, or more) of the binding affinity of the pH-dependent anti-FD antibody moiety to FD (e.g., human FD) at about pH 5.8.
In some embodiments, the anti-FD antibody moiety is affinity-matured. In some embodiments, the affinity-matured anti-FD antibody moiety has a binding affinity to FD (e.g., human FD, and/or cyno FD) at least about 2-fold (e.g., at least about any of 3, 4, 5, 10, 50, 100, 1000, or more folds) of that of the parental anti-FD antibody moiety (i.e., not affinity-matured) . In some embodiments, the affinity-matured anti-FD antibody moiety has a similar (e.g., within about 2-fold difference) binding affinity to human FD as the parental anti-FD antibody moiety, but has a binding affinity to cynomolgus monkey FD at least about 2-fold (e.g., at least about any of 3, 4, 5, 10, 50, 100, 1000, or more folds) of that of the parental anti-FD antibody moiety.
In some embodiments, the anti-FD antibody moiety is affinity-matured, and the binding of the anti-FD antibody moiety to FD (e.g., human FD) is pH-dependent.
In some embodiments, the anti-human FD antibody moiety comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) .
In some embodiments, there is provided an anti-human FD antibody moiety comprising: i) a VH comprising an H-CDR1, an H-CDR2, and an H-CDR3, respectively comprising the amino acid sequence of an H-CDR1 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ) , an H-CDR2 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ) , and an H-CDR3 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ) within a reference  VH having the amino acid sequence set forth in SEQ ID NO: 7, and ii) a VL comprising an L-CDR1, an L-CDR2, and an L-CDR3, respectively comprising the amino acid sequence of an L-CDR1 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ) , an L-CDR2 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ) , and an L-CDR3 (or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ) within a reference VL having the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the CDR positions are according to Kabat numbering. In some embodiments, the affinity of such anti-human FD antibody moiety for FD (e.g., human FD, and/or cyno FD) is comparable (e.g., within about 2-fold difference) to that of a reference antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the affinity of such anti-human FD antibody moiety for FD (e.g., human FD, and/or cyno FD) is at least about 2-fold (e.g., at least about any of 3, 4, 5, 10, 50, 100, 1000, or more folds) of that of a reference antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8.
In some embodiments, there is provided an anti-human FD antibody moiety comprising a VH and a VL, wherein: (i) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ; an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ; and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ; and (ii) the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ; an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ; and an L-CDR3 comprising the  amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) . In some embodiments, there is provided an anti-human FD antibody moiety comprising a VH and a VL, wherein: (i) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ; an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ; and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and (ii) the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ; an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to 3 (e.g., 3, 2, or 1) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) ; and an L-CDR3 comprising the amino acid sequence of any of SEQ ID NOs: 6, 49, 89, and 97.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: (i) a VH comprising an H-CDR1 comprising the amino acid sequence of D-T-Y-V-H (SEQ ID NO: 1) ; an H-CDR2 comprising the amino acid sequence of R-I-D-P-X1-X2-G-X3-T-X4-F-X5-P-R-F-Q-A (SEQ ID NO: 9) , wherein X1 is A or H, X2 is N, S, or Y, X3 is L or H, X4 is T or H, and X5 is D, V, L, or H; and an H-CDR3 comprising the amino acid sequence of A-M-E-Y (SEQ ID NO: 3) ; and (ii) a VL comprising an L-CDR1 comprising the amino acid sequence of S-A-X6-S-D-V-S-X7-M-Y (SEQ ID NO: 10) , wherein X6 is R, N, or S, and X7 is Y, D, or V; an L-CDR2 comprising the amino acid sequence of X8-T-S-N-L-A-S (SEQ ID NO: 252) , wherein X8 is D or H; and an L-CDR3 comprising the amino acid sequence of Q-Q-W-S-S-X9-P-P-W-X10 (SEQ ID NO: 11) , wherein X9 is Y or H, X10 is T, R, or H. In some embodiments, the CDR positions are according to Kabat numbering.
In some embodiments (independent of or in addition to the CDR sequences described above) , the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence homology to SEQ  ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 8. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 10 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) in the VH domain, and the VL comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 10 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) in the VL domain. In some embodiments, the one or more amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) are in one or more of the CDRs. In some embodiments, the one or more amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) are in one or more of the framework regions (FRs) . In some embodiments, the two or more amino acid variations (e.g., insertions, deletions, and/or substitutions, such as conserved substitutions) are in both CDRs and FRs. In some embodiments, i) the amino acid residue at position 24 at the VH is A or T; ii) the amino acid residue at position 74 of the VH is K or T; iii) the amino acid residue at position 77 of the VH is N or S; iv) the amino acid residue of position 97 at the VH is A or T; v) the amino acid residue at position 98 of the VH is R or Y; and/or vi) the amino acid residue at position 70 of the VL is F or Y; wherein the numbering is according to the Kabat numbering system. In some embodiments, the amino acid residue position in VH is relative to a VH comprising the amino acid sequence of SEQ ID NO: 7, and the amino acid residue position in VL is relative to a VL comprising the amino acid sequence of SEQ ID NO: 8.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 7, and L- CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 18, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80%(e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence homology to SEQ ID NO: 18; and a VL comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence homology to SEQ ID NO: 19. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 18, and a VL comprising the amino acid sequence of SEQ ID NO: 19.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 23, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 26, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 26; and a VL comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 27. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid  sequence of SEQ ID NO: 26, and a VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 34, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 34; and a VL comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 35. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 34, and a VL comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 216, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 214. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 216, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 216; and a VL comprising the amino acid sequence of SEQ ID NO: 214, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 214. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 216, and a VL comprising the amino acid sequence of SEQ ID NO: 214.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 39, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H- CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 42, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 42; and a VL comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 43. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 42, and a VL comprising the amino acid sequence of SEQ ID NO: 43.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 44, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 46; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 47, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 49. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 50, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 50, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 50; and a VL comprising the amino acid sequence of SEQ ID NO: 51, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 51. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 50, and a VL comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the anti-human FD antibody moiety possess pH-dependent binding to human and/or cyno FD. In some embodiments, the anti-human FD antibody moiety binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4) than it does  at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold of that at an acidic pH.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 52, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 54; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 58, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 59. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 58, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 58; and a VL comprising the amino acid sequence of SEQ ID NO: 59, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 59. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 58, and a VL comprising the amino acid sequence of SEQ ID NO: 59.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 63, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 64, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 66, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 67. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 66, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence  homology to SEQ ID NO: 66; and a VL comprising the amino acid sequence of SEQ ID NO: 67, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 67. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 66, and a VL comprising the amino acid sequence of SEQ ID NO: 67.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 71, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 72, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 74, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 74, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 74; and a VL comprising the amino acid sequence of SEQ ID NO: 75, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 75. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 74, and a VL comprising the amino acid sequence of SEQ ID NO: 75.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 76, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 78; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 79, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 81. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 82, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 83.  In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 82, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 82; and a VL comprising the amino acid sequence of SEQ ID NO: 83, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 83. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 82, and a VL comprising the amino acid sequence of SEQ ID NO: 83.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 84, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 85, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 86; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 90, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 91. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 90, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 90; and a VL comprising the amino acid sequence of SEQ ID NO: 91, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 91. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 90, and a VL comprising the amino acid sequence of SEQ ID NO: 91.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 92, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 93, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 94; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97. In some  embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 98, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 99. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 98, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 98; and a VL comprising the amino acid sequence of SEQ ID NO: 99, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 99. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 98, and a VL comprising the amino acid sequence of SEQ ID NO: 99.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 100, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 101, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 102; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 106, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 107. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 106, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 106; and a VL comprising the amino acid sequence of SEQ ID NO: 107, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 107. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 106, and a VL comprising the amino acid sequence of SEQ ID NO: 107.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 108, an H- CDR2 comprising the amino acid sequence of SEQ ID NO: 109, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 110; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 114, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 115. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 114, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 114; and a VL comprising the amino acid sequence of SEQ ID NO: 115, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 115. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 114, and a VL comprising the amino acid sequence of SEQ ID NO: 115.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 116, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 117, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 118; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 122, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 123. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 122, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 122; and a VL comprising the amino acid sequence of SEQ ID NO: 123, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 123.  In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 122, and a VL comprising the amino acid sequence of SEQ ID NO: 123.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 168, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 169. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 168, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 168; and a VL comprising the amino acid sequence of SEQ ID NO: 169, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 169. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 168, and a VL comprising the amino acid sequence of SEQ ID NO: 169.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and an VL comprising a L-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 184, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 185. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the  amino acid sequence of SEQ ID NO: 184, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 184; and a VL comprising the amino acid sequence of SEQ ID NO: 185, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 185. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 184, and a VL comprising the amino acid sequence of SEQ ID NO: 185.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 186, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 187. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 186, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 186; and a VL comprising the amino acid sequence of SEQ ID NO: 187, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 187. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 186, and a VL comprising the amino acid sequence of SEQ ID NO: 187.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an L-CDR2 comprising the amino acid sequence of SEQ ID  NO: 125, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 289, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 127. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 289, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 289; and a VL comprising the amino acid sequence of SEQ ID NO: 127, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 127. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 289, and a VL comprising the amino acid sequence of SEQ ID NO: 127.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 190, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 191. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 190, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 190; and a VL comprising the amino acid sequence of SEQ ID NO: 191, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 191. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 190, and a VL comprising the amino acid sequence of SEQ ID NO: 191.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, there is provided an anti-human FD antibody moiety comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 192, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 193. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 192, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 192; and a VL comprising the amino acid sequence of SEQ ID NO: 193, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 193. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 192, and a VL comprising the amino acid sequence of SEQ ID NO: 193.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 194, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 195. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 194, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 194; and a VL comprising the amino acid sequence of  SEQ ID NO: 195, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 195. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 194, and a VL comprising the amino acid sequence of SEQ ID NO: 195.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 196, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 197. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 196, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 196; and a VL comprising the amino acid sequence of SEQ ID NO: 197, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 197. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 196, and a VL comprising the amino acid sequence of SEQ ID NO: 197.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 198,  and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 199. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 198, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 198; and a VL comprising the amino acid sequence of SEQ ID NO: 199, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 199. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 198, and a VL comprising the amino acid sequence of SEQ ID NO: 199.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 156, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 157. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 156, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 156; and a VL comprising the amino acid sequence of SEQ ID NO: 157, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 157. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 156, and a VL comprising the amino acid sequence of SEQ ID NO: 157.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the  amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 158, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 159. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 158, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 158; and a VL comprising the amino acid sequence of SEQ ID NO: 159, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 159. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 158, and a VL comprising the amino acid sequence of SEQ ID NO: 159.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 160, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 161. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 160, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 160; and a VL comprising the amino acid sequence of SEQ ID NO: 161, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 161. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid  sequence of SEQ ID NO: 160, and a VL comprising the amino acid sequence of SEQ ID NO: 161.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 162, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 163. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 162, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 162; and a VL comprising the amino acid sequence of SEQ ID NO: 163, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 163. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 162, and a VL comprising the amino acid sequence of SEQ ID NO: 163.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 164, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 164, or a variant thereof having at least about 80% (e.g., at  least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 164; and a VL comprising the amino acid sequence of SEQ ID NO: 165, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 165. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 164, and a VL comprising the amino acid sequence of SEQ ID NO: 165.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 166, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 167. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 166, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 166; and a VL comprising the amino acid sequence of SEQ ID NO: 167, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 167. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 166, and a VL comprising the amino acid sequence of SEQ ID NO: 167.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 57. In some  embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 172, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 173. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 172, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 172; and a VL comprising the amino acid sequence of SEQ ID NO: 173, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 173. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 172, and a VL comprising the amino acid sequence of SEQ ID NO: 173.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 174, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 175. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 174, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 174; and a VL comprising the amino acid sequence of SEQ ID NO: 175, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 175. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 174, and a VL comprising the amino acid sequence of SEQ ID NO: 175.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 176, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 177. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 176, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 176; and a VL comprising the amino acid sequence of SEQ ID NO: 177, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 177. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 176, and a VL comprising the amino acid sequence of SEQ ID NO: 177.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 178, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 179. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 178, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 178; and a VL comprising the amino acid sequence of SEQ ID NO:  179, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 179. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 178, and a VL comprising the amino acid sequence of SEQ ID NO: 179.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 180, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 181. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 180, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 180; and a VL comprising the amino acid sequence of SEQ ID NO: 181, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 181. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 180, and a VL comprising the amino acid sequence of SEQ ID NO: 181.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 182, and L- CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 183. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 182, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 182; and a VL comprising the amino acid sequence of SEQ ID NO: 183, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 183. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 182, and a VL comprising the amino acid sequence of SEQ ID NO: 183.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 206, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 207, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 208; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 209, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 210, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 211. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 212, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 213. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 212, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 212; and a VL comprising the amino acid sequence of SEQ ID NO: 213, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 213. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 212, and a VL comprising the amino acid sequence of SEQ ID NO: 213. In some embodiments, the anti-human FD antibody moiety possess pH-dependent binding to human and/or cyno FD. In some embodiments, the anti-human FD antibody moiety binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4)  than it does at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold of that at an acidic pH.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 234, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 235, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 236; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 237, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 238, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 239. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 253, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 254. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 253, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 253; and a VL comprising the amino acid sequence of SEQ ID NO: 254, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 254. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 253, and a VL comprising the amino acid sequence of SEQ ID NO: 254. In some embodiments, the anti-human FD antibody moiety possess pH-dependent binding to human and/or cyno FD. In some embodiments, the anti-human FD antibody moiety binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold of that at an acidic pH.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 240, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 241, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 242; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 243, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 244, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 245. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H- CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 255, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 256. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 255, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 255; and a VL comprising the amino acid sequence of SEQ ID NO: 256, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 256. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 255, and a VL comprising the amino acid sequence of SEQ ID NO: 256. In some embodiments, the anti-human FD antibody moiety possess pH-dependent binding to human and/or cyno FD. In some embodiments, the anti-human FD antibody moiety binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold of that at an acidic pH.
In some embodiments, there is provided an anti-human FD antibody moiety comprising: a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 246, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 247, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 248; and a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 249, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 250, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 251. In some embodiments, there is provided an anti-human FD antibody moiety that comprises H-CDR1, H-CDR2, and H-CDR3 of a VH comprising the amino acid sequence of SEQ ID NO: 257, and L-CDR1, L-CDR2, and L-CDR3 of a VL comprising the amino acid sequence of SEQ ID NO: 258. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 257, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 257; and a VL comprising the amino acid sequence of SEQ ID NO: 258, or a variant thereof having at least about 80% (e.g., at least about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%or more) amino acid sequence homology to SEQ ID NO: 258. In some embodiments, the anti-human FD antibody moiety comprises a VH comprising the  amino acid sequence of SEQ ID NO: 257, and a VL comprising the amino acid sequence of SEQ ID NO: 258. In some embodiments, the anti-human FD antibody moiety possess pH-dependent binding to human and/or cyno FD. In some embodiments, the anti-human FD antibody moiety binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold of that at an acidic pH.
In some embodiments, the anti-human FD antibody moiety is a full-length antibody (anti-human FD full-length antibody) . In some embodiments, the anti-human FD full-length antibody comprises an Fc region, such as a human Fc region. In some embodiments, the Fc region is derived from an IgG molecule, such as any one of the IgG1, IgG2, IgG3, or IgG4 subclass. In some embodiments, the anti-human FD full-length antibody comprises a heavy chain constant region derived from IgG4 (e.g., human IgG4) . In some embodiments, the Fc region is capable of mediating an antibody effector function, such as ADCC and/or CDC. For example, antibodies of subclass IgG1, IgG2, and IgG3 with wild-type Fc sequences usually show complement activation including C1q and C3 binding, whereas IgG4 does not activate the complement system and does not bind C1q and/or C3. In some embodiments, the Fc region comprises a modification that reduces binding affinity of the Fc region to an Fc receptor (FcR) . In some embodiments, the Fc region is derived from an IgG4 Fc (e.g., human IgG4 Fc) . In some embodiments, the heavy chain constant region comprises the amino acid sequence of a wild-type human IgG4 heavy chain constant region. In some embodiments, the IgG4 heavy chain constant region or Fc region comprises mutations. See, for example, Armour KL et al., Eur J. Immunol. 1999; 29: 2613; and Shields RL et al., J. Biol. Chem. 2001; 276: 6591. In some embodiments, the heavy chain constant region comprises one or more mutations (e.g., insertion, deletion, and/or substitution) in the hinge region for improved stability. In some embodiments, the heavy chain constant region comprises an S228P substitution (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence. In some embodiments, the IgG4 heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 132. In some embodiments, the heavy chain constant region comprises one or more mutations (e.g., insertion, deletion, and/or substitution) for increased (e.g., increasing at least about any of 20%, 50%, 80%, 90%, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, or more) binding to neonatal FcR (FcRn; e.g., human and/or cyno FcRn) , herein also referred to as “FcRn variants. ” Any FcRn variants that increase  the binding of an anti-human FD full-length antibody to an FcRn (e.g., at about pH 5.8 and/or about pH 7.4) can be used herein. In some embodiments, the heavy chain constant region comprises M428L/N434A ( “LA” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence. In some embodiments, the heavy chain constant region comprises M428L/N434A ( “LA” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 133. In some embodiments, the heavy chain constant region comprises M252Y/V308P/N434Y ( “YPY” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence. In some embodiments, the heavy chain constant region comprises M252Y/V308P/N434Y ( “YPY” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 134. In some embodiments, the heavy chain constant region comprises M252Y/N286E/N434Y ( “YEY” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence. In some embodiments, the heavy chain constant region comprises M252Y/N286E/N434Y ( “YEY” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the heavy chain constant region comprises L432C/H433S/N434W/Y436L/T437CE ( “N3E” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence. In some embodiments, the heavy chain constant region comprises L432C/H433S/N434W/Y436L/T437CE ( “N3E” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132. “T437CE” designates an amino acid substitution of T437C and an insertion of E right after the 437 position (EU numbering) . In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the heavy chain constant region comprises L432C/H433S/N434W/Y436L/T437C ( “N3” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence. In some embodiments, the heavy chain constant region comprises L432C/H433S/N434W/Y436L/T437C (“N3” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the  amino acid sequence of SEQ ID NO: 132. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 137. In some embodiments, the heavy chain constant region comprises M252Y/S254T/T256E ( “YTE” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence. In some embodiments, the heavy chain constant region comprises M252Y/S254T/T256E ( “YTE” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the heavy chain constant region comprises M252Y/S254T/T256E/L432E/H433R/N434F/Y436R/T437Q ( “Y31-YTE” ) substitutions (EU numbering) relative to a wild-type human IgG4 heavy chain constant region sequence. In some embodiments, the heavy chain constant region comprises M252Y/S254T/T256E/L432E/H433R/N434F/Y436R/T437Q ( “Y31-YTE” ) substitutions (EU numbering) relative to a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 132. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 139. In some embodiments, any of the mutations (e.g., insertion, deletion, and/or substitution) that increase Fc’s binding to FcRn described herein can be combined. For example, “N3E” mutations can be combined with the “YTE” mutations to generate “N3E-YTE” FcRn variant. In some embodiments, the S228P mutation (EU numbering) can be combined with any of the FcRn mutations described herein. In some embodiments, the heavy chain constant region of an anti-human FD full-length antibody (e.g., any of the anti-human FD full-length antibodies described herein) comprises the amino acid sequence of any of SEQ ID NOs: 132-139.
In some embodiments, the anti-human FD antibody moiety is an anti-human FD full-length antibody that: i) binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold (e.g., at least about any of 2.5, 3, 3.5, 5, 10-fold, or more) of that at an acidic pH; and ii) binds more strongly to FcRn (e.g., human FcRn and/or cyno FcRn) at about pH 5.8 and/or about pH 7.4 than a reference antibody without the FcRn mutation (s) , e.g., the binding affinity to FcRn of the anti-human FD full-length antibody is at least about 2-fold (e.g., at least about any of 5, 10, 50, 100, 1000-fold, or more) of that of a reference antibody without the FcRn mutation (s) . In some embodiments, the anti-human FD antibody moiety is an  anti-human FD full-length antibody that: i) binds more strongly to FD (e.g., human FD and/or cyno FD) at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) , e.g., the binding affinity at a neutral pH is at least about 2-fold (e.g., at least about any of 2.5, 3, 3.5, 5, 10-fold, or more) of that at an acidic pH; and ii) is affinity-matured, e.g., the binding affinity to FD (e.g., human FD and/or cyno FD) is at least about 2-fold (e.g., at least about any of 3, 5, 10, 20-fold, or more) of that of its parental antibody without affinity-maturation. In some embodiments, the anti-human FD antibody moiety is an anti-human FD full-length antibody that: i) binds more strongly to FcRn (e.g., human FcRn and/or cyno FcRn) at about pH 5.8 and/or about pH 7.4 than a reference antibody without the FcRn mutation (s) , e.g., the binding affinity to FcRn of the anti-human FD full-length antibody is at least about 2-fold (e.g., at least about any of 5, 10, 50, 100, 1000-fold, or more) of that of a reference antibody without the FcRn mutation (s) ; and ii) is affinity-matured, e.g., the binding affinity to FD (e.g., human FD and/or cyno FD) is at least about 2-fold (e.g., at least about any of 2.5, 3, 3.5, 5, 10-fold, or more) of that of its parental antibody without affinity-maturation.
In some embodiments, the anti-human FD antibody moiety is an scFv (anti-human FD scFv) . In some embodiments, the anti-human FD scFv comprises from N’ to C’ : VH-optional linker-VL. In some embodiments, the anti-human FD scFv comprises from N’ to C’ : VL-optional linker-VH. Any suitable linker (e.g., see “Linker” subsection below) can be used herein, including but are not limited to any of SEQ ID NOs: 221-229. In some embodiments, there is provided an anti-human FD scFv comprising the amino acid sequence of SEQ ID NO: 219 or 220.
Also provided are anti-human FD antibody moieties and anti-human FD antibody constructs that bind to FD (e.g., human FD and/or cyno FD) competitively with any of the anti-human FD antibody moieties or anti-human FD antibody constructs described herein.
Fc domain
In some embodiments, the isolated anti-human FD antibody construct or the anti-human FD antibody moiety comprises an Fc domain. In some embodiments, the anti-human FD antibody moiety is an anti-human FD full-length antibody.
In some embodiments, the Fc domain is an Fc effector domain, namely, an Fc domain possessing some or all effector functions, including for example complement and ADCC functions. In some embodiments, the Fc effector domain is an IgG1 or IgG3 Fc region.
In some embodiments, one or more amino acid modifications may be introduced into the Fc domain of the antibody moiety, thereby generating an Fc domain variant. The Fc domain variant may comprise a human Fc domain sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc domain) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
In some embodiments, the Fc domain possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody moiety in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
In vitro and/or in vivo cytotoxicity assays can be conducted to analyze CDC and/or ADCC activities of the Fc domain. For example, Fc receptor (FcR) binding assays can be conducted to determine whether the antibody possesses FcγR binding (hence likely ADCC activity) , and/or retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991) . Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5, 500, 362 (see, e.g., Hellstrom, I. et al. Proc. Nat’ l Acad. Sci. USA 83: 7059-7063 (1986) ) and Hellstrom, I et al., Proc. Nat’ l Acad. Sci. USA 82: 1499-1502 (1985) ; 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987) ) . Alternatively, non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoToxnon-radioactive cytotoxicity assay (Promega, Madison, WI) . Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’ l Acad. Sci. USA 95: 652-656 (1998) . C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) ; Cragg, M.S. et al., Blood 101: 1045-1052 (2003) ; and Cragg, M.S. and M.J. Glennie, Blood 103: 2738-2743 (2004) ) . FcRn binding and in vivo clearance/half-life  determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’ l. Immunol. 18 (12) : 1759-1769 (2006) ) .
Antibodies with reduced effector function include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) . Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581) . In some embodiments, the Fc domain of the multispecific construct or the isolated anti-C5 antibody construct does not comprise a mutation that reduces its effector function, such as one or more mutations described herein. In some embodiments, the Fc domain of the multispecific construct or the isolated anti-C5 antibody construct comprises one or more of these mutations.
Certain antibody variants with improved or diminished binding to FcRs are described. See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9 (2) : 6591-6604 (2001) .
In some embodiments, the Fc domain is an IgG1 Fc domain. In some embodiments, the IgG1 Fc domain does not comprise L234A mutation and/or a L235A mutation. In some embodiments, the IgG1 Fc domain comprises a L234A mutation and/or a L235A mutation (“LALA” mutation) . In some embodiments, the Fc domain is an IgG3 Fc domain. In some embodiments, the Fc domain is an IgG2 or IgG4 Fc domain. In some embodiments, the Fc domain is a human IgG4 Fc comprising the amino acid sequence of SEQ ID NO: 231. In some embodiments, the Fc domain is an IgG4 Fc domain comprising S228P, L234A, and/or L235A mutation. In some embodiments, the Fc domain is an IgG4 Fc fragment comprising an S228P mutation, such as comprising the amino acid sequence of SEQ ID NO: 232. In some embodiments, the Fc domain is an IgG4 Fc fragment comprising S228P/M428L/N434A ( “PLA” ) triple mutations, such as comprising the amino acid sequence of SEQ ID NO: 230.
In some embodiments, the anti-human FD antibody moiety (or the isolated anti-human FD antibody construct) comprises an Fc domain with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc domain (EU numbering of residues) .
In some embodiments, alterations are made in the Fc domain that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC) ,  e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000) .
In some embodiments, the anti-human FD antibody moiety (or the isolated anti-human FD antibody construct) comprises a variant Fc domain comprising one or more amino acid substitutions which alters half-life and/or changes binding to FcRn. Antibodies with increased half-lives and improved binding to the FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994) ) , are described in US2005/0014934A1 (Hinton et al. ) . Those antibodies comprise an Fc domain with one or more substitutions therein which alters binding of the Fc domain to FcRn. Such Fc variants include those with substitutions at one or more of Fc domain residues, e.g., substitution of Fc domain residue 434 (US Patent No. 7,371,826) .
See also Duncan &Winter, Nature 322: 738-40 (1988) ; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 concerning other examples of Fc domain variants.
Binding affinities of anti-human FD antibody moieties
Binding affinity and specificity of the anti-human FD antibody moieties described herein can be determined experimentally by methods known in the art. For example, the binding of an antibody to a protein antigen can be detected and/or quantified using a variety of techniques such as, but not limited to, Western blot, dot blot, surface plasmon resonance (SPR) method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N. J. ) , Bio-Layer Interferometry (BLI) (e.g., Octet system, ForteBio) , RIA, ECL, IRMA, EIA, peptide scans, and enzyme-linked immunosorbent assay (ELISA) . In addition, methods for measuring the affinity (e.g., dissociation and association constants by BLI) are set forth in the working examples.
In some embodiments, the anti-human FD antibody moiety specifically binds FD (e.g., human FD and/or cyno FD) with a KD of about 10-7 M to about 10-12 M, such as any of about 10- 7 M to about 10-10 M, about 10-8 M to about 10-11 M, about 10-9 M to about 10-11 M, about 10-8 M to about 10-10 M, about 10-8 M to about 10-9 M, about 10-9 M to about 10-10 M, or about 10-10 M to about 10-11 M. In some embodiments, the anti-human FD antibody moiety specifically binds FD (e.g., human FD and/or cyno FD) with a KD of about 10-8 M to about 10-12 M, or about 1×10- 9 M to about 1×10-12 M.
In some embodiments, the anti-human FD antibody moiety binds more strongly to human FD and/or cyno FD at a neutral pH (e.g., about pH 7.4) than it does at an acidic pH (e.g., about pH 5.8) . In some embodiments, the anti-human FD antibody moiety specifically binds FD (e.g., human FD and/or cyno FD) with a KD of about 5×10-10 M to about 10×10-10 M at about pH 5.8. In some embodiments, the anti-human FD antibody moiety specifically binds FD (e.g., human FD and/or cyno FD) with a KD of about 1×10-10 M to about 5×10-10 M at about pH 7.4.
Biological properties of anti-human FD antibody moieties
The AP is thought to be constitutively active at a low level due to spontaneous hydrolysis of C3 to form C3 (H2O) . C3 (H2O) behaves like C3b in that it can associate with fB, which make fB susceptible to fD cleavage and activation. The resultant C3 (H2O) Bb then cleaves C3 to produce C3b and C3a to initiate the AP cascade by forming the C3 convertase of the AP, C3bBb. As the initial C3 convertase generates increasing amounts of C3b, an amplification loop is established. It should be noted that because the CP and LP also generate C3b, wherein C3b can bind factor B and engages the AP, the AP amplification loop also participates in the CP and LP once these pathways are activated. Thus, the AP consists of two functional entities: an independent complement activation pathway that is unrelated to CP or LP, and an amplification process that does participate and contribute to the full manifestation of CP and LP.
In some embodiments, the anti-human FD antibody moiety inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the AP. In some embodiments, the anti-human FD antibody moiety inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the AP amplification process or amplification loop. In some embodiments, the anti-human FD antibody moiety does not inhibit (or inhibit at most about any of 10%, 5%, 2%, 1%, or less) the activation of CP or LP. In some embodiments, the anti-human FD antibody moiety or use thereof preserves the ability of an individual to combat an infection through the CP and LP. In some embodiments, the anti-human FD antibody moiety inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the amplification process or amplification loop. In some embodiments, the IC50 value of the anti-FD antibody moiety in inhibiting the CP and/or LP (e.g., inhibiting sheep RBC lysis in 20%human serum) is undetectable.
In some embodiments, the activity of the AP that is inhibited using a method of the invention or an anti-human FD antibody moiety described herein is AP activation induced by one  or more of lipopolysaccharide (LPS) , lipooligosaccharide (LOS) , pathogen-associated molecular patterns (PAMPs) , and danger-associated molecular patterns (DAMPs) . In some embodiments, the activity of the AP that is inhibited using a method of invention or an anti-human FD antibody moiety described herein is the generation of C3bBb protein complex. In some embodiments, the activity of the AP that is inhibited using a method of the invention or an anti-human FD antibody moiety described herein is FD-dependent.
Methods for determining whether a particular antibody described herein inhibits human FD are known in the art. Inhibition of human FD can reduce the APC-mediated cell-lysing ability a subject’s body fluids. Such reductions of the cell-lysing ability present in the body fluid (s) can be measured by methods well known in the art such as, for example, by a conventional hemolytic assay such as the hemolysis assay in chicken erythrocyte hemolysis method as described in, e.g., Hillmen et al. (2004) N Engl. J Med 350 (6) : 552; and Yuan et al. (2017) Haematologica 102 (3) : 466-475. The concentration and/or physiologic activity of FD in a body fluid can be measured by methods well known in the art. Methods for measuring FD concentration or activity include, e.g., ELISAs (see, e.g., Corvillo et al. (2021) Int J Mol Sci 22(12) : 6608) . Inhibition of FD can result in the inhibition of alternative pathway activities, such as the production of C3b, as shown in Barratt et al. (2021) Front Immunl. 12: 712572. Thus, for example, the ELISA assays can be used to determine the inhibition of LPS-induced C3b deposition by an anti-FD antibody, as described in, e.g., Kimura et al. (2008) Blood. 111 (2) : 732-740, Li et al. (2020) Front Immunol. 11: 1123, and “Instructions for UseComplement System Alternative Pathway” Document No. LABEL-DOC-0034, 2.0, December 2018. Other assays known in the art can also be used. Using assays of these or other suitable types, candidate agents capable of inhibiting human FD can be screened.
Hemolytic assays can be used to determine the inhibitory activity of an anti-FD antibody on FD-mediated complement activation, and are thus useful for determining potential off-target binding of anti-FD antibodies. These assays include but are not limited to a rabbit red blood cell (RBC) lysis test. In order to determine the effect of the humanized anti-FD on complement pathway-mediated hemolysis in a serum test solution in vitro, for example, a rabbit RBC lysis assay may be used to examine AP regulation, or an LPS-based ELISA assay may be used to examine AP complement inhibition. The percentage of lysis is normalized by considering 100%lysis equal to the lysis occurring in the absence of the inhibitor. C3 inhibition can be  measured by methods known in the art, including, e.g., flow cytometry and commercial kits (e.g., assays) . See, for example, Mannes et al, (2021) Blood. 137 (4) : 443-455 and “Instructions for UseComplement System Alternative Pathway” Document No. LABEL-DOC-0034, 2.0, December 2018.
In some embodiments, binding of the anti-human FD antibody moiety to FD is associated with a reduction (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) in the generation of C3bBb in the complement activation pathway in an intact organism (e.g., human) . In some embodiments, the anti-human FD antibody moiety inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) LPS-induced C3b and/or C5b-9 deposition. In some embodiments, the anti-FD antibody moiety does not inhibit (or inhibit at most about any of 10%, 5%, 2%, 1%, or less) IgM-induced or mannan-induced C3b and/or C5b-9 deposition.
Methods of testing the activities (e.g., inhibition of the AP) of anti-human FD antibody moieties are well known in the art, including but not limited to, rabbit RBC lysis assay (e.g., induced by human serum) , or LPS-induced C3b and/or C5b-9 deposition assay. See, e.g., assays in US11434279, the content of which is incorporated herein by reference in its entirety. Also see Example 4 herein.
pH-dependent and/or FcRn binding optimized anti-human FD antibody moieties
In some embodiments, the anti-human FD antibody moiety described herein possess pH-dependent dissociation from factor D (e.g., human FD and/or cyno FD) . Such pH-dependent binding provides for greater persistence of administered antibody or antibody fusion protein molecules, because immune complexes (i.e., the anti-human FD antibody construct bound to factor D) taken up by cells will dissociate in the acidic environment of the endosome and allow the freed antibody or antibody fusion protein to be recycled back out of the cell through the neonatal Fc receptor (FcRn) where it is available to bind to a new factor D molecule.
As used herein, the expression “pH-dependent binding” means that the antibody exhibits reduced (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) binding to FD at acidic pH (e.g., about pH 5.8; such as in the endosome) as compared to its binding at neutral pH (e.g., about pH 7.4; such as in the blood) .
pH-dependency of the anti-human FD antibody moiety described herein can be determined experimentally by methods known in the art, such as in US20220204602,  US20220177556, US9,079,949, and US9765135, the contents of each of which are incorporated herein by reference in their entirety. Also see Example 3 herein. pH-dependency for FD binding may be reflected in the differences in binding properties such as binding affinity (e.g., dissociation constant) , kinetic parameters (e.g., association rate and dissociation rate) , and percentage dissociation, at different pH levels. In some embodiments, the pH-dependency of the anti-human FD antibody moiety may be expressed in terms of the ratio of the percentage dissociation. In some embodiments, the percentage dissociation may be expressed in terms of the low-pH dissociation factor and the neutral-pH dissociation factor. In some embodiments, the pH-dependency of the anti-human FD antibody moiety is expressed in the Koff ratio of about pH 5.8 vs.about pH 7.4. In some embodiments, the pH-dependency of the anti-human FD antibody moiety is expressed in the KD ratio of about pH 5.8 vs. about pH 7.4.
The pH dependence for FD binding of the anti-human FD antibody moiety can be assessed based on the dissociation of a FD-bound antibody at an acidic pH (e.g., about pH 5.8) or at a neutral pH (e.g., about pH 7.4) . Low-pH dissociation factor, namely, the percentage of antibody dissociated at about pH 5.8 from the antigen at about 25℃, wherein the antibody is pre-bound to the antigen at about pH 7.4, can be used to determine the dissociation of an FD-bound antibody at an acidic pH. The low-pH dissociation factor may be measured by associating an antibody and an antigen (e.g., the anti-human FD antibody construct and human/cyno factor D) at pH 7.4 for about 600 seconds, followed by a dissociation period of about 600 seconds in a buffer at about pH 5.8, and calculation of the percentage of antibody dissociated at about pH 5.8 from the antigen. In some embodiments, the low-pH dissociation factor of the anti-human FD antibody moiety (i.e., the %of dissociation of the anti-human FD antibody moiety from the bound FD under an acidic pH (e.g., about pH 5.8) ) is no less than about any of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%.
Neutral-pH dissociation factor, namely, the percentage of antibody dissociated at about pH 7.4 from the antigen at about 25 ℃, wherein the antibody is pre-bound to the antigen at about pH 7.4 and can be used to determine the dissociation of an FD-bound antibody at a neutral pH. The neutral-pH dissociation factor may be measured by associating an antibody and an antigen (e.g., the anti-human FD antibody construct and human/cyno FD) at about pH 7.4 for about 600 seconds, followed by a dissociation period of about 600 seconds in a buffer at about pH 7.4, and calculation of the percentage of antibody dissociated at about pH 7.4 from the antigen. In some  embodiments, the neutral-pH dissociation factor of the anti-human FD antibody moiety (i.e., the %of dissociation of the anti-human FD antibody moiety from the bound FD under a neutral pH (e.g., about pH 7.4) ) is no more than about any of 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.
In some embodiments, the ratio of the low-pH dissociation factor over the neutral-pH dissociation factor of the anti-human FD antibody moiety of the present invention is at least about any one of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 20, 50, or more. In some embodiments, the percentage of dissociation of the antibody for factor D at pH 5.8 over the percentage of dissociation of the antibody for factor D at pH 7.4 is at least about any of 3, 4, 5, or 6.
In some embodiments, the anti-human FD antibody moiety binds FD (e.g., human and/or cyno FD) more strongly (such as at least about any of 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, or higher, more strongly) at a neutral pH (such as about pH 7.4) than it does at an acidic pH (such as about pH 5.8) .
The FcRn variant anti-human FD antibody moiety described herein in some embodiments exhibit prolonged (e.g., increasing at least about any of 10%, 20%, 50%, 70%, 90%, 1.5-fold, 2-fold, 5-fold, 20-fold, or more) serum half-life in vivo, compared to a reference anti-human FD antibody without FcRn binding mutation (s) , such as in mice (including transgenic mice) . In some embodiments, the anti-human FD antibody moiety exhibits prolonged serum half-life in other test animals, include but are not limited to, rats, chickens, rabbits, sheep, and cyno monkeys. In some embodiments, the anti-human FD antibody moiety exhibits prolonged serum half-life in human.
Antibodies with pH-dependent binding to antigens enhance antigen degradation within the cell in endosomes or lysosomes; and more freed or unbound antibodies are rescued via FcRn binding and are ultimately recycled back into the circulation and bind to additional antigen (Igawa et al., Nat Biotechnol. 2010 Nov; 28 (11) : 1203-7) . The pH-dependent antigen binding properties can naturally occur or be created by engineering of the antigen binding interactions.
IgG Fc naturally binds to FcRn in a pH-dependent manner. Engineered antibodies with enhanced pH-dependent FcRn binding properties, e.g., increased (e.g., increasing at least about any of 2-fold, 5-fold, 20-fold, or more) binding to FcRn at acidic pH (e.g., about pH 5.8) and little (e.g., within about 2-fold difference) or no change binding to FcRn under neutral pH (e.g.,  about pH 7.4) , that can be rescued and recycled more effectively back to the serum are also known as “recycling antibodies. ” . For instance, increasing the binding affinity of an antibody’s Fc to FcRn at an acidic pH within endosomes can improve antibody serum persistence and has been used for therapeutic antibody development. Recycling antibodies can have prolonged serum half-life. Engineered antibody Fc that have increased binding affinity to FcRn at both acidic pH (e.g., about pH 5.8) within endosome and at neutral pH (e.g., about pH 7.4) can enhance antigen/antibody complex uptake from the circulation (to reduce serum antigen level) and recycle from endosome back to the circulation to bind more new antigens, thus named as “sweeping” antibody (Igawa et al., PloS One. 2013 May 7; 8 (5) : e63236) . For instances, FcRn binding mutation LA has been shown to increase antibody recycling resulting long IgG serum persistence (Yeung et al., J Immunol (2009) 182 (12) : 7663-7671) . FcRn binding mutations Y31-YTE and N3E (Borrok et al., J Biol Chem. 2015 Feb 13; 290 (7) : 4282-90) as well as YEY and YPY (Igawa et al., PloS One. 2013 May 7; 8 (5) : e63236) have been shown to significantly increasing FcRn binding at both pH 7.4, and at pH 5.8. Antibody carrying these Fc mutations observed extended serum persistence and/or reduced serum antigen levels. The contents of each of these references are incorporated herein by reference in their entireties.
In some embodiments, the anti-human FD antibody moiety has pH-dependent binding to FD (e.g., human and/or cyno FD) and has faster (e.g., at least about any of 50%, 70%, 90%, 1.5-fold, 2-fold, 5-fold, 20-fold, or more faster) dissociation from FD at acidic pH (e.g., about pH 5.8) than that at neutral pH (e.g., about pH 7.4) . In some embodiments, the anti-human FD antibody moiety (e.g., a full-length anti-human FD antibody) has improved (e.g., increasing at least about any of 10%, 20%, 50%, 70%, 90%, 1.5-fold, 2-fold, 5-fold, 20-fold, 100-fold, 1000-fold, or more) binding to FcRn, such as under about pH 7.4 and/or about pH 5.8, e.g., compared to a reference anti-human FD antibody without FcRn binding mutation (s) . In some embodiments, the anti-human FD antibody moiety is a recycling antibody moiety. In some embodiments, the anti-human FD antibody moiety is a sweeping antibody moiety. In some embodiments, the anti-human FD antibody moiety is both a recycling antibody moiety and a pH-dependent FD binding antibody moiety. In some embodiments, the anti-human FD antibody moiety is both a sweeping antibody moiety and a pH-dependent FD binding antibody moiety. In some embodiments, the binding affinity of an anti-human FD full-length antibody to FcRn at about pH 7.4 is about 10-11 M to about 10-5 M, such as about 10-10 M to about 10-6 M, or about 10-9 M to about 10-8 M. In  some embodiments, the binding affinity of an anti-human FD full-length antibody to FcRn at about pH 5.8 is about 10-11 M to about 10-7 M, such as about 10-11 M to about 10-8 M, or about 10-10 M to about 10-8 M.
Fusion Partners and Multispecific Constructs
In some embodiments, the isolated anti-human FD antibody constructs described herein further comprise a fusion partner, or a second antibody moiety that specifically recognizes a component of the complement pathway (hereinafter also referred to as “complement protein” ) . Hence in some embodiments, there is provided an isolated anti-human FD antibody construct comprising: an anti-human FD antibody moiety (e.g., any of the anti-human FD antibody moieties described herein) and a second antibody moiety specifically recognizing a component of the complement pathway (e.g., FD, or non-FD complement protein) . Any complement protein can be used herein. Exemplary fusion partners include, but are not limited to, anti-FD antibody moieties (e.g., any of the anti-human FD antibody moieties described herein) , anti-C2 antibody moieties, or anti-C5 antibody moieties. In some embodiments, the isolated anti-human FD antibody construct is monospecific. In some embodiments, the isolated anti-human FD antibody construct is multivalent. In some embodiments, the isolated anti-human FD antibody construct is multispecific ( “multispecific construct” ) .
In some embodiments, the second antibody moiety is selected from the group consisting of: a full-length antibody, Fab, Fab’ , F (ab) 2, F (ab’ ) 2, scFv, sdAb, and a combination thereof. In some embodiments, the second antibody moiety is an scFv.
The complement system plays an important role in the pathology of many autoimmune, inflammatory, and ischemic diseases. Inappropriate complement activation and its deposition on host cells can lead to complement-mediated lysis and/or injury of cells and target tissues, as well as tissue destruction due to the generation of powerful mediators of inflammation. The complement system, also known as complement cascade, is a part of the immune system that enhances (complements) the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promote inflammation, and attack the pathogen’s cell membrane. It is part of the innate immune system, which is not adaptable and does not change during an individual’s lifetime. The complement system can, however, be recruited and brought into action by antibodies generated by the adaptive immune system.
Without being bound by any theory or hypothesis, there are three known complement pathways: the alternative complement pathway (AP) , the classical pathway (CP) , and the lectin pathway (LP) . Generally, the CP is initiated by antigen-antibody complexes, the LP is activated by binding of lectins to sugar molecules on microbial surfaces, while the AP is constitutively active at a low level but can be quickly amplified on bacterial, viral, and parasitic cell surfaces due to the lack of regulatory proteins. Host cells are usually protected from AP complement activation by regulatory proteins. But in some situations, such as when the regulatory proteins are defective or missing, the AP can also be activated uncontrollably on host cells, leading to complement-mediated disease or disorder. The CP consists of components C1, C2, and C4, and converges with the AP at the C3 activation step. The LP consists of mannose-binding lectins (MBLs) and MBL-associated serine proteases (MASPs) and shares with the CP the components C4 and C2. The AP consists of components C3 and several factors, such as factor B, factor D, properdin, and the fluid phase regulator factor H. Complement activation consists of three stages: (a) recognition, (b) enzymatic activation, and (c) membrane attack leading to cell death. The first phase of CP complement activation begins with C1. C1 is made up of three distinct proteins: a recognition subunit, C1q, and the serine protease subcomponents, C1r and C1s, which are bound together in a calcium-dependent tetrameric complex, C1r2 s2. An intact C1 complex is necessary for physiological activation of C1 to result. Activation occurs when the intact C1 complex binds to immunoglobulin complexed with antigen. This binding activates C1s which then cleaves both the C4 and C2 proteins to generate C4a and C4b, as well as C2a and C2b. The C4b and C2a fragments combine to form the C3 convertase, C4b2a, which in turn cleaves C3 to form C3a and C3b. Activation of the LP is initiated by MBL binding to certain sugars on the target surface and this triggers the activation of MBL-associated serine proteases (MASPs) which then cleave C4 and C2 in a manner analogous to the activity of C1s of the CP, resulting in the generation of the C3 convertase, C4b2a. Thus, the CP and LP are activated by different mechanisms, but they share the same components C4 and C2 and both pathways lead to the generation of the same C3 convertase, C4b2a. The cleavage of C3 by C4b2a into C3b and C3a is a central event of the complement pathway for two reasons. It initiates the AP amplification loop because surface deposited C3b is a central intermediate of the AP C3 convertase C3bBb. Both C3a and C3b are biologically important. C3a is proinflammatory and together with C5a are referred to as anaphylatoxins. C3b and its further cleavage products also bind to complement receptors present  on neutrophils, eosinophils, monocytes and macrophages, thereby facilitating phagocytosis and clearance of C3b-opsonized particles. Finally, C3b can associate with C4b2a or C3bBb to form the C5 convertase of the CP and LP, and AP, respectively, to activate the terminal complement sequence, leading to the production of C5a, a potent proinflammatory mediator, and the assembly of the lytic membrane attack complex (MAC) , C5-C9.
Defective complement action is a cause of several human glomerular diseases including atypical hemolytic uremic syndrome (aHUS) , anti-neutrophil cytoplasmic antibody mediated vasculitis (ANCA) , C3 glomerulopathy, IgA nephropathy, immune complex membranoproliferative glomerulonephritis, renal ischemic reperfusion injury, lupus nephritis, membranous nephropathy, and chronic transplant mediated glomerulopathy. Aberrant complement component activation has been proposed as markers in various types of cancers and their clinical outcomes. Lung cancer patients show significantly higher plasma levels of complement proteins and activation fragments than do control donors, and elevated complement levels are correlated with lung tumor size. Complement-related proteins are also elevated in biological fluids from patients with other types of tumors. See, for example, Pio et al. Semin Immunol. 2013 Feb; 25 (1) : 54-64. Inhibition of the complement cascade has been proposed for glomerular diseases and cancer treatment.
In some embodiments, the second antibody moiety specifically recognizes FD. In some embodiments, the isolated anti-human FD antibody construct comprises two or more anti-human FD antibody moieties (e.g., scFv) , such as two or more any of the anti-human FD antibody moieties described herein. In some embodiments, the two or more anti-human FD antibody moieties are fused to each other in tandem. In some embodiments, the two or more anti-human FD antibody moieties are on different polypeptide chains within the isolated anti-human FD antibody construct. In some embodiments, the two or more anti-human FD antibody moieties bind to the same FD epitope. In some embodiments, the two or more anti-human FD antibody moieties bind to at least two different FD epitopes. In some embodiments, each of the two or more anti-human FD antibody moieties binds to a different FD epitope.
In some embodiments, the second antibody moiety specifically recognizes complement component 2 ( “C2” ) . In some embodiments, the second antibody moiety specifically recognizes complement component 5 ( “C5” ) . In some embodiments, the isolated anti-human FD antibody construct comprises an anti-human FD antibody moiety (e.g., any of the anti-human FD antibody  moieties described herein) and one or more antibody moieties (e.g., scFvs) specifically recognizing a complement protein that is not FD, such as fused to each other or with the anti-human FD antibody moiety in tandem, or on different polypeptide chains.
In some embodiments, the isolated anti-human FD antibody construct comprises (or consists essentially of, or consists of) one polypeptide chain. In some embodiments, the isolated anti-human FD antibody construct comprises (or consists essentially of, or consists of) two or more (e.g., 2) polypeptide chains, such as a heterodimer or a homodimer formed in the presence of an Fc domain.
In some embodiments, the isolated anti-human FD antibody construct comprises an anti-human FD scFv ( “scFv1” ; e.g., any of the anti-human FD scFvs described herein) fused to a second scFv (scFv2) specifically recognizing a component of the complement pathway. In some embodiments, the scFv2 is an anti-C2 scFv. In some embodiments, the scFv2 is an anti-C5 scFv. In some embodiments, the scFv (e.g., scFv1 or scFv2) comprises from N-terminus to C-terminus: VH-optional linker-VL. In some embodiments, the scFv (e.g., scFv1 or scFv2) comprises from N-terminus to C-terminus: VL-optional linker-VH.
Linkers
In some embodiments, the isolated anti-human FD antibody construct comprises one or more linkers between the anti-human FD antibody moiety (e.g., anti-FD scFv) and the second antibody moiety (e.g., scFv) specifically recognizing the complement protein. The length, the degree of flexibility, and/or other properties of the linker (s) include but not are limited to influencing the affinity, specificity, or avidity, for one or more particular antigens or epitopes. For example, longer linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another. In some embodiments, a linker (such as peptide linker) comprises flexible residues (such as glycine (G) and serine (S) ) so that anti-human FD antibody moiety (e.g., scFv) and the second antibody moiety specifically recognizing the complement protein are free to move relative to each other. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a cleavable linker.
Other linker considerations include the effect on physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as planned degradation) , rigidity, flexibility,  immunogenicity, modulation of antibody binding, the ability to be incorporated into a micelle or liposome, and the like.
The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO1996/034103.
The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, 200, or more, amino acids long. In some embodiments, the peptide linker is no more than about any of 200, 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or fewer, amino acids long. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.
In some embodiments, the peptide linker does not comprise any polymerization activity. The characteristics of a peptide linker, which comprise the absence of the promotion of secondary structures, are known in the art and described, e.g., in Dall’A cqua et al. (Biochem. (1998) 37, 9266-9273) , Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9 (1) , 73-80) . In some embodiments, the peptide linker does not promote the formation of any secondary structures. The linkage of the domains to each other can be provided by, e.g., genetic engineering. Methods for preparing fused and operatively linked bispecific single chain constructs and expressing them in mammalian cells or bacteria are well-known in the art (e.g. WO1999/054440, Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y. 1989 and 1994 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001) .
The peptide linker can be a stable linker, which is not cleavable by proteases, especially by Matrix metalloproteinases (MMPs) .
In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G) n, where n is an integer of at least one, and glycine-serine polymers (including, for example, (GS) n, where n is an integer of at least one) , glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, the linker is a GS linker.
In some embodiments, the isolated anti-human FD antibody construct (e.g., multispecific construct) comprises two polypeptide chains each comprising a subunit of the Fc domain, and the two polypeptide chains dimerize through the Fc domain.
In some embodiments, the isolated anti-human FD antibody construct (e.g., multispecific construct) comprising an Fc domain has one or more of the following properties: i) has at least about 20% (e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, or more) longer half-life in vivo; and ii) has at least about 20%(e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, or more) stronger activity in inhibiting one or more of CP, LP, AP, and TP, compared to a same isolated anti-human FD antibody construct (e.g., multispecific construct) without the Fc domain (e.g., different antibody moieties are connected via a G4S linker) .
In some embodiments, the anti-human FD antibody moiety is fused to the second antibody moiety via an Fc domain, such as an Fc domain comprising the amino acid sequence of SEQ ID NO: 230. Any of the Fc domains described under the “Fc domain” subsection above can be used herein in the linker. In some embodiments, the Fc domain is derived from human IgG4 Fc.In some embodiments, the Fc domain is flanked by GGGGS (SEQ ID NO: 221) on each end of its sequence. In some embodiments, the Fc domain is flanked by a linker (e.g., SEQ ID NO: 227) on one or each end of its sequence, then situated in between the anti-human FD antibody moiety (e.g., anti-FD scFv1) and the second antibody moiety (e.g, . scFv2) . In some embodiments, the Fc domain is flanked by GGGGSGGGGS (SEQ ID NO: 227) on each end of its sequence (hereinafter also referred to as “flanked Fc domain linker” ) , then situated in between the anti-human FD antibody moiety (e.g., anti-FD scFv1) and the second antibody moiety (e.g., scFv2) . In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 230. In some embodiments, the flanked Fc domain linker comprises the amino acid sequence of SEQ ID NO: 233.
In some embodiments, the anti-human FD antibody moiety (e.g., scFv) is fused to the fusion partner or the second antibody moiety (e.g., scFv) directly. In some embodiments, the anti-human FD antibody moiety (e.g., scFv) is fused to the fusion partner or the second antibody moiety (e.g., scFv) via a linker (e.g., peptide linker) . In some embodiments, the linker within an scFv (e.g., anti-human FD scFv and/or the scFv against the complement protein) , and/or the linker between two or more antibody moieties within the anti-human FD antibody construct, is independently is selected from the group consisting of SEQ ID NOs: 221-229, such as any of SEQ ID NOs: 221 and 226-229.
In some embodiments, the isolated anti-human FD antibody construct comprises from N terminus to C terminus: an anti-human FD scFv (e.g., any of the anti-human FD scFvs described herein) , an optional linker, and an anti-C2 scFv or an anti-C5 scFv. In some embodiments, the isolated anti-human FD antibody construct comprises from N terminus to C terminus: an anti-C2 scFv or anti-C5 scFv, an optional linker, and an anti-human FD scFv (e.g., any of the anti-human FD scFvs described herein) . In some embodiments, the isolated anti-human FD antibody construct comprises from N terminus to C terminus: an anti-human FD scFv (e.g., any of the anti-human FD scFvs described herein) , a first optional linker, an Fc domain, a second optional linker, and an anti-C2 scFv or an anti-C5 scFv. In some embodiments, the isolated anti-human FD antibody construct comprises from N terminus to C terminus: an anti-C2 scFv or an anti-C5 scFv, a first optional linker, an Fc domain, a second optional linker, and an anti-human FD scFv (e.g., any of the anti-human FD scFvs described herein) . In some embodiments, when the isolated anti-human FD antibody construct comprises an Fc domain as whole or part of a linker, the two polypeptide chains each comprising an Fc subunit dimerize through the Fc domain.
Complement factor 2 (C2)
In some embodiments, the complement factor the second antibody moiety binds to is complement factor 2 (C2) , such that the anti-human FD antibody construct is an anti-human FD/anti-C2 multispecific construct. Any anti-C2 antibody or antigen binding fragment thereof described in WO2023143583 (the content of which is incorporated herein by reference in its entirety) can be used in the anti-C2 antibody moieties described herein. In some embodiments, the anti-C2 antibody moiety is an anti-C2 scFv.
Human C2 is an 83 kDa protein (100 kDa glycosylated) that is produced as an inactive, heavily glycosylated zymogen consisting of five domains: three N-terminal complement-control-protein (CCP) domains, a von Willebrand factor A-type (VWA) domain, and a C-terminal trypsin-like serine proteinase (SP) domain. The last two domains, VWA and SP, form the C2a fragment (residues 224-732, 70 kDa) that is produced in the proteolytic activation cascade of the classical and lectin pathways. The larger fragment C2a is 57.4 kDa (70 kDa glycosylated) and provides the catalytic center to the convertase complexes of the classical and lectin-binding pathways of complement activation. C2 bound to C4b is cleaved by classical (C1s) or lectin (MASP2) proteases to produce C4bC2a, a very short-lived C3 convertase that in turn cleaves C3 to C3a and C3b, leading ultimately to formation of Membrane Attack Complex (MAC) and lysis of bacteria and damaged cells. C2 has the same serine protease domain as C4bC2a but in an inactive zymogen-like conformation, requiring cofactor-induced conformational change for activity.
The anti-C2 antibody moiety can specifically bind to C2 derived from any source, such as any organism that has a complement system, including but not limited to, dogs, cats, pigs, cows, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans. In some embodiments, the C2 is murine C2. In some embodiments, the C2 is human C2. In some embodiments, the C2 is cynomolgus monkey C2.
In some embodiments, the anti-C2 antibody moiety binds to C2. In some embodiments, the anti-C2 antibody moiety binds to C2a. In some embodiments, the anti-C2 antibody moiety has cross-species reactivity to C2 other than human C2, and/or to C2a other than human C2a. Exemplary non-human C2 and C2a include, but are not limited to, mouse C2 and C2a, rat C2 and C2a, rabbit C2 and C2a, sheep C2 and C2a, cynomolgus monkey C2 and C2a. In some embodiments, the anti-C2 antibody moiety cross-reacts with cynomolgus C2 and/or C2a.
The anti-C2 antibody moiety can be any suitable format known in the art. In some embodiments, the anti-C2 antibody moiety is selected from the group consisting of full-length antibody, Fab, Fab’ , F (ab’ ) 2, scFv, sdAb, and a combination thereof. In some embodiments, the anti-C2 antibody moiety comprises an sdAb that binds to C2. In some embodiments, the anti-C2 antibody moiety is a full-length antibody, such as a full-length antibody comprising an Fc fragment derived from IgG4 (e.g., an IgG4 Fc comprising a PLA mutation, such as SEQ ID NO: 230) . In some embodiments, the anti-C2 antibody moiety comprises (or consists essentially of, or  consists of) an anti-C2 scFv. In some embodiments, the anti-C2 antibody moiety is murine, chimeric, humanized, or human antibody. In some embodiments, the anti-C2 antibody moiety is monospecific. In some embodiments, the anti-C2 antibody moiety is multispecific. In some embodiments, the anti-C2 antibody moiety is monovalent. In some embodiments, the anti-C2 antibody moiety is multivalent.
In some embodiments, the binding of the anti-C2 antibody moiety to C2 (e.g., human C2) is pH-dependent, and wherein the anti-C2 antibody moiety binds more strongly to C2 at a neutral pH (e.g., about pH 7.4; such as that found in the blood) than it does at an acidic pH (e.g., about pH 5.8; such as that found in the endosome) . In some embodiments, the binding affinity of the pH-dependent anti-C2 antibody moiety to C2 (e.g., human C2) at about pH 7.4 is at least about 3 times (such as at least about any of 4, 5, 6, 7, 8, 9, 10, or more) times higher than the binding affinity of the pH-dependent anti-C2 antibody moiety to C2 (e.g., human C2) at about pH 5.8.
In some embodiments, the anti-C2 antibody moiety comprises: (i) a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 259, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 260, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 261; and (ii) a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 262, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 263, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 264. In some embodiments, the anti-C2 antibody moiety comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 265, or a variant thereof that has at least about 80% (e.g., at least about any of 85%, 90%, 95%, 97%, 99%, or more) amino acid homology to SEQ ID NO: 265; and a VL domain comprising the amino acid sequence of SEQ ID NO: 266, or a variant thereof that has at least about 80% (e.g., at least about any of 85%, 90%, 95%, 97%, 99%, or more) amino acid homology to SEQ ID NO: 266. In some embodiments, the anti-C2 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 265, and a VL comprising the amino acid sequence of SEQ ID NO: 266. In some embodiments, the anti-C2 antibody moiety is an scFv, such as an anti-C2 scFv comprising the amino acid sequence of SEQ ID NO: 267 or 268.
In some embodiments, the anti-C2 antibody moiety comprises: (i) a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 269, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 270, and an H-CDR3 comprising the amino acid sequence  of SEQ ID NO: 271; and (ii) a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 272, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 273, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 274. In some embodiments, the anti-C2 antibody moiety comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 275, or a variant thereof that has at least about 80% (e.g., at least about any of 85%, 90%, 95%, 97%, 99%, or more) amino acid homology to SEQ ID NO: 275; and a VL domain comprising the amino acid sequence of SEQ ID NO: 276, or a variant thereof that has at least about 80% (e.g., at least about any of 85%, 90%, 95%, 97%, 99%, or more) amino acid homology to SEQ ID NO: 276. In some embodiments, the anti-C2 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 275, and a VL comprising the amino acid sequence of SEQ ID NO: 276. In some embodiments, the anti-C2 antibody moiety is an scFv, such as an anti-C2 scFv comprising the amino acid sequence of SEQ ID NO: 277 or 278.
In some embodiments, the anti-C2 antibody moiety specifically binds C2 (e.g., human C2) with a KD of about 10-7 M to about 10-12 M, such as any of about 10-7 M to about 10-10 M, about 10-8 M to about 10-11 M, about 10-9 M to about 10-11 M, about 10-8 M to about 10-10 M, about 10-8 M to about 10-9 M, about 10-9 M to about 10-10 M, or about 10-10 M to about 10-11 M. In some embodiments, the anti-C2 antibody moiety specifically binds C2 (e.g., human C2) with a KD of about 1×10-10 M to about 5×10-10 M.
In some embodiments, the anti-C2 antibody moieties described herein inhibit (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) the CP and/or LP. In some embodiments, the anti-C2 antibody moieties described herein inhibit no more than about 70% (e.g., no more than about any of 60%, 50%, 40%, 30%, 20%, 10%, or less) of the CP and/or LP. In some embodiments, the anti-C2 antibody moiety does not inhibit (or inhibits at most about any of 10%, 5%, 2%, 1%, or less) the activation of AP. In some embodiments, the anti-C2 antibody moiety inhibits about 50%to about 80%of the CP and/or LP. In some embodiments, the IC50 value of the anti-C2 antibody moiety in inhibiting the AP (e.g., inhibiting rabbit RBC lysis in 20%human serum) is undetectable. In some embodiments, the anti-C2 antibody moiety inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) IgM-induced and/or mannan-induced C3b and/or C5b-9 deposition. In some embodiments, the anti-C2 antibody moiety does not inhibit (or inhibit at most about any of 10%, 5%, 2%, 1%, or less) LPS-induced C3b and/or C5b-9 deposition.
In some embodiments, the anti-human FD/anti-C2 multispecific construct: i) inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the CP and/or LP, such as about 50%to about 80%of the CP and/or LP, and ii) inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the AP. In some embodiments, the anti-human FD/anti-C2 multispecific construct inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) all of CP, LP, AP, and TP. In some embodiments, the anti-human FD/anti-C2 multispecific construct inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) IgM-, mannan-, and LPS-induced C3b and C5b-9 deposition. In some embodiments, the anti-human FD/anti-C2 multispecific construct inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) sheep RBC lysis and rabbit RBC lysis (e.g., induced by human serum) .
Complement factor 5 (C5)
In some embodiments, the complement factor the second antibody moiety binds to is complement factor 5 (C5) , such that the anti-human FD antibody construct is an anti-human FD/anti-C5 multispecific construct. Any anti-C5 antibody or antigen binding fragment thereof described in US20220204602, US20220177556, US11578137, WO2022134047, and PCT/US2023/063305 can be used in the anti-C5 antibody moieties described herein, the content of each of which is incorporated herein by reference in their entirety. In some embodiments, the anti-C5 antibody moiety is an anti-C5 scFv.
The C5 gene encodes a preproprotein is proteolytically processed to generate multiple protein products, including the C5 alpha chain, C5 beta chain, C5a anaphylatoxin and C5b. Human C5 is a 188 kDa protein that is comprised of the C5 alpha and beta chains, which are linked by a disulfide bridge. Cleavage of the alpha chain by a convertase enzyme results in the formation of the C5a anaphylatoxin, which possesses potent spasmogenic and chemotactic activity, and the C5b macromolecular cleavage product. The C5b macromolecular cleavage product can form a complex with the complement component C6, which then becomes the membrane attack complex (MAC) . Mutations in this gene cause complement component 5 deficiency, a disease characterized by recurrent bacterial infections.
The anti-C5 antibody moiety can specifically bind to C5 derived from any source, such as any organism that has a complement system, including but not limited to, dogs, cats, pigs,  cows, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans. In some embodiments, the C5 is murine C5. In some embodiments, the C5 is human C5.
In some embodiments, the anti-C5 antibody moiety binds to C5. In some embodiments, the anti-C5 antibody moiety binds to C5b. In some embodiments, the anti-C5 antibody moiety has cross-species reactivity to C5 other than human C5, and/or to C5b other than human C5b. Exemplary non-human C5 and C5b include, but are not limited to, mouse C5 and C5b, rat C5 and C5b, rabbit C5 and C5b, sheep C5 and C5b, cynomolgus monkey C5 and C5b. In some embodiments, the anti-C5 antibody moiety cross-reacts with cynomolgus C5 and/or C5b. In some embodiments, the anti-C5 antibody moiety does not bind to C5a.
The anti-C5 antibody moiety can be any suitable format known in the art. In some embodiments, the anti-C5 antibody moiety is selected from the group consisting of full-length antibody, Fab, Fab’ , F (ab’ ) 2, scFv, sdAb, and a combination thereof. In some embodiments, the anti-C5 antibody moiety comprises an sdAb that binds to C5. In some embodiments, the anti-C5 antibody moiety is a full-length antibody, such as a full-length antibody comprising an Fc fragment derived from IgG4 (e.g., an IgG4 Fc comprising a PLA mutation, such as SEQ ID NO: 230) . In some embodiments, the anti-C5 antibody moiety comprises (or consists essentially of, or consists of) an anti-C5 scFv. In some embodiments, the anti-C5 antibody moiety is murine, chimeric, humanized, or human antibody. In some embodiments, the anti-C5 antibody moiety is monospecific. In some embodiments, the anti-C5 antibody moiety is multispecific. In some embodiments, the anti-C5 antibody moiety is monovalent. In some embodiments, the anti-C5 antibody moiety is multivalent.
In some embodiments, the binding of the anti-C5 antibody moiety to C5 (e.g., human C5) is pH-dependent, and wherein the anti-C5 antibody moiety binds more strongly to C5 at a neutral pH (e.g., about pH 7.4; such as that found in the blood) than it does at an acidic pH (e.g., about pH 5.8; such as that found in the endosome) . In some embodiments, the binding affinity of the pH-dependent anti-C5 antibody moiety to C5 (e.g., human C5) at about pH 7.4 is at least about 3 times (such as at least about any of 4, 5, 6, 7, 8, 9, 10, or more) times higher than the binding affinity of the pH-dependent anti-C5 antibody moiety to C5 (e.g., human C5) at about pH 5.8.
In some embodiments, the anti-C5 antibody moiety binds to an epitope in the α-chain of C5, and/or an epitope in the β-chain of C5.
In some embodiments, the anti-C5 antibody moiety comprises (i) a VH comprising an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 279, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 280, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 281; and (ii) a VL comprising an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 282, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 283, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 284. In some embodiments, the anti-C5 antibody moiety comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 285, or a variant thereof that has at least about 80% (e.g., at least about any of 85%, 90%, 95%, 97%, 99%, or more) amino acid homology to SEQ ID NO: 285; and a VL domain comprising the amino acid sequence of SEQ ID NO: 286, or a variant thereof that has at least about 80% (e.g., at least about any of 85%, 90%, 95%, 97%, 99%, or more) amino acid homology to SEQ ID NO: 286. In some embodiments, the anti-C5 antibody moiety comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 285, and a VL domain comprising the amino acid sequence of SEQ ID NO: 286. In some embodiments, the anti-C5 antibody moiety is an scFv comprising the amino acid sequence of SEQ ID NO: 287 or 288.
In some embodiments, the anti-C5 antibody moiety specifically binds C5 (e.g., human C5) with a KD of about 10-7 M to about 10-12 M, such as any of about 10-7 M to about 10-10 M, about 10-8 M to about 10-11 M, about 10-9 M to about 10-11 M, about 10-8 M to about 10-10 M, about 10-8 M to about 10-9 M, about 10-9 M to about 10-10 M, or about 10-10 M to about 10-11 M. In some embodiments, the anti-C5 antibody moiety specifically binds C5 (e.g., human C5) with a KD of about 1×10-9 M to about 5×10-9 M.
In some embodiments, the anti-human FD/anti-C5 multispecific construct: i) inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the TP, and ii) inhibits (e.g., inhibit at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the AP. In some embodiments, the anti-human FD/anti-C5 multispecific construct inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) IgM-, mannan-, and LPS-induced C5b-9 deposition. In some embodiments, the anti-human FD/anti-C5 multispecific construct inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) LPS-induced C3b deposition. In some embodiments, the anti-human FD/anti-C5 multispecific construct does not inhibit (or inhibits at most about any of 10%, 5%, 2%, 1%, or less) IgM-induced or mannan-induced C3b  deposition. In some embodiments, the anti-FD/anti-C5 multispecific construct inhibits (e.g., inhibits at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) sheep RBC lysis and rabbit RBC lysis (e.g., induced by human serum) .
In some embodiments, the activity of the complement pathway that is inhibited by an anti-human FD/anti-C5 multispecific construct is complement pathway activation induced by one or more of LPS, LOS, PAMPs, and DAMPs. In some embodiments, the activity of complement signaling that is inhibited by the anti-human FD/anti-C5 multispecific construct is the generation of C5a protein, the generation of C5b protein, and/or the formation of MAC. In some embodiments, the activity of the complement pathway that is inhibited by anti-human FD/anti-C5 multispecific construct is C5-dependent.
Methods of testing the activities of the multispecific construct described herein, such as inhibition of one or more of AP, CP, LP, and TP, are well known in the art, including but not limited to, rabbit RBC lysis assay (e.g., induced by human serum) , sheep RBC lysis assay (e.g., induced by human serum) , IgM-induced C3b and/or C5b-9 deposition assay, mannan-induced C3b and/or C5b-9 deposition assay, or LPS-induced C3b and/or C5b-9 deposition assay. See, e.g., assays in US11434279, WO2023143583, US20220204602, US20220177556, US11578137, WO2022134047, and PCT/US2023/063305, the content of each of which is incorporated herein by reference in their entirety. Also see Example 4 herein.
Chimeric or Humanized Antibodies
In some embodiments, one or more of the antibody moieties of the isolated anti-human FD antibody constructs (e.g., multispecific construct) or the anti-human FD antibody moieties of the present application is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984) ) . In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from mouse) and a human constant region. In some embodiments, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are  derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived) , e.g., to restore or improve antibody specificity or affinity.
Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008) , and are further described, e.g., in Riechmann et al., Nature 332: 323-329 (1988) ; Queen et al., Proc. Nat’ l Acad. Sci. USA 86: 10029-10033 (1989) ; US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36: 25-34 (2005) (describing SDR (a-CDR) grafting) ; Padlan, Mol. Immunol. 28: 489-498 (1991) (describing “resurfacing” ) ; Dall’ Acqua et al., Methods 36: 43-60 (2005) (describing “FR shuffling” ) ; and Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al., Br. J. Cancer, 83: 252-260 (2000) (describing the “guided selection” approach to FR shuffling) .
Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 15 1 : 2296 (1993) ) ; Framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 8 9 : 4285 (1992) ; and Presta et al. J. Immunol., 151: 2623 (1993) ) ; human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008) ) ; and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996) ) .
Human Antibodies
In some embodiments, one or more of the antibody moieties of the isolated anti-human FD antibody constructs (e.g., multispecific construct) of the present application is a human antibody (known as human domain antibody, or human dAb) . Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) , Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008) , and Chen, Mol. Immunol. 47 (4) : 912-21 (2010) . Transgenic mice or rats capable of producing fully human single-domain antibodies (or dAb) are known in the art.  See, e.g., US20090307787A1, U.S. Pat. No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.
Human antibodies (e.g., human dAbs) may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005) . See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSETM technology; U.S. Patent No. 5,770,429 describingtechnology; U.S. Patent No. 7,041,870 describing K-Mtechnology, and U.S. Patent Application Publication No. US 2007/0061900, describingtechnology) . Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.
Human antibodies (e.g., human dAbs) can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (See, e.g., Kozbor J. Immunol., 133: 3001 (1984) ; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987) ; and Boerner et al., J. Immunol., 147: 86 (1991) ) . Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006) . Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26 (4) : 265-268 (2006) (describing human-human hybridomas) . Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3) : 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27 (3) : 185-91 (2005) .
Human antibodies (e.g., human dAbs) may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain.
Substitution, Insertion, and Deletion Variants
In some embodiments, antibody variants comprising one or more amino acid substitutions are included in the isolated anti-human FD antibody constructs (e.g., multispecific construct) or anti-human FD antibody moieties described herein. Sites of interest for substitutional mutagenesis include the HVRs (or CDRs) and FRs. Conservative substitutions are shown in Table B under the heading of “Preferred substitutions. ” More substantial changes are provided in Table B under the heading of “exemplary substitutions, ” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
Table B. Amino acid substitutions
Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody) . Generally, the resulting variant (s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity) .
Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR “hotspots, ” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008) ) , and/or SDRs (a-CDRs) , with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O’ Brien et al., ed., Human Press, Totowa, NJ, (2001) ) . In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis) . A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to  bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots” or CDRs. In some embodiments of the variant VHH sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
Amino acid sequence insertions include amino-and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N-or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
Glycosylation variants
In some embodiments, one or more antibody moieties of the isolated anti-human FD antibody constructs (e.g., multispecific construct) or the anti-human FD antibody moieties of the present application is altered to increase or decrease the extent to which the construct is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
Where the antibody moiety (or the isolated anti-human FD antibody construct) comprises an Fc domain, the carbohydrate attached thereto may be altered. Native antibodies  produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc domain. See, e.g., Wright et al. TIBTECH 15: 26-32 (1997) . The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc) , galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in the antibody moiety may be made in order to create antibody variants with certain improved properties.
In some embodiments, the antibody moiety (or the isolated anti-human FD antibody construct) has a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc domain. For example, the amount of fucose in such antibody may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc domain (EU numbering of Fc domain residues) ; however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L. ) ; US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd) . Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004) ; Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) . Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986) ; US Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially at Example 11) , and knockout cell lines, such as alpha-1, 6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) ; Kanda, Y. et al., Biotechnol. Bioeng., 94 (4) : 680-688 (2006) ; and WO2003/085107) .
In some embodiments, the antibody moiety (or the isolated anti-human FD antibody construct) has bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc domain of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al. ) ; US Patent No. 6, 602, 684 (Umana et al. ) ; and US 2005/0123546 (Umana et al. ) . Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc domain are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.) ; WO 1998/58964 (Raju, S. ) ; and WO 1999/22764 (Raju, S. ) .
Cysteine Engineered Antibody Variants
In some embodiments, it may be desirable to create cysteine engineered antibody moieties, e.g., “thioMAbs, ” in which one or more residues of one or more of the antibody moieties in an isolated anti-human FD antibody construct herein are substituted with cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In some embodiments, any one or more of the following residues may be substituted with cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc domain. Cysteine engineered antibody moieties may be generated as described, e.g., in U.S. Patent No. 7, 521, 541.
III. Nucleic Acids, Vectors, and host cells
Nucleic acid molecules encoding any of the isolated anti-human FD antibody constructs (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) , or various antibody moieties described herein are also contemplated. In some embodiments, there is provided a nucleic acid (e.g., isolated nucleic acid) encoding (e.g., one or more polypeptide components of) the anti-human FD antibody constructs or various antibody moieties therein. Also provided are mRNAs comprising such nucleic acids. In some embodiments, the mRNA is formulated in liposomes or lipid nanoparticles. Further provided are vectors comprising any of the nucleic acids (e.g., isolated nucleic acid) described herein, such as viral vectors (e.g., AAV vector or lentiviral vector) .
Also contemplated here are isolated host cell comprising or expressing (e.g., one or more polypeptide components of) any of the isolated anti-human FD antibody constructs (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) described herein. Also provided are isolated host cells comprising a nucleic acid encoding (e.g., one or more polypeptide components of) the isolated anti-human FD antibody construct described herein. Also provided are isolated host cells comprising a vector comprising a nucleic acid encoding (e.g., one or more polypeptide components of) the isolated anti-human FD antibody construct described herein.
The present application also includes variants to these nucleic acid sequences. For example, the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding (e.g., one or more polypeptide components of) the isolated anti-human FD antibody constructs (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) or various antibody moieties described herein under at least moderately stringent hybridization conditions.
The present application also provides vectors in which a nucleic acid of the present application is inserted.
The nucleic acid can be clone into a number of types of vectors. For example, the nucleic acid can be clone into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) , and in other virology and molecular biology manuals. Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193) .
In some embodiments, the nucleic acid encoding the isolated anti-human FD antibody construct (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific  constructs, or anti-human FD/anti-C5 multispecific constructs) comprises sequences or features in addition to the coding sequence encoding the isolated anti-human FD antibody construct. For example, the nucleic acid can be an mRNA (or a DNA sequence encoding the mRNA) comprising optional features that improve targeting of the isolated anti-human FD antibody construct encoded by the mRNA and to increase stability of the mRNA.
In some embodiments, the mRNA encoding the anti-human FD antibody moiety or the isolated anti-human FD antibody construct comprises a nucleic acid sequence encoding a signal peptide. Signal peptides are short peptides that may be present at the N-terminus or the C-terminus of a newly synthesized protein, that may function to properly translocate the protein. In some embodiments, the signal peptide assists with translocating the anti-human FD antibody moiety or the isolated anti-human FD antibody construct encoded by the coding sequence of the mRNA. In some embodiments, the signal peptide translocates the anti-human FD antibody moiety or the isolated anti-human FD antibody construct to the cellular membrane.
In some embodiments, the mRNA comprises one or more untranslated regions (UTRs) . The UTR of the mRNA may be involved in various regulatory aspects of gene expression. It should be understood that the UTRs (e.g., the 5’ UTRs and/or the 3’ UTRs) provided herein are examples, and that the mRNA may comprise any UTR from any gene. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide synthetic (e.g., artificial UTRs) which are not variants of wild type genes. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5’ or 3’ , UTR may be inverted, shortened, lengthened, made chimeric with one or more other 5’ UTRs or 3’ UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3’ or 5’ UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3’ or 5’ ) comprise a variant UTR.
In some embodiments, a double, triple or quadruple UTR, such as a 5’ or 3’ UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. It is also within the scope of the present  invention to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level. The UTRs provided herein may also include translation enhancer elements (TEE) .
In some embodiments, the mRNA comprises a 5’ UTR. The 5’ UTRs provided herein may be recognized by the ribosome, thereby allowing the ribosome to bind and initiate translation of the mRNA (e.g., translation of the coding sequence and/or nucleic acid encoding a signal peptide of the mRNA) . In some embodiments, the 5’ UTR is upstream from the coding sequence of the mRNA.
In some embodiments, the mRNA comprises a 3’ UTR. The 3’ UTRs provided herein may be involved in translation termination (e.g., translation of the coding sequence and/or nucleic acid encoding a signal peptide of the mRNA) , and can also be important for post-transcriptional modifications. In some embodiments, the 3’ UTR is downstream from the coding sequence of the mRNA. In some embodiments, the 3’ UTR immediately follows the translation stop codon of the coding sequence of the mRNA. In some embodiments, the mRNA comprises one or more stop codons before the 3’ UTR.
In some embodiments, the mRNA comprises a 5’ UTR and a 3’ UTR, such as any of the 5’ UTRs and 3’ UTRs provided herein. In some embodiments, the 5’ UTR and the 3’ UTR are derived from the same species. In some embodiments, the 5’ UTR and the 3’ UTR are not derived from the same species. In some embodiments, the 5’ UTR is synthetic, and the 3’ UTR is not synthetic. In some embodiments, the 5’ UTR is not synthetic, and the 3’ UTR is synthetic.
In some embodiments, the mRNA comprises one or more additional features, such as but not limited to a poly (A) sequence, one or more chemical modifications, a 5’ cap, or a combination thereof.
In some embodiments, the mRNA comprises a poly (A) sequence (e.g., a polyadenylation sequence) . Poly (A) sequences consist of multiple adenosine monophosphates in succession. In some embodiments, the poly (A) sequence is crucial for translation of the mRNA. In some embodiments, the poly (A) sequence is downstream of the coding sequence of the mRNA. In some embodiments, the poly (A) sequence is downstream of a 3’ UTR of the mRNA. In some embodiments, the poly (A) sequence has a length of about 50 nucleotides or longer, such  as about 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, or longer. In some embodiments, the poly (A) sequence has a length of about 150 nucleotides or shorter, such as about 100 nucleotides, 90 nucleotides, 80 nucleotides, 70 nucleotides, 50 nucleotides, or shorter. In some embodiments, the poly (A) sequence has a length of about 105 nucleotides.
Vectors and delivery of vectors
The present application also contemplates vectors comprising a nucleic acid encoding any of the isolated anti-human FD antibody constructs described herein (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) . The term “vector” as used herein refers to DNA or RNA molecules that comprise a coding nucleotide sequence that can be transcribed into RNAs or expressed into proteins. In some embodiments, the vector contains one or more regulatory elements operably linked to the nucleotide sequence encoding the isolated anti-human FD antibody construct. When the vector is introduced into a host cell, under suitable conditions, the coding nucleotide sequence in the construct can be transcribed or expressed.
In some embodiments, the vector comprises a promoter that is operably linked to the coding nucleotide sequence, such that the promoter controls the transcription or expression of the coding nucleotide sequence. A promoter may be positioned 5’ (upstream) of a coding nucleotide sequence under its control. The distance between the promoter and the coding sequence may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function. In some embodiments, the vector comprises a 5’ UTR and/or a 3’ UTR that regulates the transcription or expression of the coding nucleotide sequence. In some embodiments, the promoter is a U6 promoter. In some embodiments, the promoter is a Poly II promoter as discussed in the sections described above.
Suitable vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular) ; nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid, ” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Certain vectors are capable of  autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) . Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the transcription or expression of coding nucleotide sequences to which they are operatively linked. Such vectors are referred to herein as “expression vectors” .
In some embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector, and a mammalian cell vector.
Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012) , and in Ausubel et al. (1999) , and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses (AAV) , herpes viruses, and lentiviruses. In some embodiments, a murine stem cell virus (MSCV) vector is used to express a desired nucleic acid. MSCV vectors have been demonstrated to efficiently express desired nucleic acids in cells. Other examples of viral vectors are those based upon Moloney Murine Leukemia Virus (MoMuLV) and human immunodeficiency virus (HIV) . In some embodiments, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193) .
Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for transcription or expression of the nucleic acid in a host cell. In some embodiments, the recombinant expression vector includes one or more regulatory elements, which may be selected on the basis of the host cells to be used for transcription or expression, which is operatively linked to the nucleic acid sequence to be transcribed or expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element (s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell) .
In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the rAAV vector is a vector derived from an AAV serotype,  including without limitation, AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV capsid serotype or the like. In some embodiments, the nucleic acid in the AAV comprises an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV capsid serotype or the like. In some embodiments, the nucleic acid in the AAV further encodes an isolated anti-human FD antibody construct (e.g., anti-human FD antibody moiety, anti-human FD/anti-C2 multispecific construct, or anti-human FD/anti-C5 multispecific construct) as described herein. Use of any AAV serotype is considered within the scope of the present disclosure. In some embodiments, the vector is encapsidated in a rAAV particle. In some embodiments, the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV2 V708K, AAV2-HBKO, AAVDJ8, AAVPHP. B, AAVPHP. eB, AAVBR1, AAVHSC15, AAVHSC17, goat AAV, AAV1/AAV2 chimeric, bovine AAV, mouse AAV, or raAV2/HboV1 serotype capsid.
In some embodiments, the vector is a lentiviral vector.
The nucleic acids described herein or the vectors (e.g., viral vectors) can be delivered directly into a host cell or an individual (e.g., human) for the purpose of gene therapy. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, electroporation, nanoparticles, exosomes, microvesicles, or gene-gun, naked DNA and artificial virions.
The use of RNA or DNA viral based systems for the delivery of nucleic acids has high efficiency in targeting a virus to specific cells and trafficking the viral payload to the cellular nuclei.
In certain embodiments, there is provided a method of i) expressing any of the isolated anti-human FD antibody constructs (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) described herein, and/or ii) treating a complement-mediated disease in an individual (e.g., human) , comprising introducing a viral vector (such as an AAV or a lentiviral vector) comprising (or encoding) a  nucleic acid encoding any of the isolated anti-human FD antibody constructs described herein to a host cell or the individual (e.g., human) . In some embodiments, the vector is an rAAV vector. In some embodiments, the construct is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the construct is flanked by two AAV ITRs. In some embodiments, the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, the vector further comprises a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid is located upstream or downstream of the nucleic acid encoding the isolated anti-human FD antibody construct. In some embodiments, the vector is a self-complementary rAAV vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding the anti-human FD antibody construct and a second nucleic acid sequence encoding a complement of the anti-human FD antibody construct, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal resolution sequence. In some embodiments, the vector is encapsidated in a rAAV particle. In some embodiments, the AAV viral particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV2 V708K, AAV2-HBKO, AAVDJ8, AAVPHP. B, AAVPHP. eB, AAVBR1, AAVHSC15, AAVHSC17, goat AAV, AAV1/AAV2 chimeric, bovine AAV, mouse AAV, or raAV2/HboV1 serotype capsid.
In some embodiments, the vector (e.g., viral vector) is directly injected into the eye. In some embodiments, the vector allows delivery of the nucleic acid to the eye. Suitable viral vectors for eye delivery include, but are not limited to, AAV2, AAV4, AAV5, and AAV8. In some embodiments, the delivery method includes, but is not limited to nanoparticles, nanospheres, microparticles, microspheres, liposomes, nonbiodegradable implantable devices, biodegradable implantable devices, microcatheters, microneedles, micropumps, or port drug delivery system.
IV. Methods of treatment
Also provided herein are methods of inhibiting complement activation or reducing the activity of a complement system, and/or treating diseases or conditions (such as complement-mediated diseases or disorders) in an individual (e.g., human) . In some embodiments, the methods comprise administering an effective amount of an anti-human FD antibody construct described herein (or pharmaceutical composition thereof) , a nucleic acid encoding the anti-human FD antibody construct, or a vector (e.g., an AAV vector or lentiviral vector) comprising a nucleic acid encoding the anti-human FD antibody construct, to an individual (e.g., human) . In some embodiments, the individual is a mammal (e.g., human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. ) . In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc. In various embodiments, the complement-mediated disease or disorder is an AP-mediated disease or AP-mediated disorder. AP-mediated pathologies and conditions that can be treated with the anti-human FD antibody constructs (or pharmaceutical compositions thereof) and methods of the invention include, but are not limited to, macular degeneration (MD) , age-related macular degeneration (AMD) , ischemia reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, post-surgery systemic inflammatory syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD) , paroxysmal nocturnal hemoglobinuria (PNH) syndrome, autoimmune hemolytic anemia (AIHA) , Gaucher disease, myasthenia gravis, neuromyelitis optica (NMO) , multiple sclerosis, delayed graft function, antibody-mediated rejection, atypical hemolytic uremic syndrome (aHUS) , central retinal vein occlusion (CRVO) , central retinal artery occlusion (CRAO) , epidermolysis bullosa, sepsis, septic shock, organ transplantation, inflammation (including, but not limited to, inflammation associated with cardiopulmonary bypass surgery and kidney dialysis) , C3 glomerulopathy (C3G) , membranous nephropathy, IgA nephropathy (IgAN) , glomerulonephritis (including, but not limited to, anti-neutrophil cytoplasmic antibody (ANCA) -mediated glomerulonephritis, lupus nephritis, and combinations thereof) , ANCA-mediated vasculitis, thrombotic microangiopathies secondary to systemic lupus erythematosus (SLE-TMA) , Shiga toxin induced HUS, and antiphospholipid antibody-induced pregnancy loss, graft versus host disease (GvHD) , bullous pemphigoid, hidradenitis suppurativa, dermatitis herpetiformis, sweets syndrome, pyoderma gangrenosum, palmo-plantar pustulosis &pustular psoriasis, rheumatoid neutrophilic dermatoses, subcorneal  pustular dermatosis, bowel-associated dermatosis-arthritis syndrome, neutrophilic eccrine hidradenitis, linear IgA disease, or any combinations thereof.
In some embodiments, there is provided a method of treating a complement-mediated disease in an individual (e.g., human) , comprising administering to the individual an effective amount of any of anti-human FD antibody constructs (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) described herein (or pharmaceutical composition thereof) . In some embodiments, there is provided a method of treating a complement-mediated disease in an individual (e.g., human) , comprising administering to the individual an effective amount of a vector (e.g., viral vector, such as AAV vector or lentiviral vector) encoding any of the anti-human FD antibody constructs (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) described herein (or pharmaceutical composition thereof) . In some embodiments, the anti-human FD antibody construct is a multispecific construct (e.g., an anti-human FD/anti-C2 multispecific construct, or an anti-human FD/anti-C5 multispecific construct) , or pharmaceutical compositions thereof) . In some embodiments, there is provided a method of treating a complement-mediated disease in an individual (e.g., human) , comprising administering to the individual an effective amount of a vector (e.g., viral vector, such as AAV vector or lentiviral vector) encoding any of the multispecific anti-human FD antibody constructs described herein or pharmaceutical compositions thereof. In some embodiments, the complement-mediated disease is AP-and/or FD-mediated disease. In some embodiments, the complement-mediated disease is CP-and/or LP-and/or C2-mediated disease (e.g., anti-human FD/anti-C2 multispecific constructs can be used) . In some embodiments, the complement-mediated disease is TP-and/or C5-mediated disease (e.g., anti-human FD/anti-C5 multispecific constructs can be used) . In some embodiments, the complement-mediated disease is an eye disease. In some embodiments, the eye disease is selected from the group selecting of: macular degeneration, AMD, glaucoma, diabetic retinopathy, autoimmune uveitis, dry eye disorder, neuromyelitis optica (NMO) , central retinal vein occlusion (CRVO) , and subcorneal pustular dermatosis, and any combinations thereof. In some embodiments, the anti-human FD antibody construct (or pharmaceutical composition thereof) , or the vector (e.g., viral vector) encoding the anti-human FD antibody construct (or pharmaceutical composition thereof) , is directly administered to the eye.
The anti-human FD antibody constructs provided herein (or pharmaceutical composition thereof) can be used in combination with other treatment modalities, such as, for example anti-inflammatory therapies, and the like. Examples of anti-inflammatory therapies that can be used in combination with the anti-human FD antibody constructs or methods of the invention include, for example, therapies that employ steroidal drugs, as well as therapies that employ non-steroidal drugs.
In some embodiments, there is provided a method of inhibiting complement activation or reducing the activity of a complement system (e.g., activity of AP) in an individual (e.g., human) , comprising administering (such as systemically administering, for example by subcutaneous or intravenous administration) to the individual an effective amount of: i) an anti-human FD antibody construct, such as any of the anti-human FD antibody constructs described herein (e.g., anti-human FD antibody moiety, anti-human FD/anti-C2 multispecific construct, or anti-human FD/anti-C5 multispecific construct) , or a pharmaceutical composition thereof; or ii) a vector (e.g., an AAV vector or lentiviral vector) comprising a nucleic acid encoding an anti-human FD antibody construct, or a pharmaceutical composition thereof. In some embodiments, the anti-human FD antibody construct, the vector, or pharmaceutical composition thereof, is administered by subcutaneous administration. In some embodiments, the method inhibits complement activation (e.g., AP activation) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method reduces the activity of a complement system (e.g., activity of AP) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.
Dosage and routes of administration
Dosages and desired drug concentrations of pharmaceutical compositions of the present application may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics, ” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46.
Typically, dosages which may be administered in a method of the invention to a subject, in some embodiments a human, range in amount from 0.5 μg to about 50 mg per kilogram of body weight of the subject. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of subject and type of disease state being treated, the age of the subject and the route of administration.
In some embodiments, the anti-human FD antibody construct or a nucleic acid (e.g., viral vector) encoding thereof (or pharmaceutical composition thereof) is administered for a single time. In some embodiments, the anti-human FD antibody construct or a nucleic acid (e.g., viral vector) encoding thereof (or pharmaceutical composition thereof) is administered for multiple times (such as any of 2, 3, 4, 5, 6, or more times) . In some embodiments, the anti-human FD antibody construct or a nucleic acid (e.g., viral vector) encoding thereof (or pharmaceutical composition thereof) is administered once per week, once 2 weeks, once per month, once per 2 months, once per 3 months, once per 6 months, once per year, or the like. In some embodiments, the anti-human FD antibody construct or a nucleic acid (e.g., viral vector) encoding thereof (or pharmaceutical composition thereof) is administered at a dose of between about 0.1 mg/dose and about 20 mg/dose. In some embodiments, the anti-human FD antibody construct or a nucleic acid (e.g., viral vector) encoding thereof (or pharmaceutical composition thereof) is administered to the individual (such as to the eye of the individual) once. The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, include but not limited to, the type and severity of the disease being treated, the type and age of the subject, etc.
In some embodiments, the anti-human FD antibody construct is formulated in a reconstituted and liquid formulation. Parenteral administration of the anti-human FD antibody construct or a nucleic acid (e.g., viral vector) encoding thereof (or pharmaceutical composition thereof) includes any route of administration characterized by physical breaching of a tissue of an individual and administration of the pharmaceutical composition through the breach in the tissue. Parental administration can be local, regional or systemic. Parenteral administration thus includes, but is not limited to, administration by injection of the composition, by application of the composition through a surgical incision or through a tissue-penetrating non-surgical wound,  and the like. Parenteral administration is contemplated to include, but is not limited to, intravenous, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, or intrasternal injection.
In some embodiments, the vector, e.g., plasmid or viral vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the delivery is via intravenous, transdermal, mucosal, or other delivery methods. Such delivery may be either via a single dose, or multiple doses. One skilled in the art understands that the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choice, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc..
The anti-human FD antibody construct or nucleic acid (e.g., viral vector) encoding thereof of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. A unit dose is discrete amount of the anti-human FD antibody construct or the nucleic acid (e.g., viral vector) encoding thereof comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to an individual or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the individual treated and further depending upon the route by which the composition is to be administered.
V. Methods of preparation
Also provided are methods of preparing any of the isolated anti-human FD antibody constructs described herein (e.g., anti-human FD antibody moiety, anti-human FD/anti-C2 multispecific construct, or anti-human FD/anti-C5 multispecific construct) .
The anti-human FD antibody constructs described herein can be made by introducing a nucleic acid into a host cell, allowing expression of the protein, and purifying the protein from the host cell extract or supernatant. Methods of making proteins comprising antibody moieties and constructing nucleic acids or vectors encoding thereof, as well as purifying proteins comprising antibody moieties are known in the art. E.g., see US20220204602.
Proteins comprising antibody moieties can be obtained using methods known in the art, such as by immunizing a non-human mammal and obtaining hybridomas therefrom, or by cloning a library of antibodies using molecular biology techniques known in the art and subsequence selection or by using phage display. Nucleic acid constructs encoding any one of the antibodies or antigen-binding fragments described herein, vectors, and host cells for preparation are also provided.
The polypeptide portion of the constructs or targeting moieties described herein may be prepared by any of the known protein expression and purification methods in the art. DNA sequence encoding the polypeptide portion of the constructs or targeting moieties can be fully synthesized. After obtaining such sequence, it is cloned into a suitable expression vector, then transfected into a suitable host cell. The transfected host cells are cultured, and the supernatant is harvested and purified to obtain the polypeptide portion of the constructs or targeting moieties described herein. The polypeptide portion of the constructs or targeting moieties described herein can also be obtained by conventional immunization methods, such as by immunizing a mammal (e.g., mouse, llama) , and obtaining the constructs or targeting moieties from the serum; or from a hybridoma, such as by fusing B cells from lymph nodes and/or spleen of immunized animal with myeloma cells. Purification can be followed.
Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975) , or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567) . DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) . The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, in order to synthesize monoclonal antibodies in such recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pliickthun, Immunol. Revs. 130: 151-188 (1992) .
In a further embodiment, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990) .  Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology, 10: 779-783 (1992) ) , as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993) ) . Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.
In some embodiments, the invention is a cell or cell line (such as host cells) that produces at least one of the anti-human FD antibody constructs described herein. In one embodiment, the cell or cell line is a genetically modified cell. In one embodiment, the cell or cell line is a hybridoma.
Hybrid cells (hybridomas) are generally produced from mass fusions between murine splenocytes, which are highly enriched for B-lymphocytes, and myeloma “fusion partner cells” (Alberts et al., Molecular Biology of the Cell (Garland Publishing, Inc. 1994) ; Harlow et al., Antibodies. A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988) . The cells in the fusion are subsequently distributed into pools that can be analyzed for the production of antibodies with the desired specificity. Pools that test positive can be further subdivided until single cell clones are identified that produce antibodies of the desired specificity. Antibodies produced by such clones are referred to as monoclonal antibodies.
Nucleic acids encoding the anti-human FD antibody constructs described herein, or the heavy chain or light chain or fragments thereof, can be obtained and used in accordance with recombinant nucleic acid techniques for the production of the specific immunoglobulin, immunoglobulin chain, or a fragment or variant thereof, in a variety of host cells or in an in vitro translation system. For example, the antibody-encoding nucleic acids, or fragments thereof, can be placed into suitable prokaryotic or eukaryotic vectors, e.g., expression vectors, and introduced into a suitable host cell by an appropriate method, e.g., transformation, transfection, electroporation, infection, such that the nucleic acid is operably linked to one or more expression control elements, e.g., in the vector or integrated into the host cell genome.
The invention provides isolated nucleic acid molecules comprising a nucleic acid sequence encoding a heavy chain and/or a light chain, as well as fragments thereof. A nucleic  acid molecule comprising sequences encoding both the light and heavy chain, or fragments thereof, can be engineered to contain a synthetic signal sequence for secretion of the antibody, or fragment, when produced in a cell. Furthermore, the nucleic acid molecule can contain specific DNA links which allow for the insertion of other antibody sequences and maintain the translational reading frame so to not alter the amino acids normally found in antibody sequences.
The antibody-encoding nucleic acids, or fragments thereof, can be subjected to various recombinant nucleic acid techniques known to those skilled in the art such as site-directed mutagenesis.
The expression vectors may contain a variety of elements for controlling expression, including without limitation, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be selected as appropriate by a person of ordinary skill in the art.
A promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression may be employed. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2012) . The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and fragments thereof.
An example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) , human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney virus promoter, the avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the muscle creatine promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter in the invention provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is  operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. Further, the invention includes the use of a tissue-specific promoter or cell-type specific promoter, which is a promoter that is active only in a desired tissue or cell. Tissue-specific promoters are well known in the art and include, but are not limited to, the HER-2 promoter and the PSA associated promoter sequences.
Selectable markers may be selected to allow selection of the host cells inserted with the vector from those not, for example, the selectable markers may be genes that confer antibiotic resistance.
In some embodiments, the two or more polypeptides of a construct or a targeting moiety are encoded by a single vector. In some embodiments, the two or more polypeptides of a construct or a targeting moiety are encoded by two or more vectors. Host cells containing the vector may be useful in expression or cloning of the isolated nucleic acids. Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. The expression of antibodies and antigen-binding fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pluckthun, A. BioTechnology 9: 545-551 (1991) . Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of antibodies or antigen-binding fragments thereof, see recent reviews, for example Ref, M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560. Higher eukaryotic cells, in particular, those derived from multicellular organisms can be used for expression of glycosylated polypeptides. Suitable higher eukaryotic cells include, without limitation, invertebrate cells and insect cells, and vertebrate cells. In some embodiments, the host cell is E. coli. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell or an HEK293 cell.
The vector can be introduced to the host cell using any suitable methods known in the art, including, but not limited to, DEAE-dextran mediated delivery, calcium phosphate precipitate method, cationic lipids mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of a vector of interest are well known in the art. In some  embodiments, the host cells comprise two or more vectors each encoding a polypeptide of any of the protein constructs or targeting moieties described herein. In some embodiments, the host cells comprise a single vector comprising isolated nucleic acids encoding two or more polypeptides of any of the protein constructs or targeting moieties described herein.
In some embodiments, the present application provides methods of making the polypeptide portion of any of the isolated anti-human FD antibody constructs described herein, comprising i) culturing a host cell comprising any of the isolated nucleic acids described herein or any of the vectors described herein, or any of the isolated host cells described herein, under a condition suitable for the expression of the polypeptide portion of any of the anti-human FD antibody constructs described herein, and ii) obtaining the expressed polypeptide portion of any of the anti-human FD antibody constructs described herein from said host cell (e.g., from the cell culture) . The isolated host cells are cultured under conditions that allow expression of the isolated nucleic acids inserted in the vectors. Suitable conditions for expression of polynucleotides may include, without limitation, suitable medium, suitable density of host cells in the culture medium, presence of necessary nutrients, presence of supplemental factors, suitable temperatures and humidity, and absence of microorganism contaminants. A person with ordinary skill in the art can select the suitable conditions as appropriate for the purpose of the expression. In some embodiments, the methods of making further comprises purifying any of the obtained polypeptide portion of any of the anti-human FD antibody constructs described herein.
In some embodiments, the polypeptides expressed by the host cell can assemble together (e.g., form a polypeptide complex such as a dimer) and produce any of the polypeptide portion of any of the anti-human FD antibody constructs described herein. In some embodiments, the polypeptide complex may be formed inside the host cell. For example, the polypeptide complex may be formed inside the host cell with the aid of relevant enzymes and/or cofactors. In some embodiments, the polypeptide complex may be secreted out of the cell. In some embodiments, individual polypeptides may be secreted out of the host cell then form a polypeptide complex outside of the host cell.
In some embodiments, the two or more polypeptides of any of the anti-human FD antibody constructs described herein may be separately expressed and allowed to form (e.g., dimerize) a protein complex under suitable conditions. For example, the first polypeptide and the second polypeptide may be combined in a suitable buffer and allow the first protein monomer  and the second protein monomer to dimerize through appropriate interactions such as hydrophobic interactions. In some embodiments, the first polypeptide and the second polypeptide may be combined in a suitable buffer containing an enzyme and/or a cofactor which can promote the dimerization of the first polypeptide and the second polypeptide. In some embodiments, the first polypeptide and the second polypeptide may be combined in a suitable vehicle and allow them to react with each other in the presence of a suitable reagent and/or catalyst.
The expressed polypeptide (s) and/or the polypeptide complex can be collected using any suitable methods. The polypeptide (s) and/or the polypeptide complex can be expressed intracellularly, in the periplasmic space or be secreted outside of the cell into the medium. If the polypeptide and/or the polypeptide complex are expressed intracellularly, the host cells containing the polypeptide and/or the polypeptide complex may be lysed and polypeptide and/or the polypeptide complex may be isolated from the lysate by removing the unwanted debris by centrifugation or ultrafiltration. If the polypeptide and/or the polypeptide complex is secreted into periplasmic space of E. coli, the cell paste may be thawed in the presence of agents such as sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) for about 30 min, and cell debris can be removed by centrifugation (Carter et al., BioTechnology 10: 163-167 (1992) ) . If the polypeptide and/or the polypeptide complex is secreted into the medium, the supernatant of the cell culture may be collected and concentrated using a commercially available protein concentration filter, for example, an Amincon or Millipore Pellicon ultrafiltration unit. A protease inhibitor and/or an antibiotic may be included in the collection and concentration steps to inhibit protein degradation and/or growth of contaminated microorganisms.
The expressed polypeptide (s) and/or the polypeptide complex can be further purified by a suitable method, such as without limitation, affinity chromatography, hydroxylapatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, ion exchange fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column) , chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation (see, for review, Bonner, P. L., Protein purification, published by Taylor &Francis. 2007; Janson, J. C., et al., Protein purification: principles, high resolution methods and applications, published by Wiley-VCH, 1998) .
In some embodiments, the polypeptides and/or polypeptide complexes can be purified by affinity chromatography. In some embodiments, protein A chromatography or protein A/G (fusion protein of protein A and protein G) chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising a component derived from antibody CH2 domain and/or CH3 domain (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983) ) ; Zettlit, K. A., Antibody Engineering, Part V, 531-535, 2010) . In some embodiments, protein G chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising IgG γ3 heavy chain (Guss et al., EMBO J. 5: 1567 1575 (1986) ) . In some embodiments, protein L chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising κ light chain (Sudhir, P., Antigen engineering protocols, Chapter 26, published by Humana Press, 1995; Nilson, B. H. K. et al., J. Biol. Chem., 267, 2234-2239 (1992) ) . The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where any of the isolated anti-human FD antibody constructs comprises an additional CH3 domain, the bakerbond ABX resin (J.T. Baker, Phillipsburg, N.J. ) is useful for purification. In some embodiments, MabSelect SuRe is used for purification. In some embodiments, the method further comprises analyzing the purified protein product, such as by SDS-PAGE and/or SEC-HPLC.
VI. Methods of identifying and analyzing antibody moieties
The antibody moieties described herein can be obtained, for example, by molecular cloning the antibody sequences and identifying antibodies suitable for making the anti-human FD antibody constructs (e.g., anti-human FD antibody moiety, or multispecific construct) .
Methods of predicting protein tertiary structure based on amino acid sequences are known in the art. See, for example, Jumper et al. (2021) Nature 596 (7873) : 583-589; Kelley et al. (2015) Nature Protocols 10: 845-858; Roy et al. (2010) Nature Protocols 5: 725-738; Lambert et al. (2002) Bioinformatics 18: 1250-1256; Kim et al (2004) Nucleic Acids Res. 32 (Web Server issue) : W526-31; Lamiable et al (2016) Nucleic Acids Res. 44 (W1) : W449-54; Huang et al. (2015) Nucleic Acids Res. 43 (W1) : W338-W342; and Zemla et al. (2005) Nucleic Acids Res. 33: W111-W115.
Methods of analyzing activities of antibodies and antibody moieties described herein are known in the art. Binding affinity and specificity of the antibody or antibody moieties described herein can be determined experimentally by methods known in the art. For example, the binding of an antibody to a protein antigen can be detected and/or quantified using a variety of techniques such as, but not limited to, Western blot, dot blot, surface plasmon resonance (SPR) method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J. ) , Bio-Layer Interferometry (BLI) (e.g. Octet system, ForteBio) , RIA, ECL, IRMA, EIA, peptide scans, and enzyme-linked immunosorbent assay (ELISA) . See, e.g., Benny K. C. Lo (2004) “Antibody Engineering: Methods and Protocols. ” Humana Press (ISBN: 1588290921) ; Borrebaek (1992) “Antibody Engineering, A Practical Guide. ” W.H. Freeman and Co., N.Y. ; Borrebaek (1995) “Antibody Engineering. ” 2nd Edition, Oxford University Press, N.Y., Oxford; Johne et al. (1993) . J Immunol Meth 160: 191-198; Jonssonetal. (1993) Ann Biol Clin 51: 19-26; and Jonsson et al. (1991) Biotechniques 11: 620-627. In addition, methods for measuring the affinity (e.g., dissociation and association constants by BLI) are set forth in the working examples.
Binding of an anti-FD antibody moiety to FD, or the binding of the second antibody moiety to the complement protein (e.g., binding of an anti-C2 antibody moiety to C2, binding of an anti-C5 antibody moiety to C5) , can be measured by using a variety of techniques such as, but not limited to, Western blot, dot blot, SPR method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J. ) , BLI (e.g. Octet system, ForteBio) , RIA, ECL, IRMA, EIA, peptide scans, and ELISA. See, e.g., Benny K. C. Lo (2004) “Antibody Engineering: Methods and Protocols. ” Humana Press (ISBN: 1588290921) ; Borrebaek (1992) “Antibody Engineering, A Practical Guide. ” W.H. Freeman and Co., N.Y. ; Borrebaek (1995) “Antibody Engineering. ” 2nd Edition, Oxford University Press, N. Y., Oxford; Johne et al. (1993) J Immunol Meth 160: 191-198; Jonssonetal. (1993) Ann Biol Clin 51: 19-26; and Jonsson et al. (1991) Biotechniques 11: 620-627.
The activity of the anti-C2 antibody can also be carried out by biological assays. Methods for determining whether a particular antibody described herein inhibits C2 cleavage are known in the art. Inhibition of human C2 can reduce the cell-lysing ability of complement in a subject’s body fluids. Such reductions of the cell-lysing ability of complement present in the body fluid (s) can be measured by methods well known in the art such as, for example, by a  conventional hemolytic assay such as the hemolysis assay in chicken erythrocyte hemolysis method as described in, e.g., Hillmen et al. (2004) N Engl. J Med 350 (6) : 552. In order to determine the effect of the anti-C2 antibody on classical complement pathway-mediated hemolysis in a serum test solution in vitro, for example, sheep erythrocytes coated with hemolysin or chicken erythrocytes sensitized with anti-chicken erythrocyte antibody are used as target cells. The percentage of lysis is normalized by considering 100%lysis equal to the lysis occurring in the absence of the inhibitor.
IgM-mediated C3b formation assays can be used to determine the inhibitory activity of the anti-C2 antibodies on classical pathway induced C3b formation. Plate-bound and immobilized IgM can resemble immune complex and activate the classical pathway complement. In this assay, human IgM is coated onto ELISA plate, and after washing with PBS buffer, normal human serum (1%or 50%) in GVB++ buffer is added to the plate and incubated at room temperature for 60 min. After washing, the amount of C3b generated and bound to the plate is detected by anti-C3b/iC3b antibodies.
Inhibition of the lectin pathway by the anti-C2 antibodies may be assessed by assays targeting the mannan-binding lectin (MBL) pathway of complement activation. MBL is a carbohydrate-binding serum protein, which circulates in complex with serine proteases known as mannan-binding lectin associated serine proteases (MASPs) . When bound to microorganisms, the MBL complex activates, through activities of MASP-1 and MASP-2, the complement components C4 and C2, thereby generating the C3 convertase and leading to opsonisation by the deposition of C4b and C3b fragments. See, for example, Petersen et al. J Immunol Methods. 2001 Nov 1; 257 (1-2) : 107-16.
Methods for determining whether a candidate compound inhibits the cleavage of human C2 into forms C2a and C2b are known in the art and described in, e.g., Thomas et al. (1996) Mol Immunol 33 (17-18) : 1389-401; and Evans et al. (1995) Mol Immunol 32 (16) : 1183-95. For example, the concentration and/or physiologic activity of C2a and C2b in a body fluid can be measured by methods well known in the art. Methods for measuring C2a concentration or activity include, e.g., biolayer interferometry, RIAs, or ELISAs (see, e.g., Wurzner et al. (1991) Complement Inflamm 8: 328-340) . Using assays of these or other suitable types, candidate agents capable of inhibiting human complement component C2 can be screened.
The activity of the anti-C5 antibody can also be carried out by biological assays. Methods for determining the inhibitory activity of anti-C5 antibodies on the classical pathway are known in the art and described in, e.g., Zelek et al, (2020) Immunology. 161 (2) : 103-113. Inhibition of C5 prevents the generation of C5a and C5b, which subsequently inhibits leukocyte and platelet activation. For example, anti-C5 complement inhibition during extracorporeal circulation can be measured by methods known in the art. See, for example, Rinder et al. (1995) J Clin Invest. 96 (3) : 1564-1572. Generation of C5a can be measured by methods known in the art. See, for example, Volokina et al. (2015) Blood. 126 (2) : 278-279.
VII. Pharmaceutical Compositions, Kits, and Articles of Manufacture
Further provided by the present application are pharmaceutical compositions comprising i) any one of the anti-human FD antibody constructs (e.g., anti-human FD antibody moieties, anti-human FD/anti-C2 multispecific constructs, or anti-human FD/anti-C5 multispecific constructs) , or any of the isolated nucleic acids encoding the anti-human FD antibody constructs, or any of the vectors (e.g., a viral vector such as an AAV) comprising such isolated nucleic acids, or any mRNA comprising or encoded by such isolated nucleic acids, and ii) an optional pharmaceutically acceptable carrier.
Suitable formulations (e.g., pharmaceutical composition) of the anti-human FD antibody construct described herein, a nucleic acid encoding the anti-human FD antibody construct, or a vector (e.g., viral vector) comprising a nucleic acid encoding the anti-human FD antibody construct, can be obtained by mixing the anti-human FD antibody construct (or the nucleic acid encoding the anti-human FD antibody construct, or the vector comprising a nucleic acid encoding the anti-human FD antibody construct) having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ) . The formulations can be lyophilized formulations or aqueous solutions. In some embodiments, the formulation is suitable for application to the eye.
In some embodiments, the pharmaceutical composition further comprises additional ingredients. Additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and  solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA) , which is incorporated herein by reference. Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall (e.g., surfactant, such as polysorbate (e.g., polysorbate 80) or poloxamer) .
For example, acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes) ; and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG) . In some embodiments, lyophilized formulations are provided.
In order for the pharmaceutical compositions to be used for in vivo administration, they must be sterile. The pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes. The pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
The formulations of the pharmaceutical compositions may be prepared by any method known or hereafter developed in the art of pharmacology. Preparations include but are not  limited to, bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-or multi-dose unit. The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1, 3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for parenteral, intramuscular, intradermal, subcutaneous, or intravenous routes of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. In some embodiments, the pharmaceutical composition is for eye (e.g., intraocular) administration.
A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. A unit dose is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to an individual or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
Also provided are kits comprising any one of the anti-human FD antibody constructs (or the nucleic acid encoding the anti-human FD antibody construct, or the vector (e.g., viral vector) comprising a nucleic acid encoding the anti-human FD antibody construct) described herein.
The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags) , and the like. Kits may optionally provide additional components such as buffers and interpretative information.
The present application thus also provides articles of manufacture. The article of manufacture can comprise a container and a label or package insert on or associated with the container. Suitable containers include vials (such as sealed vials) , bottles, jars, flexible packaging, and the like. Generally, the container holds a composition, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) .
Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention. The invention will now be described in greater detail by reference to the following non-limiting examples. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
EXAMPLES
The examples below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.
Example 1: Humanization of mouse anti-human FD antibody 118A1
A mouse anti-human FD IgG4 monoclonal antibody comprising the VH amino acid sequence of SEQ ID NO: 140 and VL amino acid sequence of SEQ ID NO: 141 was used to generate humanized anti-human FD antibodies and variants thereof. CDR-grafting was used to generate humanized anti-human FD antibodies. To minimize the potential impact of CDR glycosylation on antibody properties, the conserved N-linked glycosylation sequence motif in H-CDR2 was removed by site-specific mutation. The humanized 118A1 with (indicated with “p2” ) or without the H-CDR2 mutation were expressed and purified from CHO cells. The binding affinities of the recombinant, humanized anti-FD antibodies were determined using SPR. Several humanized anti-FD antibodies showed the same or less than 2-fold reduction of binding KD compared to the murine “parental” anti-FD antibody 118A1 (achimeric form of 118A1 clone with human Fc served as reference control) . The binding kinetics and affinity of the humanized  anti-FD antibodies to human FD were determined by SPR (Table 1) . Also, removing conserved N-linked glycosylation sequence motif in H-CDR2 did not affect much of the binding affinity of the humanized antibodies (e.g., compare z16 and z16-p2, compare z17 and z17-p2) .
Clone z16-p2 was selected for further sequence engineering for affinity maturation.
Table 1. Binding affinity of humanized 118A1 mAbs to human FD protein.
Example 2: Binding affinity optimization of z16-p2 humanized clone
To improve binding affinity of the humanized antibody to human and monkey FD, each amino acid of the six CDRs of the anti-FD scFv clone comprising VH and VL sequences of z16-p2 (SEQ ID NOs: 7 and 8) was individually mutated to all 20 amino acids using site-directed mutagenesis. DNA primers containing an NNS codon encoding 20 amino acids were used to introduce mutations at each targeted CDR position. The degenerate primers were used in site-directed mutagenesis reactions. Briefly, each degenerate primer was phosphorylated. The PCR products were purified, then electroporated into BL21 for colony formation and production of scFv fragments.
To identify variants with improved binding containing point mutation in the CDRs, a single point scFv capture ELISA (SPE) was carried out. Clones exhibiting an optical density (OD) signal at 450 nm that was two-fold greater than that of the parental clone z16-p2 were picked and sequenced. Representative CDR sequences with single amino acid substitution that show binding improvements are shown in Table 2 (SEQ ID NOs: 61, 69, 79, 89, 97, 103, 111, 119, and 129 are nine unique CDR sequences containing single amino acid substitutions. One clone (1L8-B6 × 3L10E9 (mAb-18) ) has two combined CDR mutations included SEQ ID NOs: 124 and 126) . As shown in FIG. 1, FIG. 2A, and FIG. 2B, compared to the parental humanized  z16-p2 clone, several clones with single amino acid substitutions significantly improved binding to both human FD and cynomolgus FD. z16-p2 served as reference anti-human FD control and an irrelevant scFv clone served as negative control.
Table 2. Representative anti-FD scFv variants with single amino acid substitutions in either H-CDR or V-CDR regions that show binding improvement to human FD.
Four heavy chains and eight light chains containing the single or double mutations for improved binding were selected for cloning into IgG expression vectors. The vectors were co-transfected into HEK293 cells and the expression supernatants were purified. Monoclonal antibodies derived from the combination of the heavy and light chains (SEQ ID NOs: 142-197 (VH and VL sequences) ) as shown in Table 3 were evaluated for their binding activity to human and cynomolgus FD using ELISA. FIGs. 3A and 3B showed binding of the representative IgG4 variants to human and cynomolgus FD. As shown in FIGs. 3A-3B, compared to parental humanized z16-p2 clone in IgG4 format, mAb-12, mAb-22, mAb-28, mAb-32, mAb-38, and mAb-42 clones all showed much higher binding to cynomolgus monkey FD, and similar or higher binding to human FD.
Table 3. Monoclonal antibodies derived from combinations of heavy and light chains containing single mutations for improved binding.
Example 3: Engineering of pH-dependent binding of anti-FD mAbs
To engineer mAbs for pH-dependent binding, a histidine substitution was introduced to each position of the six CDRs of clone mAb-42 (IgG4 format) bymutagenesis, a method known as histidine scanning. For each variant, HEK293 cells were transiently transfected with the expression plasmids encoding a mutated mAb-42 heavy chain and a wild-type light chain, and cultivated with serum-free medium in shaking flasks for 5-7 days. The light chain Ab variants were obtained in a similar way by co-transfecting HEK293 cells with expression plasmids encoding a mutated light chain and a wild-type heavy chain. The expression supernatant was collected by centrifugation for binding characterization. IgG from the expression supernatant was used to measure FD binding under either pH 7.4 or pH 5.8 using GatorTM (Probe Life, Inc. ) .
Mutant anti-FD antibodies that maintained the same binding dissociation constant at pH 7.4 but had reduced binding dissociation constant at pH 5.8, compared to parental mAb-42, were identified. The pH-differential binding variants with multiple histidine mutations were further created by co-transfecting HEK293 cells with expression plasmids encoding heavy or light chain  carrying binding beneficial, single histidine mutation. Variants with multiple binding beneficial histidine mutations within an antibody chain were created bymutagenesis by introducing the beneficial mutations into a same antibody chain. The pH-dependent binding variants were purified from the expression supernatant by Protein-Aaffinity chromatography for affinity determinations. Purified antibodies were dialyzed into 20 mM histidine buffer with 150 mM NaCl and quantitated by Nanodrop (Thermo Fisher Scientific, Inc., USA) before affinity measurement using GatorTM (Probe Life, Inc. ) .
The binding sensorgrams of exemplary pH-binding engineered anti-FD variants to human and cyno FD are shown in FIG. 4. The binding affinity and pH-dependent binding activities of representative anti-FD variants are shown in Table 4. mAb-42 served as control. As shown in FIG. 4 and Table 4, pH-binding engineered anti-FD variants maintained similar binding dissociation constant at pH 7.4 to human and/or cyno FD, but had reduced binding dissociation constant at pH 5.8, compared to parental mAb-42. Sequences of 5 of the pH-binding engineered variants are shown in Table 5.
Table 4. Binding kinetics of pH-binding engineered variants to human FD.
Table 5. CDR sequences of the pH-binding engineered variants.

Example 4. Functional characterization of affinity-matured and/or pH-dependent binding variants
Inhibition of rabbit RBC lysis in the presence of human and/or cyno serum.
Rabbit red blood cells (rRBC) were prepared by centrifuging buffer diluted with rabbit blood at 1500 rpm for 5 min at 4℃. After washing, the cells were resuspended in AP buffer, and the cell density was adjusted to 4 × 108 cells/ml. Serially diluted anti-FD antibodies were added to the assay wells containing Normal Human Serum (NHS) to a final concentration of 50%or 30%. Distilled water or EDTA was used as the positive control (PC) or negative control (NC) , respectively. Rabbit RBC was added at last to the antibody and NHS mixture (1 × 107 cells per well) and incubated at 37℃ for 40 min. The lysis reaction was stopped by addition of ice-cold 40 mM EDTA in PBS. The reaction mixture was centrifuged for 5 min at 1500 rpm. The supernatant was collected and transferred to a flat-bottom 96-well plate. Color of the supernatant was measured at OD 405 nm on a Microplate reader. The %lysis of each sample was calculated with the following formula: %lysis = (ODsample -ODNC) / (ODPC -ODNC) × 100%, and the data were analyzed with GraphPad Prism (version 7.0, GraphPad Software, Inc) .
Inhibition of LPS-C3b deposition in ELISA
An LPS-C3b ELISA was used to assess the inhibition of the alternative pathway by anti-FD antibodies. In the LPS-C3b ELISA, NHS samples (final concentration 10%) were preincubated with serially diluted anti-FD antibodies at 4℃ for 1.5 hours, added to an ELISA 96-well plate coated with LPS (500 ng/well) , and incubated at 37℃ for 1 hour. The AP complement activation was stopped by adding 100 μl/well pre-chilled 40 mM EDTA. After washing, the plate was incubated with 100 μl/well of anti-C3 antibody-HRP (1: 4,000) at room temperature for 1 hour, followed by washing, adding 100 μl/well of TMB to develop color, and then adding 50 μl/well of 2N H2SO4 to stop the reaction. The absorbance at 450 nm was read on a densitometric plate reader. A sample with only the EDTA buffer added was used as the negative control (NC, 0%deposition) , and a sample with only AP buffer (APB) added was used  as the positive control (PC, 100%deposition) . The %deposition of C3b of each sample was calculated with the following formula: %deposition (C3b) = (ODsample -ODNC) / (ODPC -ODNC) × 100%.
Anti-FD affinity-matured IgG4 variants were evaluated for inhibition of NHS-induced rabbit RBC lysis (FIG. 5) and LPS-induced C3b deposition (FIG. 6) . As shown in FIG. 5, all the representative affinity-matured anti-FD IgG4 variants completely inhibited 50%NHS-induced rabbit RBC lysis, and exhibited stronger inhibition than the parental z16-p2 clone. mAb-42 was the most potent mAb with an IC50 value of 1.49 μg/ml (Table 6) . The same constructs were assessed for inhibition of the alternative pathway using an LPS-induced C3b deposition assay. All constructs inhibited the alternative pathway (FIG. 6) , and the affinity-matured variants showed stronger AP inhibition than the parental z16-p2 clone. The corresponding IC50 values for both lysis and deposition assay are listed in Table 6.
Anti-FD pH-binding engineered variants were also evaluated for inhibition of NHS-induced rabbit RBC lysis (FIG. 7) and LPS-induced C3b deposition (FIG. 8) . As shown in FIG. 7, all pH-binding engineered variants completely inhibited 30%NHS-induced rabbit RBC lysis (FIG. 7) . The IC50 values of inhibition of rRBC lysis by mAb-42-2536 and mAb-42-2821 in 30%NHS were 0.71 μg/ml and 0.74 μg/ml, respectively, which were comparable to the parental mAb-42 clone. All pH-dependent constructs also inhibited the alternative pathway with similar potency as the parental mAb-42 clone (FIG. 8) . The IC50 values of inhibition by mAb-42-2536 and mAb-42-2821 in 10%NHS were 0.13 μg/ml and 0.14 μg/ml, respectively. Further, as shown in Table 7, pH-dependent engineering did not greatly affect binding affinities of the variants to human and cyno FD, compared to the parental mAb-42.
Table 6. Binding to human and cyno FD and potency of affinity-matured anti-FD constructs*
*Average Molecular Weight of mAbs (IgG4) are about 150 kDa.
Table 7. Binding to human and cyno FD and potency of pH-binding engineered anti-FD constructs*
*Average Molecular Weight of mAbs (IgG4) are about 150 kDa.
Example 5: Combined pH-dependent target binding and optimized FcRn binding at pH 7.4 for effective target removal from circulation
Because antibodies bound to soluble FD can form a stable complex in serum and increase total FD serum concentration, antigen-sweeping property for anti-FD antibody was sought. A panel of FcRn binding mutations that increase FcRn binding at pH 5.8 and at pH 7.4 were engineered into an exemplary mAb-42 pH-dependent anti-FD variant from Example 3 (herein also referred to as “mAb-42pH” ) , to reduce total FD concentration in serum.
The affinity-matured mAb-42 clone was engineered with pH-dependent FD binding mutations (mAb42-pH) and/or with various FcRn binding mutations that show increased FcRn binding affinity at both pH 5.8 and 7.4 (FIG. 9) . All antibodies were in an IgG4 backbone with an S228P mutation (herein referred to as “IgG4P” , SEQ ID NO: 132) in the hinge region for improved stability. The variants containing different FcRn binding mutations were tested for antigen sweeping activity. “LA” variant (SEQ ID NO: 133) : S228P, M428L+N434A. “YPY” variant (SEQ ID NO: 134) : S228P, M252Y+V308P+N434Y. “YEY” variant (SEQ ID NO: 135) : S228P, M252Y+N286E+N434Y. “N3E” variant (SEQ ID NO: 136) : S228P, L432C+H433S+N434W+Y436L+T437CE. “N3” variant (SEQ ID NO: 137) : S228P, L432C+H433S+N434W+Y436L+T437C. “YTE” variant (SEQ ID NO: 138) : S228P, M252Y+S254T+T256E. “Y31-YTE” variant (SEQ ID NO: 139) : S228P, M252Y+S254T+T256E+L432E+H433R+N434F+Y436R+T437Q.
FcRn binding affinities of mAb-42pH variants containing various FcRn binding mutations were determined using BLI (GatorTM, Probe Life Inc., USA) . Briefly, biotinylated FcRn (AcroBio) was captured on streptavidin (SA) -coupled sensor probes and equilibrated in kinetic (K) buffer (phosphate-buffered saline containing 0.02%bovine serum albumin and 0.002%Tween 20, pH 7.4) . Antibodies were captured onto the SA-sensors by dipping them into 200 μL of transfection supernatant for 600 seconds at pH 7.4. The biosensors were then incubated with  human FD prepared in the K buffer at pH 7.4 (40 nM) for 600 seconds followed by a 600-second dissociation period in K Buffer, pH 7.4 or pH 5.8. The data were processed and analyzed by the Gator evaluation software.
The binding kinetics of the FcRn binding variants are shown in FIG. 10 and the binding affinities of the FcRn binding variants under pH 7.4 and pH 5.8 are summarized in Table 8. mAb-42pH with S228P mutation in IgG4 served as control ( “mAb42pH-IgG4P” ) . As shown in FIG. 10 and Table 8, mAb42pH-IgG4P showed expected FcRn binding affinity under pH 5.8 but no detectable binding ( “N/A” ) under pH 7.4. FcRn binding variants LA, Y31-YTE, N3E, YEY and YPY all showed improved FcRn binding affinity under both pH 5.8 and pH 7.4, compared to that of the parental IgG4P backbone. Particularly, compared to mAb42pH-IgG4P-LA, which had a KD of 1270 nM at pH 7.4, variants with FcRn binding mutations N3E, YPY, and YEY showed greater than 225-to 1000-fold increase in FcRn binding at pH 7.4, with KDs of 5.63 nM, 1.03 nM, and 3.68 nM, respectively. Affinity-enhanced pH-dependent FcRn binding that is double-digit KD (nM) at pH 7.4 and single-digit KD (nM) at pH 5.8 has been suggested to achieve maximal target clearance when combined with similar target binding affinities in reverse pH directions (Yang et al., MAbs. 2017 Oct; 9 (7) : 1105-1117) .
Table 8. Summary of FcRn binding affinity of mAb-42pH-FcRn binding variants
Example 6: In vivo evaluation of FD sweeping effect in hFD/hFcRn transgenic mice
Generation of a humanized FcRn transgenic mouse expressing matured human FD
To characterize the pharmacokinetics and FD serum level of exemplary mAb-42pH FcRn binding variants (from Example 5) with the potential antigen sweeping properties in vivo, a  humanized FD/FcRn transgenic mouse model was generated. Mature human FD (hFD) cDNA was cloned into the pCAGGS vector at an EcoRI restriction site by the infusion cloning method (kit from Takara) . After confirming hFD protein expression in transfected HEK cells, the entire expression cassette with CMV IE enhancer to rabbit beta globin polyA was ligated into an AAV vector with TBG promoter at a BstXI restriction site. Positive clones were screened by Sma I digestion. Super-coiled endotoxin-free plasmid was prepared by the EndoFree plasmid kit (Cat. No. 12362, Qiagen) , and was used for AAV8 virus production. The packaging, purification, and titer determination of AAV8 virus was accomplished by using standard procedures. Scid/FcRn humanized mice (Strain number: 018441, Jackson Laboratory, Bar Harbor, Maine, USA) were injected by retro-orbital intravenous (I.V.) route with an AAV8 virus containing human mature FD (3×1011 gene copies/mouse) . After two weeks, blood samples were collected and processed for human FD protein detection.
To detect human FD expression in mouse blood, 96-well plates were coated with 50 μl of a capture anti-human FD mAb (generated in-house) at a final concentration of 2 μg/ml in PBS at 37℃ for 1 hr. Following three washes with washing buffer, the plates were incubated with 200 μl of blocking buffer at room temperature (RT) for 1 hr. After washing, the plates were incubated with 50 μl of diluted mouse plasma samples or standards in the blocking solution at RT for 1 hr. Plasma dilution started at 1/25, followed by a two-fold serial dilution. Recombinant hFD standard dilution started at 125 ng/ml and was serially diluted by 2-fold. After washing, the plates were incubated with 50 μl of biotin-conjugated detection anti-human FD mAb (generated in-house) at a final concentration of 2 μg/ml in blocking solution at RT for 1 hr. After washing, the plates were developed with 100 μl of HRP substrate (Cat. No. 34029, Thermo Scientific) for 3 min. The reaction was stopped with 50 μl of 2N H2SO4 and the plate was read at 450 nm in a micro plate reader.
Characterization of pharmacokinetics and FD sweeping effect of pH-dependent human FD binding and human FcRn binding mutations in the SCID/hFD mice
To study pharmacokinetics and serum FD accumulation, mAb-42 (not antigen pH-dependent engineering) constructed in IgG4P-LA backbone (SEQ ID NO: 133; mAb42-IgG4P-LA, or “42WT” ) and its pH-dependent FD-binding and/or FcRn binding variants were administered to hFD/FcRn-Scid mice via retro-orbital I.V. infusion at 40 mg/kg dosage.  Following antibody injection, plasma was collected at specific time points (2 hrs, day 1 (d1) , d3, d5, d7, d11 or d12/13, and d15) and total anti-hFD antibody (measured as human IgG4) and total hFD (free plus antibody-bound) concentrations were determined by ELISA. To measure total human IgG4 in mouse plasma, 96-well plates were coated with 50 μl of an anti-human kappa light chain antibody (Antibody Solutions, AS75-P) at a final concentration of 2 μg/ml in bicarbonate buffer at 37℃ for 1 hr. Following three washes with PBS containing 0.05%Tween-20 (washing buffer) , the plates were incubated with 200 μl of 1%BSA/PBS (blocking buffer) at RT for 1 hr. After washing, the plates were incubated with 50 μl of diluted mouse plasma samples or the appropriate anti-hFD mAb standards (the same pre-injection mAb was used as standard in each mAb PK study) in the blocking solution at RT for 1 hr. Five-fold serial dilutions starting at 1/160 were made for plasma samples whereas two-fold serial dilutions starting at 800 ng/ml were made for standards. After washing, the plates were incubated with 50 μl of anti-human IgG4 HRP (1: 2000 dilution, Invitrogen, A10654) in blocking solution at RT for 1 hr. After washing, the plates were developed with 100 μl of HRP substrate (Thermo Scientific, 34029) for 3 min. The reaction was stopped with 50 μl of 2N H2SO4 and the plate was read at 450 nm in a microplate reader. To measure free anti-hFD mAbs, 96-well plates were coated with 50 μl of recombinant hFD at a final concentration of 2 μg/ml in bicarbonate buffer at 37℃ for 1 hr. Following three washes with washing buffer, the plates were incubated with 200 μl of blocking buffer at RT for 1 hr. After washing, the plates were incubated with 50 μl of diluted plasma samples or standards in the blocking solution at RT for 1 hr. Two-fold serial dilutions starting at 1/200-1/500 dilution of mouse plasma were made. Two-fold dilutions were also made for antibody standards starting at 500ng/ml-1000 ng/ml. After washing, the plates were incubated with 50 μl of anti-human IgG4 HRP (1: 2000 dilution, Invitrogen, A10654) in blocking solution at RT for 1 hr. After washing, the plates were developed with 100 μl of HRP substrate (Thermo Scientific, 34029) for 3 min. The reaction was stopped with 50 μl of 2N H2SO4 and the plate was read at 450 nm in a microplate reader.
The following mAbs were studied: mAb42-IgG4P-LA (42WT (no anti-FD pH-dependent binding engineering) ) , mAb42pH-IgG4P-LA (42Mut) , mAb42pH-IgG4P-YEY (YEY) , mAb42pH-IgG4P-YPY (YPY) , mAb42pH-IgG4P-N3E (N3E) , and mAb42pH-IgG4P-Y31-YTE (YTE) . See FIG. 9.40 mg/kg of each mAb was separately injected into hFD/FcRn-Scid mice. As  shown in FIG. 11A, the anti-hFD pH-dependent binding mutation did not impact serum persistence since 42WT and 42Mut showed similar PK profiles in the hFD/FcRn-Scid mice. MAbs containing mutations that increased FcRn binding at both pH 7.4 and 5.8 include mAb42pH-IgG4P-Y31-YTE (lines with asterisk, FIG. 11A) , mAb42pH-IgG4P-YEY (lines with triangles, FIG. 11A) , and mAb42pH-IgG4P-YPY (lines with filled circles, FIG. 11A) . While FcRn binding sweeping mutation N3E showed a similar PK profile as 42Mut and 42WT, YTE, YEY, and YPY showed faster clearance in mice than the other FcRn binding mutant mAbs (FIG. 11A) . Human IgG4 levels of mice injected with YPY variant were consistently lower than that of mice injected with other mAbs, even starting from 2 hrs post-injection. When the mice were measured for total plasma hFD levels, plasma hFD level increased following mAb administration, as expected (FIG. 11B) . As shown in FIG. 11B, mAbs containing both pH-dependent hFD-binding mutations in the Fab domain and FcRn-binding mutations in the Fc domain accumulated overall less total plasma hFD by day 7, compared to mAb42-IgG4P-LA (42WT) , which does not contain pH-dependent hFD-binding mutations in the Fab domain. For example, compare mAb42-IgG4P-LA (42WT) and mAb42pH-IgG4P-LA (42Mut) . This suggests that pH-dependent hFD binding contributed to plasma hFD reduction. Among all FcRn binding variants, FcRn binding sweeping mutations YPY produced the strongest hFD reduction effect, or antigen-sweeping effect, compared with other Fc mutations; particularly, the YPY variant completely ablated hFD accumulation by day 15 (FIG. 11B) . When the ratios of total plasma IgG4 mAb and total hFD in the hFD Scid/FcRn mice were compared for each mAb, higher IgG4/hFD ratios were observed for FcRn binding sweeping mutations YEY and N3E from day 5 onwards (FIG. 11C) . The YEY and N3E mutations showed higher binding affinity to FcRn at both pH 5.8 and pH 7.4. Their higher IgG4/hFD ratios may have been due to higher binding affinity to FcRn at pH 5.8 which improved antibody recycling (or high total IgG4) (see FIG. 11A) and higher binding affinity to FcRn at pH 7.4 which promoted antigen sweeping activity (or low hFD level) (see FIG. 11B) .
40 mg/kg of either mAb42-IgG4P-LA (42WT) or mAb42pH-IgG4P-N3E (N3E) was injected into hFD/FcRn-Scid mice. The total (FIG. 12A) and free (FIG. 12B) plasma IgG4 levels were compared. There was no significant difference between the total IgG4 levels of mice injected with 42WT or N3E mAbs (FIG. 12A) . When free plasma IgG4 levels were measured, mice injected with 42WT showed a sudden drop of free IgG4 concentration between day 5 and  day 7 and remained low thereafter, while mice injected with N3E showed a slow, gradual decrease in free plasma IgG4 levels over time (FIG. 12B) . The pH-dependent FD and FcRn binding properties of N3E may account for the enhanced release and recycling of free mAb from the mAb/hFD complex. Free IgG4 can continue to bind, neutralize, and sweep circulating hFD into endosomes. When total hFD levels in hFD-expressing Scid/FcRn mice were measured following treatment with either 42WT or N3E mAb, treatment with N3E showed a much lower concentration of total hFD in plasma than 42WT, demonstrating that N3E contributed to strong antigen-sweeping effects by the pH-dependent FD-and FcRn-binding mutations in the Fab and Fc domains, respectively (FIG. 12C) . When the ratios of total IgG4 mAb to total hFD in the injected hFD Scid/FcRn mice were compared, mice injected with N3E had a higher IgG4/hFD ratio than 42WT after day 3. These data show that pH-dependent binding to hFD and high binding affinity to FcRn at pH 7.4 of mAb42pH-IgG4P-N3E resulted in longer mAb persistence (recycling effect) and lower hFD accumulation (antigen sweeping effect) (FIG. 12D) .
SEQUENCE TABLE












Claims (31)

  1. An isolated antibody construct (anti-human FD antibody construct) comprising an antibody moiety specifically binding to human factor D (anti-human FD antibody moiety) , wherein the anti-human FD antibody moiety comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL) , wherein:
    (i) the VH comprises a heavy chain CDR1 ( “H-CDR1” ) comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof comprising up to 3 amino acid variations; a heavy chain CDR2 ( “H-CDR2” ) comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to 3 amino acid variations; and a heavy chain CDR3 ( “H-CDR3” ) comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to 3 amino acid variations; and
    (ii) the VL comprises a light chain CDR1 ( “L-CDR1” ) comprising the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to 3 amino acid variations; a light chain CDR2 ( “L-CDR2” ) comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to 3 amino acid variations; and a light chain CDR3 ( “L-CDR3” ) comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to 3 amino acid variations.
  2. The isolated anti-human FD antibody construct of claim 1, wherein:
    (i) the VH comprises an H-CDR1 comprising the amino acid sequence of D-T-Y-V-H (SEQ ID NO: 1) ; an H-CDR2 comprising the amino acid sequence of R-I-D-P-X1-X2-G-X3-T-X4-F-X5-P-R-F-Q-A (SEQ ID NO: 9) , wherein X1 is A or H, X2 is N, S, or Y, X3 is L or H, X4 is T or H, and X5 is D, V, L, or H; and an H-CDR3 comprising the amino acid sequence of A-M-E-Y (SEQ ID NO: 3) ; and
    (ii) the VL comprises an L-CDR1 comprising the amino acid sequence of S-A-X6-S-D-V-S-X7-M-Y (SEQ ID NO: 10) , wherein X6 is R, N, or S, and X7 is Y, D, or V; an L-CDR2 comprising the amino acid sequence of X8-T-S-N-L-A-S (SEQ ID NO: 252) , wherein X8 is D or H; and an L-CDR3 comprising the amino acid sequence of Q-Q-W-S-S-X9-P-P-W-X10 (SEQ ID NO: 11) , wherein X9 is Y or H, X10 is T, R, or H.
  3. The isolated anti-human FD antibody construct of claim 1 or 2, wherein:
    (1) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6;
    (2) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 23, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 25;
    (3) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 36, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 39, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 41;
    (4) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 44, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 46; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 47, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 49;
    (5) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 52, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 54; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, an L-CDR2 comprising the amino acid  sequence of SEQ ID NO: 56, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 57;
    (6) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 63, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 64, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 65;
    (7) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 71, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 72, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 73;
    (8) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 76, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 78; the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 79, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 81;
    (9) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 84, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 85, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 86; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89;
    (10) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 92, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 93, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 94; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid  sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97;
    (11) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 100, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 101, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 102; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105;
    (12) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 108, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 109, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 110; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113;
    (13) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 116, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 117, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 118; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121;
    (14) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 126;
    (15) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an L-CDR2 comprising the amino acid  sequence of SEQ ID NO: 125, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 126;
    (16) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6;
    (17) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 124, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 125, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 126;
    (18) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89;
    (19) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105;
    (20) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid  sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97;
    (21) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113;
    (22) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 128, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 130; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121;
    (23) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 57;
    (24) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89;
    (25) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid  sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105;
    (26) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97;
    (27) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113;
    (28) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 60, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 62; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121;
    (29) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 57;
    (30) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, an L-CDR2 comprising the amino acid  sequence of SEQ ID NO: 88, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 89;
    (31) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 103, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 104, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 105;
    (32) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 95, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 96, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 97;
    (33) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 113;
    (34) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 68, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 70; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 119, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 120, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 121;
    (35) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 206, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 207, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 208; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 209, an L-CDR2 comprising the amino acid  sequence of SEQ ID NO: 210, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 211;
    (36) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 234, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 235, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 236; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 237, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 238, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 239;
    (37) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 240, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 241, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 242; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 243, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 244, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 245; or
    (38) the VH comprises an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 246, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 247, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 248; and the VL comprises an L-CDR1 comprising the amino acid sequence of SEQ ID NO: 249, an L-CDR2 comprising the amino acid sequence of SEQ ID NO: 250, and an L-CDR3 comprising the amino acid sequence of SEQ ID NO: 251.
  4. The isolated anti-human FD antibody construct of any one of claims 1-3, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 80%amino acid sequence homology to SEQ ID NO: 7; and the VL comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 80%amino acid sequence homology to SEQ ID NO: 8.
  5. The isolated anti-human FD antibody construct of claim 4, wherein:
    i) the amino acid residue at position 24 of the VH is A or T;
    ii) the amino acid residue at position 74 of the VH is K or T;
    iii) the amino acid residue at position 77 of the VH is N or S;
    iv) the amino acid residue at position 97 of the VH is A or T;
    v) the amino acid residue at position 98 of the VH is R or Y; and/or
    vi) the amino acid residue at position 70 of the VL is F or Y,
    wherein the numbering is according to the Kabat numbering system.
  6. The isolated anti-human FD antibody construct of any one of claims 1-5, wherein:
    (1) the VH comprises the amino acid sequence of SEQ ID NO: 7, and the VL comprises the amino acid sequence of SEQ ID NO: 8;
    (2) the VH comprises the amino acid sequence of SEQ ID NO: 18, and the VL comprises the amino acid sequence of SEQ ID NO: 19;
    (3) the VH comprises the amino acid sequence of SEQ ID NO: 26, and the VL comprises the amino acid sequence of SEQ ID NO: 27;
    (4) the VH comprises the amino acid sequence of SEQ ID NO: 34, and the VL comprises the amino acid sequence of SEQ ID NO: 35;
    (5) the VH comprises the amino acid sequence of SEQ ID NO: 42, and the VL comprises the amino acid sequence of SEQ ID NO: 43;
    (6) the VH comprises the amino acid sequence of SEQ ID NO: 50, and the VL comprises the amino acid sequence of SEQ ID NO: 51;
    (7) the VH comprises the amino acid sequence of SEQ ID NO: 58, and the VL comprises the amino acid sequence of SEQ ID NO: 59;
    (8) the VH comprises the amino acid sequence of SEQ ID NO: 66, and the VL comprises the amino acid sequence of SEQ ID NO: 67;
    (9) the VH comprises the amino acid sequence of SEQ ID NO: 74, and the VL comprises the amino acid sequence of SEQ ID NO: 75;
    (10) the VH comprises the amino acid sequence of SEQ ID NO: 82, and the VL comprises the amino acid sequence of SEQ ID NO: 83;
    (11) the VH comprises the amino acid sequence of SEQ ID NO: 90, and the VL comprises the amino acid sequence of SEQ ID NO: 91;
    (12) the VH comprises the amino acid sequence of SEQ ID NO: 98, and the VL comprises the amino acid sequence of SEQ ID NO: 99;
    (13) the VH comprises the amino acid sequence of SEQ ID NO: 106, and the VL comprises the amino acid sequence of SEQ ID NO: 107;
    (14) the VH comprises the amino acid sequence of SEQ ID NO: 114, and the VL comprises the amino acid sequence of SEQ ID NO: 115;
    (15) the VH comprises the amino acid sequence of SEQ ID NO: 122, and the VL comprises the amino acid sequence of SEQ ID NO: 123;
    (16) the VH comprises the amino acid sequence of SEQ ID NO: 156, and the VL comprises the amino acid sequence of SEQ ID NO: 157;
    (17) the VH comprises the amino acid sequence of SEQ ID NO: 158, and the VL comprises the amino acid sequence of SEQ ID NO: 159;
    (18) the VH comprises the amino acid sequence of SEQ ID NO: 160, and the VL comprises the amino acid sequence of SEQ ID NO: 161;
    (19) the VH comprises the amino acid sequence of SEQ ID NO: 162, and the VL comprises the amino acid sequence of SEQ ID NO: 163;
    (20) the VH comprises the amino acid sequence of SEQ ID NO: 164, and the VL comprises the amino acid sequence of SEQ ID NO: 165;
    (21) the VH comprises the amino acid sequence of SEQ ID NO: 166, and the VL comprises the amino acid sequence of SEQ ID NO: 167;
    (22) the VH comprises the amino acid sequence of SEQ ID NO: 168, and the VL comprises the amino acid sequence of SEQ ID NO: 169;
    (23) the VH comprises the amino acid sequence of SEQ ID NO: 172, and the VL comprises the amino acid sequence of SEQ ID NO: 173;
    (24) the VH comprises the amino acid sequence of SEQ ID NO: 174, and the VL comprises the amino acid sequence of SEQ ID NO: 175;
    (25) the VH comprises the amino acid sequence of SEQ ID NO: 176, and the VL comprises the amino acid sequence of SEQ ID NO: 177;
    (26) the VH comprises the amino acid sequence of SEQ ID NO: 178, and the VL comprises the amino acid sequence of SEQ ID NO: 179;
    (27) the VH comprises the amino acid sequence of SEQ ID NO: 180, and the VL comprises the amino acid sequence of SEQ ID NO: 181;
    (28) the VH comprises the amino acid sequence of SEQ ID NO: 182, and the VL comprises the amino acid sequence of SEQ ID NO: 183;
    (29) the VH comprises the amino acid sequence of SEQ ID NO: 184, and the VL comprises the amino acid sequence of SEQ ID NO: 185;
    (30) the VH comprises the amino acid sequence of SEQ ID NO: 186, and the VL comprises the amino acid sequence of SEQ ID NO: 187;
    (31) the VH comprises the amino acid sequence of SEQ ID NO: 190, and the VL comprises the amino acid sequence of SEQ ID NO: 191;
    (32) the VH comprises the amino acid sequence of SEQ ID NO: 192, and the VL comprises the amino acid sequence of SEQ ID NO: 193;
    (33) the VH comprises the amino acid sequence of SEQ ID NO: 194, and the VL comprises the amino acid sequence of SEQ ID NO: 195;
    (34) the VH comprises the amino acid sequence of SEQ ID NO: 196, and the VL comprises the amino acid sequence of SEQ ID NO: 197;
    (35) the VH comprises the amino acid sequence of SEQ ID NO: 198, and the VL comprises the amino acid sequence of SEQ ID NO: 199;
    (36) the VH comprises the amino acid sequence of SEQ ID NO: 212, and the VL comprises the amino acid sequence of SEQ ID NO: 213;
    (37) the VH comprises the amino acid sequence of SEQ ID NO: 253, and the VL comprises the amino acid sequence of SEQ ID NO: 254;
    (38) the VH comprises the amino acid sequence of SEQ ID NO: 255, and the VL comprises the amino acid sequence of SEQ ID NO: 256;
    (39) the VH comprises the amino acid sequence of SEQ ID NO: 257, and the VL comprises the amino acid sequence of SEQ ID NO: 258;
    (40) the VH comprises the amino acid sequence of SEQ ID NO: 289, and the VL comprises the amino acid sequence of SEQ ID NO: 127; or
    (41) the VH comprises the amino acid sequence of SEQ ID NO: 216, and the VL comprises the amino acid sequence of SEQ ID NO: 214.
  7. The isolated anti-human FD antibody construct of any one of claims 1-6, wherein the anti-human FD antibody moiety cross-reacts with a cynomolgus monkey factor D (cyno FD) .
  8. The isolated anti-human FD antibody construct of any one of claims 1-7, wherein the anti-human FD antibody moiety is pH-dependent, and wherein the anti-human FD antibody moiety binds more strongly to human FD at a neutral pH than it does at an acidic pH.
  9. The isolated anti-human FD antibody construct of any one of claims 1-8, wherein the anti-human FD antibody moiety is selected from the group consisting of: a full-length antibody, Fab, Fab’, F (ab) 2, F (ab’) 2, scFv, and a combination thereof.
  10. The isolated anti-human FD antibody construct of any one of claims 1-9, wherein the anti-human FD antibody moiety is a full-length antibody (anti-human FD full-length antibody) .
  11. The isolated anti-human FD antibody construct of claim 10, wherein the anti-human FD full-length antibody comprises a heavy chain constant region derived from IgG4.
  12. The isolated anti-human FD antibody construct of claim 11, wherein the heavy chain constant region comprises the amino acid sequence of any of SEQ ID NOs: 132-139.
  13. The isolated anti-human FD antibody construct of any one of claims 1-9, wherein the anti-human FD antibody moiety is an scFv (anti-human FD scFv) .
  14. The isolated anti-human FD antibody construct of claim 14, wherein the anti-human FD scFv comprises the amino acid sequence of SEQ ID NO: 219 or 220.
  15. The isolated anti-human FD antibody construct of any one of claims 1-14, wherein the isolated anti-human FD antibody construct further comprises a second antibody moiety specifically recognizing a component of the complement pathway.
  16. The isolated anti-human FD antibody construct of claim 15, wherein the second antibody moiety is selected from the group consisting of: a full-length antibody, Fab, Fab’, F (ab) 2, F (ab’) 2, scFv, sdAb, and a combination thereof.
  17. The isolated anti-human FD antibody construct of claim 15 or 16, wherein the component of the complement pathway is complement component 2 (C2) .
  18. The isolated anti-human FD antibody construct of claim 15 or 16, wherein the component of the complement pathway is complement component 5 (C5) .
  19. The isolated anti-human FD antibody construct of any one of claims 15-18, wherein the anti-human FD antibody moiety is an scFv (scFv1) , and wherein the second antibody moiety is an scFv (scFv2) .
  20. The isolated anti-human FD antibody construct of claim 19, wherein the isolated anti-human FD antibody construct comprises from N-terminus to C-terminus:
    (i) scFv1-optional linker-scFv2;
    (ii) scFv2-optional linker-scFv1;
    (iii) scFv1-optional linker-Fc domain-optional linker-scFv2; or
    (iv) scFv2-optional linker-Fc domain-optional linker-scFv1.
  21. The isolated anti-human FD antibody construct of claim 20, wherein:
    i) the linker comprises the amino acid sequence of any of SEQ ID NOs: 221-229; and/or ii) the Fc domain comprises the amino acid sequence of SEQ ID NO: 230.
  22. An isolated nucleic acid encoding the isolated anti-human FD antibody construct of any one of claims 1-21.
  23. A vector comprising the isolated nucleic acid of claim 22.
  24. The vector of claim 23, wherein the vector is a viral vector.
  25. The vector of claim 24, wherein the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector.
  26. A host cell comprising the isolated nucleic acid of claim 22, or the vector of any one of claims 23-25.
  27. A method of making an isolated anti-human FD antibody construct, comprising:
    i) culturing a host cell comprising the isolated nucleic acid of claim 22, or the vector of any one of claims 23-25, or the host cell of claim 26, under a condition suitable for the expression of the isolated anti-human FD antibody construct; and
    ii) obtaining the expressed isolated anti-human FD antibody construct from said host cell.
  28. A pharmaceutical composition comprising the isolated anti-human FD antibody construct of any one of claims 1-21, the isolated nucleic acid of claim 22, or the vector of any one of claims 23-25, and a pharmaceutically acceptable carrier.
  29. A method for treating a complement-mediated disease in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of claim 28.
  30. The method of claim 29, wherein the complement-mediated disease is selected from the group consisting of: macular degeneration (MD) , age-related macular degeneration (AMD) , ischemia reperfusion injury, arthritis, rheumatoid arthritis, lupus, ulcerative colitis, stroke, post-surgery systemic inflammatory syndrome, asthma, allergic asthma, chronic obstructive pulmonary disease (COPD) , paroxysmal nocturnal hemoglobinuria (PNH) syndrome, autoimmune hemolytic anemia (AIHA) , Gaucher disease, myasthenia gravis, neuromyelitis optica (NMO) , multiple sclerosis, delayed graft function, antibody-mediated rejection, atypical hemolytic uremic syndrome (aHUS) , central retinal vein occlusion (CRVO) , central retinal artery occlusion (CRAO) , epidermolysis bullosa, sepsis, septic shock, organ transplantation, inflammation, C3 glomerulopathy (C3G) , membranous nephropathy, IgA nephropathy (IgAN) , glomerulonephritis, thrombotic microangiopathies secondary to systemic lupus erythematosus (SLE-TMA) , anti-neutrophil cytoplasmic antibody (ANCA) -mediated vasculitis, Shiga toxin induced HUS, antiphospholipid antibody-induced pregnancy loss, graft versus host disease  (GvHD) , bullous pemphigoid, hidradenitis suppurativa, dermatitis herpetiformis, sweets syndrome, pyoderma gangrenosum, palmo-plantar pustulosis &pustular psoriasis, rheumatoid neutrophilic dermatoses, subcorneal pustular dermatosis, bowel-associated dermatosis-arthritis syndrome, neutrophilic eccrine hidradenitis, linear IgA disease, and any combinations thereof.
  31. A method for reducing the activity of a complement system in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of claim 28.
PCT/CN2024/116356 2023-09-03 2024-09-02 Anti-human factor d antibody constructs and uses thereof WO2025045250A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN2023116638 2023-09-03
CN2023116639 2023-09-03
CNPCT/CN2023/116639 2023-09-03
CNPCT/CN2023/116638 2023-09-03

Publications (1)

Publication Number Publication Date
WO2025045250A1 true WO2025045250A1 (en) 2025-03-06

Family

ID=92925690

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/CN2024/116358 WO2025045251A2 (en) 2023-09-03 2024-09-02 Multispecific constructs comprising anti-factor d moiety
PCT/CN2024/116356 WO2025045250A1 (en) 2023-09-03 2024-09-02 Anti-human factor d antibody constructs and uses thereof

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/116358 WO2025045251A2 (en) 2023-09-03 2024-09-02 Multispecific constructs comprising anti-factor d moiety

Country Status (1)

Country Link
WO (2) WO2025045251A2 (en)

Citations (84)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0054951A1 (en) 1980-12-24 1982-06-30 Chugai Seiyaku Kabushiki Kaisha Dibenz(b,f)(1,4)oxazepine derivatives, process for preparing the same, and pharmaceutical compositions comprising the same
EP0239400A2 (en) 1986-03-27 1987-09-30 Medical Research Council Recombinant antibodies and methods for their production
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
EP0404097A2 (en) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Bispecific and oligospecific, mono- and oligovalent receptors, production and applications thereof
WO1991010741A1 (en) 1990-01-12 1991-07-25 Cell Genesys, Inc. Generation of xenogeneic antibodies
WO1993011161A1 (en) 1991-11-25 1993-06-10 Enzon, Inc. Multivalent antigen-binding proteins
WO1994029351A2 (en) 1993-06-16 1994-12-22 Celltech Limited Antibodies
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
WO1996034103A1 (en) 1995-04-25 1996-10-31 Vrije Universiteit Brussel Variable fragments of immunoglobulins - use for therapeutic or veterinary purposes
WO1996034096A1 (en) 1995-04-28 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1996033735A1 (en) 1995-04-27 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
WO1997030087A1 (en) 1996-02-16 1997-08-21 Glaxo Group Limited Preparation of glycosylated antibodies
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
WO1998024893A3 (en) 1996-12-03 1998-08-20 Abgenix Inc TRANSGENIC MAMMALS HAVING HUMAN IG LOCI INCLUDING PLURAL VH AND Vλ REGIONS AND ANTIBODIES PRODUCED THEREFROM
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1998058964A1 (en) 1997-06-24 1998-12-30 Genentech, Inc. Methods and compositions for galactosylated glycoproteins
WO1999022764A1 (en) 1997-10-31 1999-05-14 Genentech, Inc. Methods and compositions comprising glycoprotein glycoforms
WO1999051642A1 (en) 1998-04-02 1999-10-14 Genentech, Inc. Antibody variants and fragments thereof
WO1999054440A1 (en) 1998-04-21 1999-10-28 Micromet Gesellschaft Für Biomedizinische Forschung Mbh CD19xCD3 SPECIFIC POLYPEPTIDES AND USES THEREOF
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO2000061739A1 (en) 1999-04-09 2000-10-19 Kyowa Hakko Kogyo Co., Ltd. Method for controlling the activity of immunologically functional molecule
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
WO2001029246A1 (en) 1999-10-19 2001-04-26 Kyowa Hakko Kogyo Co., Ltd. Process for producing polypeptide
WO2001029058A1 (en) 1999-10-15 2001-04-26 University Of Massachusetts Rna interference pathway genes as tools for targeted genetic interference
US6326193B1 (en) 1999-11-05 2001-12-04 Cambria Biosciences, Llc Insect control agent
WO2001096584A2 (en) 2000-06-12 2001-12-20 Akkadix Corporation Materials and methods for the control of nematodes
WO2002031140A1 (en) 2000-10-06 2002-04-18 Kyowa Hakko Kogyo Co., Ltd. Cells producing antibody compositions
US20020164328A1 (en) 2000-10-06 2002-11-07 Toyohide Shinkawa Process for purifying antibody
WO2003011878A2 (en) 2001-08-03 2003-02-13 Glycart Biotechnology Ag Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity
WO2003048731A2 (en) 2001-12-03 2003-06-12 Abgenix, Inc. Antibody categorization based on binding characteristics
US20030115614A1 (en) 2000-10-06 2003-06-19 Yutaka Kanda Antibody composition-producing cell
US6602684B1 (en) 1998-04-20 2003-08-05 Glycart Biotechnology Ag Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US20030157108A1 (en) 2001-10-25 2003-08-21 Genentech, Inc. Glycoprotein compositions
WO2003084570A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. DRUG CONTAINING ANTIBODY COMPOSITION APPROPRIATE FOR PATIENT SUFFERING FROM FcϜRIIIa POLYMORPHISM
WO2003085107A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Cells with modified genome
WO2003085119A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. METHOD OF ENHANCING ACTIVITY OF ANTIBODY COMPOSITION OF BINDING TO FcϜ RECEPTOR IIIa
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
WO2004042072A2 (en) 2002-11-01 2004-05-21 The Regents Of The University Of Colorado, A Body Corporate Quantitative analysis of protein isoforms using matrix-assisted laser desorption/ionization time of flight mass spectrometry
US20040110282A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost
US20040109865A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-containing medicament
WO2004049794A2 (en) 2002-12-03 2004-06-17 The Babraham Institute Single chain antibodies produced in a transgenic mouse
US20040132140A1 (en) 2002-04-09 2004-07-08 Kyowa Hakko Kogyo Co., Ltd. Production process for antibody composition
WO2004056312A2 (en) 2002-12-16 2004-07-08 Genentech, Inc. Immunoglobulin variants and uses thereof
WO2004092219A2 (en) 2003-04-10 2004-10-28 Protein Design Labs, Inc Alteration of fcrn binding affinities or serum half-lives of antibodies by mutagenesis
US20050014934A1 (en) 2002-10-15 2005-01-20 Hinton Paul R. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
WO2005035778A1 (en) 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. PROCESS FOR PRODUCING ANTIBODY COMPOSITION BY USING RNA INHIBITING THE FUNCTION OF α1,6-FUCOSYLTRANSFERASE
WO2005035586A1 (en) 2003-10-08 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Fused protein composition
US20050123546A1 (en) 2003-11-05 2005-06-09 Glycart Biotechnology Ag Antigen binding molecules with increased Fc receptor binding affinity and effector function
WO2005053742A1 (en) 2003-12-04 2005-06-16 Kyowa Hakko Kogyo Co., Ltd. Medicine containing antibody composition
WO2005100402A1 (en) 2004-04-13 2005-10-27 F.Hoffmann-La Roche Ag Anti-p-selectin antibodies
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
WO2006029879A2 (en) 2004-09-17 2006-03-23 F.Hoffmann-La Roche Ag Anti-ox40l antibodies
US7041870B2 (en) 2000-11-30 2006-05-09 Medarex, Inc. Transgenic transchromosomal rodents for making human antibodies
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody
US7189826B2 (en) 1997-11-24 2007-03-13 Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
US20070061900A1 (en) 2000-10-31 2007-03-15 Murphy Andrew J Methods of modifying eukaryotic cells
US7371826B2 (en) 1999-01-15 2008-05-13 Genentech, Inc. Polypeptide variants with altered effector function
WO2008077546A1 (en) 2006-12-22 2008-07-03 F. Hoffmann-La Roche Ag Antibodies against insulin-like growth factor i receptor and uses thereof
US7521541B2 (en) 2004-09-23 2009-04-21 Genetech Inc. Cysteine engineered antibodies and conjugates
US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies
US20090307787A1 (en) 2006-01-25 2009-12-10 Franklin Gerardus Grosveld Generation of heavy-chain only antibodies in transgenic animals
US20100122358A1 (en) 2008-06-06 2010-05-13 Crescendo Biologics Limited H-Chain-only antibodies
US8754287B2 (en) 2009-12-10 2014-06-17 Regeneron Pharmaceuticals, Inc. Mice that make heavy chain antibodies
US9079949B1 (en) 2014-03-07 2015-07-14 Alexion Pharmaceuticals, Inc. Anti-C5 antibodies having improved pharmacokinetics
US20150289489A1 (en) 2014-03-21 2015-10-15 Regeneron Pharmaceuticals, Inc. Non-human animals that make single domain binding proteins
WO2015168468A1 (en) * 2014-05-01 2015-11-05 Genentech, Inc. Anti-factor d antibody variants and uses thereof
US9765135B2 (en) 2014-12-19 2017-09-19 Chugai Seiyaku Kabushiki Kaisha Anti-C5 antibodies
WO2018148486A1 (en) * 2017-02-10 2018-08-16 The Trustees Of The University Of Pennsylvania Anti-factor d antibodies and uses thereof
WO2018191548A2 (en) * 2017-04-14 2018-10-18 Kodiak Sciences Inc. Complement factor d antagonist antibodies and conjugates thereof
US20220177556A1 (en) 2018-09-06 2022-06-09 The Trustees Of The University Of Pennsylvania Humanized anti-c5 antibodies and uses thereof
US20220204602A1 (en) 2019-04-24 2022-06-30 The Trustees Of The University Of Pennsylvania Bi-functional humanized anti-c5 antibodies and factor h fusion proteins and uses thereof
WO2022134047A1 (en) 2020-12-25 2022-06-30 The Trustees Of The University Of Pennsylvania Humanized anti-c5 antibodies and factor h fusion proteins and uses thereof
WO2022215054A1 (en) * 2021-04-09 2022-10-13 Takeda Pharmaceutical Company Limited Antibodies targeting complement factor d and uses therof
US11578137B2 (en) 2017-03-06 2023-02-14 The Trustees Of The University Of Pennsylvania Anti-C5 antibodies and uses thereof
WO2023143583A1 (en) 2022-01-29 2023-08-03 Kira Pharmaceuticals (Suzhou) Ltd. Anti-c2 antibodies and uses thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021226442A2 (en) * 2020-05-08 2021-11-11 Ngm Biopharmaceuticals, Inc. Therapeutic uses of c3-binding agents

Patent Citations (88)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0054951A1 (en) 1980-12-24 1982-06-30 Chugai Seiyaku Kabushiki Kaisha Dibenz(b,f)(1,4)oxazepine derivatives, process for preparing the same, and pharmaceutical compositions comprising the same
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
EP0239400A2 (en) 1986-03-27 1987-09-30 Medical Research Council Recombinant antibodies and methods for their production
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5648260A (en) 1987-03-18 1997-07-15 Scotgen Biopharmaceuticals Incorporated DNA encoding antibodies with altered effector functions
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
EP0404097A2 (en) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Bispecific and oligospecific, mono- and oligovalent receptors, production and applications thereof
WO1991010741A1 (en) 1990-01-12 1991-07-25 Cell Genesys, Inc. Generation of xenogeneic antibodies
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1993011161A1 (en) 1991-11-25 1993-06-10 Enzon, Inc. Multivalent antigen-binding proteins
WO1994029351A2 (en) 1993-06-16 1994-12-22 Celltech Limited Antibodies
WO1996034103A1 (en) 1995-04-25 1996-10-31 Vrije Universiteit Brussel Variable fragments of immunoglobulins - use for therapeutic or veterinary purposes
WO1996033735A1 (en) 1995-04-27 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1996034096A1 (en) 1995-04-28 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1997030087A1 (en) 1996-02-16 1997-08-21 Glaxo Group Limited Preparation of glycosylated antibodies
WO1998024893A3 (en) 1996-12-03 1998-08-20 Abgenix Inc TRANSGENIC MAMMALS HAVING HUMAN IG LOCI INCLUDING PLURAL VH AND Vλ REGIONS AND ANTIBODIES PRODUCED THEREFROM
WO1998058964A1 (en) 1997-06-24 1998-12-30 Genentech, Inc. Methods and compositions for galactosylated glycoproteins
WO1999022764A1 (en) 1997-10-31 1999-05-14 Genentech, Inc. Methods and compositions comprising glycoprotein glycoforms
US7189826B2 (en) 1997-11-24 2007-03-13 Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody
WO1999051642A1 (en) 1998-04-02 1999-10-14 Genentech, Inc. Antibody variants and fragments thereof
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
US6602684B1 (en) 1998-04-20 2003-08-05 Glycart Biotechnology Ag Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
WO1999054440A1 (en) 1998-04-21 1999-10-28 Micromet Gesellschaft Für Biomedizinische Forschung Mbh CD19xCD3 SPECIFIC POLYPEPTIDES AND USES THEREOF
US7332581B2 (en) 1999-01-15 2008-02-19 Genentech, Inc. Polypeptide variants with altered effector function
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US7371826B2 (en) 1999-01-15 2008-05-13 Genentech, Inc. Polypeptide variants with altered effector function
WO2000061739A1 (en) 1999-04-09 2000-10-19 Kyowa Hakko Kogyo Co., Ltd. Method for controlling the activity of immunologically functional molecule
WO2001029058A1 (en) 1999-10-15 2001-04-26 University Of Massachusetts Rna interference pathway genes as tools for targeted genetic interference
WO2001029246A1 (en) 1999-10-19 2001-04-26 Kyowa Hakko Kogyo Co., Ltd. Process for producing polypeptide
US6326193B1 (en) 1999-11-05 2001-12-04 Cambria Biosciences, Llc Insect control agent
WO2001096584A2 (en) 2000-06-12 2001-12-20 Akkadix Corporation Materials and methods for the control of nematodes
WO2002031140A1 (en) 2000-10-06 2002-04-18 Kyowa Hakko Kogyo Co., Ltd. Cells producing antibody compositions
US20030115614A1 (en) 2000-10-06 2003-06-19 Yutaka Kanda Antibody composition-producing cell
US20020164328A1 (en) 2000-10-06 2002-11-07 Toyohide Shinkawa Process for purifying antibody
US20070061900A1 (en) 2000-10-31 2007-03-15 Murphy Andrew J Methods of modifying eukaryotic cells
US7041870B2 (en) 2000-11-30 2006-05-09 Medarex, Inc. Transgenic transchromosomal rodents for making human antibodies
WO2003011878A2 (en) 2001-08-03 2003-02-13 Glycart Biotechnology Ag Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity
US20030157108A1 (en) 2001-10-25 2003-08-21 Genentech, Inc. Glycoprotein compositions
WO2003048731A2 (en) 2001-12-03 2003-06-12 Abgenix, Inc. Antibody categorization based on binding characteristics
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
US20040110704A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells of which genome is modified
US20040109865A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-containing medicament
US20040110282A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost
US20040132140A1 (en) 2002-04-09 2004-07-08 Kyowa Hakko Kogyo Co., Ltd. Production process for antibody composition
WO2003085119A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. METHOD OF ENHANCING ACTIVITY OF ANTIBODY COMPOSITION OF BINDING TO FcϜ RECEPTOR IIIa
WO2003085107A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Cells with modified genome
WO2003084570A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. DRUG CONTAINING ANTIBODY COMPOSITION APPROPRIATE FOR PATIENT SUFFERING FROM FcϜRIIIa POLYMORPHISM
US20050014934A1 (en) 2002-10-15 2005-01-20 Hinton Paul R. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
WO2004042072A2 (en) 2002-11-01 2004-05-21 The Regents Of The University Of Colorado, A Body Corporate Quantitative analysis of protein isoforms using matrix-assisted laser desorption/ionization time of flight mass spectrometry
WO2004049794A2 (en) 2002-12-03 2004-06-17 The Babraham Institute Single chain antibodies produced in a transgenic mouse
WO2004056312A2 (en) 2002-12-16 2004-07-08 Genentech, Inc. Immunoglobulin variants and uses thereof
WO2004092219A2 (en) 2003-04-10 2004-10-28 Protein Design Labs, Inc Alteration of fcrn binding affinities or serum half-lives of antibodies by mutagenesis
WO2005035586A1 (en) 2003-10-08 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Fused protein composition
WO2005035778A1 (en) 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. PROCESS FOR PRODUCING ANTIBODY COMPOSITION BY USING RNA INHIBITING THE FUNCTION OF α1,6-FUCOSYLTRANSFERASE
US20050123546A1 (en) 2003-11-05 2005-06-09 Glycart Biotechnology Ag Antigen binding molecules with increased Fc receptor binding affinity and effector function
WO2005053742A1 (en) 2003-12-04 2005-06-16 Kyowa Hakko Kogyo Co., Ltd. Medicine containing antibody composition
US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies
WO2005100402A1 (en) 2004-04-13 2005-10-27 F.Hoffmann-La Roche Ag Anti-p-selectin antibodies
WO2006029879A2 (en) 2004-09-17 2006-03-23 F.Hoffmann-La Roche Ag Anti-ox40l antibodies
US7521541B2 (en) 2004-09-23 2009-04-21 Genetech Inc. Cysteine engineered antibodies and conjugates
US20090307787A1 (en) 2006-01-25 2009-12-10 Franklin Gerardus Grosveld Generation of heavy-chain only antibodies in transgenic animals
WO2008077546A1 (en) 2006-12-22 2008-07-03 F. Hoffmann-La Roche Ag Antibodies against insulin-like growth factor i receptor and uses thereof
US20100122358A1 (en) 2008-06-06 2010-05-13 Crescendo Biologics Limited H-Chain-only antibodies
US8754287B2 (en) 2009-12-10 2014-06-17 Regeneron Pharmaceuticals, Inc. Mice that make heavy chain antibodies
US9079949B1 (en) 2014-03-07 2015-07-14 Alexion Pharmaceuticals, Inc. Anti-C5 antibodies having improved pharmacokinetics
US20150289489A1 (en) 2014-03-21 2015-10-15 Regeneron Pharmaceuticals, Inc. Non-human animals that make single domain binding proteins
WO2015168468A1 (en) * 2014-05-01 2015-11-05 Genentech, Inc. Anti-factor d antibody variants and uses thereof
US9765135B2 (en) 2014-12-19 2017-09-19 Chugai Seiyaku Kabushiki Kaisha Anti-C5 antibodies
WO2018148486A1 (en) * 2017-02-10 2018-08-16 The Trustees Of The University Of Pennsylvania Anti-factor d antibodies and uses thereof
US11434279B2 (en) 2017-02-10 2022-09-06 The Trustees Of The University Of Pennsylvania Anti-factor D antibodies and uses thereof
US11578137B2 (en) 2017-03-06 2023-02-14 The Trustees Of The University Of Pennsylvania Anti-C5 antibodies and uses thereof
WO2018191548A2 (en) * 2017-04-14 2018-10-18 Kodiak Sciences Inc. Complement factor d antagonist antibodies and conjugates thereof
US20220177556A1 (en) 2018-09-06 2022-06-09 The Trustees Of The University Of Pennsylvania Humanized anti-c5 antibodies and uses thereof
US20220204602A1 (en) 2019-04-24 2022-06-30 The Trustees Of The University Of Pennsylvania Bi-functional humanized anti-c5 antibodies and factor h fusion proteins and uses thereof
WO2022134047A1 (en) 2020-12-25 2022-06-30 The Trustees Of The University Of Pennsylvania Humanized anti-c5 antibodies and factor h fusion proteins and uses thereof
WO2022215054A1 (en) * 2021-04-09 2022-10-13 Takeda Pharmaceutical Company Limited Antibodies targeting complement factor d and uses therof
WO2023143583A1 (en) 2022-01-29 2023-08-03 Kira Pharmaceuticals (Suzhou) Ltd. Anti-c2 antibodies and uses thereof

Non-Patent Citations (148)

* Cited by examiner, † Cited by third party
Title
"Instructions for Use WIESLABOO Complement System Alternative Pathway", DOCUMENT NO. LABEL-DOC-0034, December 2018 (2018-12-01)
"Remington's Pharmaceutical Sciences", 1985, MACK PUBLISHING CO.
ABHINANDANMARTIN, MOL. IMMUNOL., vol. 45, 2008, pages 3832 - 3839
ADOLF-BRYFOGLE ET AL., NUCLEIC ACIDS RES., vol. 43, 2015, pages D432 - D438
ALBERTS ET AL.: "Molecular Biology of the Cell", 1994, GARLAND PUBLISHING, INC
AL-LAZIKANI B ET AL., J. MOL. BIOL., vol. 273, 1997, pages 927 - 948
ALMAGROFRANSSON, FRONT. BIOSCI., vol. 13, 2008, pages 1619 - 1633
ARMOUR KL ET AL., EUR J. IMMUNOL., vol. 29, 1999, pages 2613
BACA ET AL., J. BIOL. CHEM., vol. 272, 1997, pages 10678 - 10684
BARBAS ET AL., PROC NAT. ACAD. SCI. USA, vol. 91, 1994, pages 3809 - 3813
BARRATT ET AL., FRONT IMMUNL, vol. 12, 2021, pages 712572
BENNY K. C. LO: "Antibody Engineering: Methods and Protocols", 2004, HUMANA PRESS
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
BOERNER ET AL., J. IMMUNOL., vol. 147, no. 1, 1991, pages 86 - 95
BONNER, P. L.: "Protein purification", 2007, TAYLOR & FRANCIS
BORREBAEK: "Antibody Engineering, A Practical Guide", 1992, W.H. FREEMAN AND CO.
BORROK ET AL., J BIOL CHEM., vol. 290, no. 7, 13 February 2015 (2015-02-13), pages 4282 - 90
BRODEUR ET AL.: "Monoclonal Antibody Production Techniques and Applications", 1987, MARCEL DEKKER, INC., pages: 51 - 63
BRUGGEMANN ET AL., YEAR IN IMMUNOL, vol. 7, 1993, pages 33
BRUGGEMANN, M ET AL., J. EXP. MED., vol. 166, 1987, pages 1351 - 1361
BURTON, MOLEC IMMUNOL, vol. 22, 1985, pages 161 - 206
BURTON, MOLEC. IMMUNOL., vol. 22, 1985, pages 161 - 206
CAPEL ET AL., IMMUNOMETHODS, vol. 4, 1994, pages 25 - 34
CARTER ET AL., BIOTECHNOLOGY, vol. 10, 1992, pages 163 - 167
CARTER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 4285
CHEADLE ET AL., MOL IMMUNOL, vol. 29, 1992, pages 21 - 30
CHEN, MOL. IMMUNOL., vol. 47, no. 4, 2010, pages 912 - 21
CHOTHIA ET AL., J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CHOWDHURY, METHODS MOL. BIOL., vol. 207, 2008, pages 179 - 196
CLACKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628
CLYNES ET AL., PNAS USA, vol. 95, 1998, pages 652 - 656
CLYNES ET AL., PROC. NAT'L ACAD. SCI. USA, vol. 95, 1998, pages 652 - 656
CORVILLO ET AL., INT J MOL SCI, vol. 22, no. 12, 2021, pages 6608
CRAGG, M.S. ET AL., BLOOD, vol. 101, 2003, pages 1045 - 1052
CRAGG, M.S.M.J. GLENNIE, BLOOD, vol. 103, 2004, pages 2738 - 2743
CUNNINGHAMWELLS, SCIENCE, vol. 244, 1989, pages 1081 - 1085
DALL' ACQUA ET AL., BIOCHEM., vol. 37, 1998, pages 9266 - 9273
DALL' ACQUA ET AL., METHODS, vol. 36, 2005, pages 61 - 68
DE HAAS ET AL., J. LAB. CLIN. MED., vol. 126, 1995, pages 330 - 41
EDGAR, R.C., BMC BIOINFORMATICS, vol. 5, no. 1, 2004, pages 113
EDGAR, R.C., NUCLEIC ACIDS RESEARCH, vol. 32, no. 5, 2004, pages 1792 - 1797
EHRENMANN F ET AL., NUCLEIC ACIDS RES., vol. 38, 2010, pages D301 - D307
EVANS ET AL., MOL IMMUNOL, vol. 32, no. 16, 1995, pages 1183 - 95
FELLOUSE, PROC. NATL. ACAD. SCI. USA, vol. 101, no. 34, 2004, pages 12467 - 12472
FISHWILD ET AL., NATURE BIOTECHNOL., vol. 14, 1996, pages 826 - 851
GARRED PETER ET AL: "Therapeutic Targeting of the Complement System: From Rare Diseases to Pandemics", PHARMACOLOGICAL REVIEWS, vol. 73, no. 2, 1 April 2021 (2021-04-01), US, pages 792 - 827, XP055945241, ISSN: 0031-6997, Retrieved from the Internet <URL:https://pharmrev.aspetjournals.org/content/pharmrev/73/2/792.full.pdf> DOI: 10.1124/pharmrev.120.000072 *
GAZZANO-SANTORO ET AL., J. IMMUNOL. METHODS, vol. 202, 1996, pages 163
GHETIE ET AL., NATURE BIOTECHNOLOGY, vol. 15, no. 7, 1997, pages 637 - 40
GHETIEWARD, IMMUNOL. TODAY, vol. 18, no. 12, 1997, pages 592 - 8
GUSS ET AL., EMBO J, vol. 5, 1986, pages 1567 - 1575
GUYER ET AL., J. IMMUNOL., vol. 117, 1976, pages 587
HAMMERLING ET AL.: "Monoclonal Antibodies and T-Cell Hybridomas", 1981, ELSEVIER, pages: 563 - 681
HARRIS, BIOCHEM. SOC. TRANSACTIONS, vol. 23, 1995, pages 1035 - 1038
HAWKINS ET AL., J. MAL. BIOL., vol. 226, 1992, pages 889 - 896
HELLSTROM, I ET AL., PROC. NAT'LACAD. SCI. USA, vol. 82, 1985, pages 1499 - 1502
HELLSTROM, I ET AL., PROC. NAT'LACAD. SCI. USA, vol. 83, 1986, pages 7059 - 7063
HILLMEN ET AL., N ENGL. J MED, vol. 350, no. 6, 2004, pages 552
HINTON ET AL., J. BIOL. CHEM., vol. 279, no. 8, 2004, pages 6213 - 6
HONEGGERPLUCKTHUN, J. MOL. BIOL., vol. 309, 2001, pages 657 - 670
HONGO ET AL., HYBRIDOMA, vol. 14, no. 3, 1995, pages 253 - 260
HOOGENBOOM ET AL.: "Methods in Molecular Biology", vol. 178, 2001, HUMAN PRESS, pages: 1 - 37
HOOGENBOOMWINTER, J. MOL. BIOL., vol. 222, 1991, pages 581 - 597
HOUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883
HUANG ET AL., NUCLEIC ACIDS RES, vol. 43, no. W1, 2015, pages W338 - W342
HURLEGROSS, CURR. OP. BIOTECH., vol. 5, 1994, pages 428 - 433
IDUSOGIE ET AL., J. IMMUNOL., vol. 164, 2000, pages 4178 - 4184
IGAWA ET AL., NAT BIOTECHNOL., vol. 28, no. 11, November 2010 (2010-11-01), pages 1203 - 7
IGAWA ET AL., PLOS ONE, vol. 8, no. 5, 7 May 2013 (2013-05-07), pages e63236
JACKSON ET AL., J. IMMUNOL., vol. 154, no. 7, 1995, pages 3310 - 2004
JAKOBOVITS ET AL., NATURE, vol. 362, 1993, pages 255 - 258
JAKOBOVITS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
JANSON, J. C. ET AL.: "Protein purification: principles, high resolution methods and applications", 1998, WILEY-VCH
JOHNE ET AL., J IMMUNOL METH, vol. 160, 1993, pages 191 - 198
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
JONSSON ET AL., ANN BIOL CLIN, vol. 51, 1993, pages 19 - 26
JONSSON ET AL., BIOTECHNIQUES, vol. 11, 1991, pages 620 - 627
JUMPER ET AL., NATURE, vol. 596, no. 7873, 2021, pages 583 - 589
KABAT ET AL., J. BIOL. CHEM., vol. 252, 1977, pages 6609 - 6616
KANDA, Y ET AL., BIOTECHNOL. BIOENG., vol. 94, no. 4, 2006, pages 680 - 688
KASSA ENOCH ET AL: "Complement inhibition as a therapeutic strategy in retinal disorders", EXPERT OPINION ON BIOLOGICAL THERAPY, vol. 19, no. 4, 11 February 2019 (2019-02-11), pages 335 - 342, XP055814646, ISSN: 1471-2598, DOI: 10.1080/14712598.2019.1575358 *
KELLEY, NATURE PROTOCOLS, vol. 10, 2015, pages 845 - 858
KIM ET AL., J. IMMUNOL., vol. 24, 1994, pages 249
KIM ET AL., NUCLEIC ACIDS RES., vol. 32, 2004, pages W526 - 31
KIMURA ET AL., BLOOD, vol. 111, no. 2, 2008, pages 732 - 740
KLIMKA ET AL., BR. J. CANCER, vol. 83, 2000, pages 252 - 260
KOHLERMILSTEIN, NATURE, vol. 256, 1975, pages 495 - 97
KOZBOR, J. IMMUNOL., vol. 133, 1984, pages 3001
LAMBERT ET AL., BIOINFORMATICS, vol. 18, 2002, pages 1250 - 1256
LAMIABLE ET AL., NUCLEIC ACIDS RES, vol. 44, no. W1, 2016, pages W449 - 54
LEE ET AL., J. IMMUNOL. METHODS, vol. 284, no. 1-2, 2004, pages 119 - 132
LEFRANC M.P. ET AL., DEV. COMP. IMMUNOL., vol. 27, 2003, pages 55 - 77
LI ET AL., FRONT IMMUNOL, vol. 11, 2020, pages 1123
LI ET AL., PROC. NATL. ACAD. SCI. USA, vol. 103, 2006, pages 3557 - 3562
LINDMARK ET AL., J. IMMUNOL. METH., vol. 62, 1983, pages 1 - 13
LONBERG ET AL., NATURE, vol. 368, 1994, pages 812 - 813
LONBERG, CURR. OPIN. IMMUNOL., vol. 20, 2008, pages 450 - 459
LONBERG, NAT. BIOTECH., vol. 23, 2005, pages 1117 - 1125
LONBERGHUSZAR, INTERN. REV. IMMUNOL., vol. 13, 1995, pages 65 - 93
M. DAËRON, ANNU. REV. IMMUNOL., vol. 15, 1997, pages 203 - 234
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745
MANNES ET AL., BLOOD, vol. 137, no. 4, 2021, pages 443 - 455
MARKS ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 779 - 783
MARKS ET AL., J. MOL. BIOL., vol. 222, 1992, pages 581 - 597
MCCAFFERTY ET AL., NATURE, vol. 348, 1990, pages 552 - 554
MORDENTI, JCHAPPELL, W ET AL.: "Toxicokinetics and New Drug Development", 1989, PERGAMON PRESS, article "The Use of Interspecies Scaling in Toxicokinetics", pages: 42 - 46
MORRISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 - 6855
NI, XIANDAI MIANYIXUE, vol. 26, no. 4, 2006, pages 265 - 268
NILSON, B. H. K. ET AL., J. BIOL. CHEM., vol. 267, 1992, pages 2234 - 2239
OKAZAKI ET AL., J. MOL. BIOL., vol. 336, no. 5, 2004, pages 1239 - 1249
PADLAN, MOL. IMMUNOL., vol. 28, 1991, pages 489 - 498
PETERSEN ET AL., J IMMUNOL METHODS, vol. 257, no. 1-2, 1 November 2001 (2001-11-01), pages 107 - 16
PETKOVA, S.B. ET AL., INT'L. IMMUNOL., vol. 18, no. 12, 2006, pages 1759 - 1769
PIO ET AL., SEMIN IMMUNOL, vol. 25, no. 1, February 2013 (2013-02-01), pages 54 - 64
PLIICKTHUN, IMMUNOL. REVS., vol. 130, 1992, pages 151 - 188
PLUCKTHUN, A, BIOTECHNOLOGY, vol. 9, 1991, pages 545 - 551
PRESTA ET AL., J. IMMUNOL., vol. 151, 1993, pages 2623 - 90
PRESTA, CURR. OP. STRUCT. BIOL., vol. 2, 1992, pages 593 - 596
QUEEN ET AL., PROC. NAT'LACAD. SCI. USA, vol. 86, 1989, pages 10029 - 10033
RAAGWHITLOW, FASEB, vol. 9, no. 1, 1995, pages 73 - 80
RAVETCHKINET, ANNU. REV. IMMUNOL., vol. 9, 1991, pages 457 - 492
REF, M. E., CURR. OPINION BIOTECH., vol. 4, 1993, pages 573 - 576
RIECHMANN ET AL., NATURE, vol. 322, 1988, pages 738 - 329
RINDER ET AL., J CLIN INVEST, vol. 96, no. 3, 1995, pages 1564 - 1572
RIPKA ET AL., BIOCHEM. BIOPHYS., vol. 249, 1986, pages 533 - 545
ROSOK ET AL., J. BIOL. CHEM., vol. 271, 1996, pages 22611 - 22618
ROY ET AL., NATURE PROTOCOLS, vol. 5, 2010, pages 725 - 738
SAMBROOK ET AL.: "A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY PRESS, article "Molecular Cloning"
SCHIER ET AL., GENE, vol. 169, 1995, pages 147 - 155
SHIELDS RL ET AL., J. BIOL. CHEM., vol. 276, no. 2, 2001, pages 6591 - 6604
SHINKAWA ET AL., J. BIOL. CHEM., vol. 278, no. 5, 2003, pages 3466 - 3473
SKERRA ET AL., CURR. OPINION IN IMMUNOL., vol. 5, 1993, pages 256 - 262
THOMAS ET AL., MOL IMMUNOL, vol. 33, no. 17-18, 1996, pages 1389 - 401
TRILL J. J. ET AL., CURR. OPINION BIOTECH, vol. 6, 1995, pages 553 - 560
VAN DIJKVAN DE WINKEL, CURR. OPIN. PHARMACOL., vol. 5, 2001, pages 368 - 74
VASWANIHAMILTON, ANN. ALLERGY, ASTHMA & IMMUNOL., vol. 1, 1998, pages 105 - 115
VOLLMERSBRANDLEIN, HISTOLOGY AND HISTOPATHOLOGY, vol. 20, no. 3, 2005, pages 927 - 937
VOLLMERSBRANDLEIN, METHODS AND FINDINGS IN EXPERIMENTAL AND CLINICAL PHARMACOLOGY, vol. 27, no. 3, 2005, pages 185 - 91
VOLOKINA ET AL., BLOOD, vol. 126, no. 2, 2015, pages 278 - 279
WATERHOUSE ET AL., NUCL. ACIDS RES., vol. 21, 1993, pages 2265 - 2266
WRIGHT ET AL., TIBTECH, vol. 15, 1997, pages 26 - 32
WURZNER ET AL., COMPLEMENT INFLAMM, vol. 8, 1991, pages 328 - 340
YAMANE-OHNUKI ET AL., BIOTECH. BIOENG., vol. 87, 2004, pages 614
YANG ET AL., MABS, vol. 9, no. 7, October 2017 (2017-10-01), pages 1105 - 1117
YEUNG ET AL., J IMMUNOL, vol. 182, no. 12, 2009, pages 7663 - 7671
YUAN, HAEMATOLOGICA, vol. 102, no. 3, 2017, pages 466 - 475
ZELEK ET AL., IMMUNOLOGY, vol. 161, no. 2, 2020, pages 103 - 113
ZEMLA ET AL., NUCLEIC ACIDS RES, vol. 33, 2005, pages W111 - W115
ZETTLIT, K. A., ANTIBODY ENGINEERING, 2010, pages 531 - 535

Also Published As

Publication number Publication date
WO2025045251A2 (en) 2025-03-06
WO2025045251A3 (en) 2025-04-03

Similar Documents

Publication Publication Date Title
US11746153B2 (en) Binding molecules specific for FcγRIIA and uses thereof
JP6114326B2 (en) Antibodies against tissue factor pathway inhibitor
EP2654790B1 (en) Modified antibody with improved half-life
EP3424953B1 (en) Therapeutic antibodies
AU2015384281B2 (en) Novel antibody binding to TFPI and composition comprising the same
CA2689680A1 (en) Fc receptor-binding polypeptides with modified effector functions
CN114423785A (en) Anti-CD154 antibody and use thereof
WO2020221451A1 (en) Antibodies binding to plasmodium circumsporozoite protein and uses thereof
TW202229337A (en) Canine antibody variants
AU2021294687C1 (en) Antibodies specifically recognizing C5A and uses thereof
WO2025045250A1 (en) Anti-human factor d antibody constructs and uses thereof
JP2024511610A (en) Anti-TMEM106B antibody for treatment and prevention of coronavirus infection
CN115925918A (en) Antibody specifically recognizing C5A and application thereof
JP2023515480A (en) ANTI-IL-2 ANTIBODY, ANTIGEN-BINDING FRAGMENT THEREOF AND MEDICINAL USE THEREOF
WO2020260315A1 (en) Deimmunized antibodies binding to alpha-4 integrin and uses thereof
CN118786143A (en) Anti-CCR6 antibodies and uses thereof
WO2023245078A1 (en) Anti-parvovirus antibodies and uses thereof
KR20230004739A (en) Humanized Anti-Human CD89 Antibodies and Uses Thereof
EA049650B1 (en) ANTIBODIES SPECIFICALLY RECOGNIZING C5a AND THEIR APPLICATION OPTIONS
HK1191216B (en) Modified antibody with improved half-life
HK1191216A (en) Modified antibody with improved half-life

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24782176

Country of ref document: EP

Kind code of ref document: A1