Multi-specific constructs comprising anti-factor D moieties

Abstract

The present invention provides multispecific (e.g., bispecific) constructs that target complement factor D and another complement protein (e.g., C2, C5), nucleic acids encoding such multispecific constructs, and their use for the treatment of complement mediated diseases. Also provided are novel antibody constructs that specifically recognize C5, nucleic acids encoding the antibody constructs, and uses thereof.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to International Patent Application No. PCT / CN2023 / 116638 and International Patent Application No. PCT / CN2023 / 116639, filed on September 3, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Reference to the electronic sequence list

[0004] The contents of the electronic serial number (792252001341SEQLIST.xml; size: 120,453 bytes; and creation date: August 29, 2024) are incorporated herein by reference in their entirety. Technical Field

[0005] This application relates to multispecific constructs, such as anti-complement C5 / anti-FD bispecific antibodies and anti-complement C2 / anti-FD bispecific antibodies; nucleic acids encoding these multispecific constructs; and their uses, including the treatment of complement-mediated diseases or conditions. Background Technology

[0006] The complement system is part of the innate immune system and plays a crucial role in host defense. Complement also plays a pathogenic role in human inflammatory diseases. Activation of the complement system occurs through three distinct pathways: the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). CP is triggered by antigen-antibody binding. LP is triggered when mannose-binding lectin (MBL) interacts with sugar molecules on the surface of microorganisms. Activation of both pathways leads to the assembly of CP C3 convertase C4b2a, but direct cleavage of C3 by MBL-associated serine proteases may also occur. AP is a self-amplifying loop driven by AP C3 convertase C3bBb. AP activation can be secondary to CP or LP activation, or it can be initiated independently. In the latter case, low levels of spontaneous C3 "tick-over" produce initial C3bBb, which, in the absence of proper regulation, rapidly triggers AP. Therefore, it is generally accepted that AP activation on non-self surfaces with little or no negative regulation is considered a default process, and autologous cells typically avoid this outcome by utilizing various membrane-bound and liquid-phase complement repressor proteins. Under certain conditions, altered, damaged, or stressed autologous cells and tissues can also activate AP and cause inflammatory damage.

[0007] Factor D (FD) is an essential enzyme for complement activation in acute prostatitis (AP). Upon binding of factor B to C3b, FD cleaves factor B, producing the active C3 convertase C3bBb. Factor D is a serine protease of approximately 24 kDa, generated from a factor D precursor via the enzymatic action of mannose-binding lectin-associated serine protease-3 (MASP-3), and circulates in the blood as a constitutively active enzyme after generation. Compared to other complement proteins in the blood, the concentration of factor D in the blood is relatively low (approximately 2 μg / ml), with a high turnover rate. The relatively low serum concentration suggests the potential for therapeutic inhibition of factor D activity in the blood, but the rapid turnover rate of FD presents a challenge for sustained inhibition of this enzyme. Small molecule antagonists of FD have been tested in clinical trials and have shown good AP inhibitory effects and efficacy. However, this treatment regimen requires high doses and frequent administration, leading to reports of hepatotoxicity. Anti-FD biologics for the treatment of macular degeneration have been evaluated in clinical trials. Topical application of the anti-FD Fab fragment to the vitreous humor was used to maintain favorable pharmacokinetics. Clinical trials showed promising efficacy in Phase II, but failed to meet the primary endpoint in Phase III.

[0008] The eye is an immune-immune site due to the presence of complement regulators on its surface that can prevent or rapidly shut down complement activation. The complement system contributes to the eye's immune immunity through low-level complement conversion within the eye. Conversely, the complement system in a healthy cornea is suppressed to protect it from persistent immune damage from continuous exposure to pathogens. Therefore, the delicate balance between complement activation and suppression is crucial for maintaining a healthy ocular environment. Dysregulation of the complement pathway is a major pathogenic mechanism for many ocular diseases, including but not limited to age-related macular degeneration (AMD), glaucoma, diabetic retinopathy, and autoimmune uveitis. Given the role of the complement system in ocular immune homeostasis and immune tolerance, there is a need in the art for antibodies capable of modulating complement activity and thereby treating complement-dependent ocular diseases.

[0009] All publications, patents, patent applications, and published patent applications mentioned herein are incorporated herein in their entirety by reference. Summary of the Invention

[0010] In one respect, this article provides a multispecific construct comprising a first antibody portion that specifically recognizes complement factor D (“FD”) and a second antibody portion that specifically recognizes components of the complement pathway.

[0011] In some embodiments of the multispecific construct described above, the first antibody portion that specifically recognizes FD comprises an immunoglobulin heavy chain variable domain (“VH1”) and an immunoglobulin light chain variable domain (“VL1”).

[0012] In some embodiments of any of the multispecific constructs described above, the first antibody portion is scFv. In some embodiments, the first antibody portion includes a VH1-optional linker-VL1 from the N-terminus to the C-terminus. In some embodiments, the first antibody portion includes a VL1-optional linker-VH1 from the N-terminus to the C-terminus.

[0013] In some embodiments of any of the above multispecific constructs, (i) the VH1 comprises: a heavy chain CDR1 (“H-CDR1”), the H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); a heavy chain CDR2 (“H-CDR2”), the H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and a heavy chain CDR3 (“H-CDR3”), the H-CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and (ii) the VL1 comprises: a light chain CDR1 (“L-CDR1”), the L-CDR1 comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and a light chain CDR2 (“L-CDR2”), the L-CDR2 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and a light chain CDR2 (“L-CDR2”), the L-CDR2 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and a heavy ...3 (“H-CDR3”), the H-CDR2 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and a heavy chain CDR2 (“L-CDR2”), the L-CDR2 comprising the amino acid sequence of SEQ ID NO: The amino acid sequence of SEQ ID NO: 5 or a variant thereof containing up to 3 amino acid variations (e.g., substitutions); and a light chain CDR3 (“L-CDR3”), said L-CDR3 containing the amino acid sequence of SEQ ID NO: 6 or a variant thereof containing up to 3 amino acid variations (e.g., substitutions). In some embodiments, (i) said VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and (ii) said VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0014] In some embodiments of any of the above-described multispecific constructs, VH1 comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 7; and VL1 comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 8. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8.

[0015] In some embodiments of any of the above-described multispecific constructs, the first antibody portion is scFv, which contains the amino acid sequence of SEQ ID NO: 9 or 10.

[0016] In some embodiments of the multispecific construct described above, the second antibody portion that specifically recognizes components of the complement pathway includes an immunoglobulin heavy chain variable domain (“VH2”) and an immunoglobulin light chain variable domain (“VL2”).

[0017] In some embodiments of any of the multispecific constructs described above, the second antibody portion is scFv. In some embodiments, the second antibody portion includes VH2-optional linker-VL2 from the N-terminus to the C-terminus. In some embodiments, the second antibody portion includes VL2-optional linker-VH2 from the N-terminus to the C-terminus.

[0018] In some embodiments of any of the above multispecific constructs, the complement pathway component is complement component 2 (“C2”) (hereinafter also referred to as the “anti-FD / anti-C2 multispecific construct”).

[0019] In some embodiments of any of the above-described anti-FD / anti-C2 multispecific constructs, (i) the VH2 comprises: H-CDR1, which comprises the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); H-CDR2, which comprises the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and H-CDR3, which comprises the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and the VL2 comprises: L-CDR1, which comprises the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); L-CDR2, which comprises the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and L-CDR2, which comprises the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and H ... The VH2 comprises: SEQ ID NO: 15, an amino acid sequence thereof, or a variant thereof containing up to 3 amino acid changes (e.g., substitutions); and L-CDR3, the L-CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof containing up to 3 amino acid changes (e.g., substitutions); or (ii) the VH2 comprises: H-CDR1, the H-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof containing up to 3 amino acid changes (e.g., substitutions); H-CDR2, the H-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, or a variant thereof containing up to 3 amino acid changes (e.g., substitutions); and H-CDR3, the H-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, or a variant thereof containing up to 3 amino acid changes (e.g., substitutions); and the VL2 comprises: L-CDR1, the L-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an amino acid sequence ... The amino acid sequence of SEQ ID NO: 24, or a variant thereof containing up to 3 amino acid changes (e.g., substitutions); L-CDR2, wherein the L-CDR2 contains the amino acid sequence of SEQ ID NO: 25, or a variant thereof containing up to 3 amino acid changes (e.g., substitutions); and L-CDR3, wherein the L-CDR3 contains the amino acid sequence of SEQ ID NO: 26, or a variant thereof containing up to 3 amino acid changes (e.g., substitutions).In some embodiments, (i) the VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and the VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16; or (ii) the VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and the VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, H-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. L-CDR2 containing the amino acid sequence of SEQ ID NO: 25 and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26.

[0020] In some embodiments of any of the above-described anti-FD / anti-C2 multispecific constructs, (i) the VH2 comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 17; and the VL2 comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 18; or (ii) the VH2 comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 27; and the VL2 comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 28. In some embodiments, (i) the VH2 comprises the amino acid sequence of SEQ ID NO: 17 and the VL2 comprises the amino acid sequence of SEQ ID NO: 18; or (ii) the VH2 comprises the amino acid sequence of SEQ ID NO: 27 and the VL2 comprises the amino acid sequence of SEQ ID NO: 28.

[0021] In some embodiments of any of the above-described anti-FD / anti-C2 multispecific constructs, the second antibody portion is scFv, which contains the amino acid sequence of any one of SEQ ID NO: 19, 20, 29, and 30.

[0022] In some embodiments of the multispecific construct according to any of the above-described multispecific constructs containing a second antibody portion that specifically recognizes a complement pathway component, the complement pathway component is complement component 5 (“C5”) (hereinafter also referred to as “anti-FD / anti-C5 multispecific construct”).

[0023] In some embodiments of any of the above-described anti-FD / anti-C5 multispecific constructs, (i) the VH2 comprises: H-CDR1, which comprises the amino acid sequence of SEQ ID NO: 31, or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); H-CDR2, which comprises the amino acid sequence of SEQ ID NO: 32, or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and H-CDR3, which comprises the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and (ii) the VL2 comprises: L-CDR1, which comprises the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); L-CDR2, which comprises the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and L-CDR2, which comprises the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to 3 amino acid variations (e.g., substitutions); and H ... The amino acid sequence of SEQ ID NO: 35, or a variant thereof comprising up to three amino acid changes (e.g., substitutions); and L-CDR3, said L-CDR3 comprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof comprising up to three amino acid changes (e.g., substitutions). In some embodiments, (i) said VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and (ii) said VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36.

[0024] In some embodiments of any of the above-described anti-FD / anti-C5 multispecific constructs, (i) the VH2 comprises the amino acid sequence of SEQ ID NO: 37, or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 37; and (ii) the VL2 comprises the amino acid sequence of SEQ ID NO: 38, or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 38. In some embodiments, the VH2 comprises the amino acid sequence of SEQ ID NO: 37, and the VL2 comprises the amino acid sequence of SEQ ID NO: 38.

[0025] In some embodiments of any of the above-described anti-FD / anti-C5 multispecific constructs, the second antibody portion is scFv, which contains the amino acid sequence of SEQ ID NO: 39 or 40.

[0026] In some embodiments of any of the multispecific constructs described above, the first antibody portion is directly linked to the second antibody portion.

[0027] In some embodiments of any of the above-described multispecific constructs, the first antibody portion is linked to the second antibody portion via a peptide linker.

[0028] In some embodiments of any of the multispecific constructs described above, the multispecific construct comprises, from the N-terminus to the C-terminus: (i) VH1-optional linker-VL1-optional linker-VH2-optional linker-VL2; (ii) VL1-optional linker-VH1-optional linker-VL2-optional linker-VH2; (iii) VH1-optional linker-VL1-optional linker-VL2-optional linker-VH2; (iv) VL1-optional linker-VH1-optional linker-VH2-optional linker-VL2; (v) VH2-optional linker-VL2-optional linker-VH1-optional linker-VL1; (vi) VL2-optional linker-VH2-optional linker-VH1-optional linker-VL1; (vii) VH2-optional connector-VL2-optional connector-VL1-optional connector-VH1; or (viii) VL2-optional connector-VH2-optional connector-VL1-optional connector-VH1.

[0029] In some embodiments of any of the multispecific constructs described above, the multispecific construct comprises the amino acid sequence of any of the following: SEQ ID NO: 42-55, 59-63, 65, 67, 69, 71-75, 77, 78, 81, 82, 85-88, 106, and 108. In some embodiments, the multispecific construct comprises the amino acid sequence of any of the following: SEQ ID NO: 42-50, 63, 67, 71, 73, 77, 81, 85, 87, 106, and 108.

[0030] In some embodiments of any of the multispecific constructs described above, the first antibody portion is linked to the second antibody portion via an Fc domain. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41.

[0031] In some embodiments of any multispecific construct according to the above-described first antibody portion linked to a second antibody portion via an Fc domain, the multispecific construct comprises, from the N-terminus to the C-terminus, (i) VH1-optional linker-VL1-optional linker-Fc-optional linker-VH2-optional linker-VL2; (ii) VL1-optional linker-VH1-optional linker-Fc-optional linker-VL2-optional linker-VH2; (iii) VH1-optional linker-VL1-optional linker-Fc-optional linker-VL2-optional linker-VH2; (iv) VL1-optional linker-VH1-optional linker-Fc-optional linker-VH2-optional linker-VL2; (v) VH2-optional linker-VL2-optional linker-Fc-optional linker-VH1-optional linker-VL1; (vi) VL2-optional connector-VH2-optional connector-Fc-optional connector-VH1-optional connector-VL1; (vii) VH2-optional connector-VL2-optional connector-Fc-optional connector-VL1-optional connector-VH1; or (viii) VL2-optional connector-VH2-optional connector-Fc-optional connector-VL1-optional connector-VH1.

[0032] In some embodiments of any multispecific construct according to the first antibody portion linked to the second antibody portion via an Fc domain, the multispecific construct comprises an amino acid sequence of any of the following: SEQ ID NO: 51-55, 59-62, 65, 69, 72, 74, 75, 78, 82, 86, and 88.

[0033] On the other hand, this paper provides an isolated nucleic acid that encodes any of the above-mentioned multispecific constructs.

[0034] On the other hand, this document provides a vector containing any of the isolated nucleic acids described above. In some embodiments, the vector is a viral vector, such as an adeno-associated virus (AAV) vector or a lentiviral vector.

[0035] On the other hand, this document provides an mRNA comprising any of the aforementioned nucleic acids. In some embodiments, the mRNA is formulated in liposomes or lipid nanoparticles.

[0036] Pharmaceutical compositions are also provided comprising any of the above-described multispecific constructs, any of the above-described isolated nucleic acids, any of the above-described vectors, or any of the above-described mRNAs, and optionally, pharmaceutically acceptable carriers.

[0037] On the other hand, this document provides a method for treating a complement-mediated disease in an individual (e.g., a human), the method comprising administering an effective amount of any of the above-described pharmaceutical compositions to the individual. In some embodiments, the complement-mediated disease is an ocular disease. In some embodiments, the ocular disease is selected from the group consisting of: macular degeneration (MD), age-related macular degeneration (AMD), glaucoma, diabetic retinopathy, autoimmune uveitis, dry eye syndrome, neuromyelitis optica, central retinal vein occlusion, and subkeratotic pustular dermatosis. In some embodiments, the pharmaceutical composition is applied directly to the eye.

[0038] On the other hand, the present invention provides a host cell containing any of the isolated nucleic acids or any of the vectors described above.

[0039] This application also provides a kit comprising the multispecific construct described herein, a nucleic acid encoding the multispecific construct, a vector (e.g., a viral vector) containing the nucleic acid, and / or a pharmaceutical composition containing or encoding the multispecific construct described herein, as well as instructions for use or product information relating to the treatment of complement-mediated diseases or disorders using the multispecific construct (or pharmaceutical composition), nucleic acid, or vector.

[0040] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the present invention. Attached Figure Description

[0041] Figure 1 SDS-PAGE of purified scFv for FD (42WT), C2 (3.2GL, 69.44) or C5 (102-1) under reducing conditions was depicted.

[0042] Figures 2A - 2D The inhibitory effects of anti-FD (42WT), anti-C2 (3.2GL, 69.44), and anti-C5 (102-1) scFv and their corresponding parental monoclonal antibodies on 20% human serum-induced lysis of rabbit or sheep RBCs were described.

[0043] Figure 3A A diagram depicting an exemplary bispecific scFv molecule having a peptide (e.g., G4S) linker. Figure 3B A diagram depicting an exemplary bispecific scFv molecule with an Fc linker.

[0044] Figure 4SDS-PAGE images depicting purified bispecific 42WT-scFv and 102-1-scFv molecules (αFD / αC5) with G4S linkers (left panel) and purified bispecific 42WT-scFv and 3.2GL-scFv molecules (αFD / αC2) with G4S linkers (right panel) are shown.

[0045] Figure 5A The bispecific anti-FD and anti-C5 scFv inhibition of sheep RBC lysis (exposed to 20% human serum) with G4S linker molecules was described. Figure 5B The bispecific anti-FD and anti-C5 scFv inhibitory activity against rabbit RBC lysis (exposed to 20% human serum) with a G4S linker molecule was demonstrated. Parental scFv was used as a control.

[0046] Figures 6A - 6B The bispecific anti-FD and anti-C2 scFv inhibitory effects on sheep RBC lysis (exposed to 20% human serum) with G4S linker molecules were described. Figures 6C - 6D The bispecific anti-FD and anti-C2 scFv with a G4S linker molecule was used to depict the inhibition of rabbit RBC lysis (exposed to 20% human serum). The parental scFv was used as a control.

[0047] Figures 7A - 7C Characterization of G4S-linked bispecific anti-C5 and anti-FD molecules in a modified Wieslab assay used to study the inhibitory effects of IgM or LPS-induced C5b-9 or C3b deposition is described.

[0048] Figures 8A - 8E Characterization of G4S-linked bispecific anti-C2 and anti-FD molecules in a modified Wieslab assay used to study the inhibitory effects of IgM, mannan, or LPS-induced C5b-9 or C3b deposition is described.

[0049] Figure 9A The bispecific anti-FD and anti-C5 inhibition of sheep RBC lysis was described using Fc linker molecules. Figure 9B The bispecific anti-FD and anti-C5 inhibition of rabbit RBC lysis was described using an Fc linker molecule. The parental scFv was used as a control.

[0050] Figures 10A - 10B The bispecific anti-FD and anti-C2 scFv inhibitory effects on sheep RBC lysis (exposed to 20% human serum) with Fc linker molecules were described. Figures 10C - 10D The bispecific anti-FD and anti-C2 scFv with an Fc linker molecule was used to describe the inhibition of rabbit RBC lysis (exposed to 20% human serum). The parental scFv was used as a control.

[0051] Figures 11A - 11B Characterization of Fc-linked bispecific scFv anti-C5 and anti-FD molecules in a modified Wieslab assay used to study the inhibitory effects of IgM or LPS-induced C5b-9 or C3b deposition is described.

[0052] Figures 12A - 12D Characterization of Fc-linked bispecific scFv anti-FD and anti-C2 molecules in a modified Wieslab assay used to study the inhibitory effects of IgM, mannan, or LPS-induced C5b-9 or C3b deposition is described.

[0053] Figure 13 The expression titers, purity, IC50 in sheep erythrocyte lysis assay (exposed to 50% human serum) and rabbit erythrocyte assay (exposed to 50% human serum) of G4S-linked bispecific anti-FD (42WT) and anti-C2 (3.2GL, 69.44) scFv, as well as G4S-linked bispecific anti-FD and anti-C5 (102-1) scFv, were characterized. Detailed Implementation

[0054] This application provides novel multispecific (e.g., bispecific) molecules that bind to and inhibit factor D and a second component (e.g., C2 or C5) in the complement pathway. These multispecific molecules are particularly effective in controlling unwanted, uncontrolled, or excessive complement activation and are therefore useful for treating complement-mediated conditions and diseases, particularly ocular diseases, including but not limited to macular degeneration, age-related macular degeneration, glaucoma, diabetic retinopathy, and autoimmune uveitis.

[0055] Therefore, this application provides a novel multispecific molecule in which a first antibody partially and specifically binds to and inhibits the function of FD, and a second antibody partially and specifically binds to a complement pathway component.

[0056] On the other hand, methods are provided for inhibiting complement activation and / or treating complement-related diseases by administering any multispecific antibody or construct thereof, or a nucleic acid or vector (e.g., a viral vector such as AAV) encoding said multispecific antibody or construct thereof.

[0057] Novel isolated anti-C5 antibody constructs are also provided, said constructs comprising an antibody moiety that specifically recognizes C5 (anti-C5 antibody moiety), such as multispecific anti-C5 antibody constructs comprising an anti-C5 antibody moiety and an antibody moiety that specifically recognizes FD (anti-FD antibody moiety), and multispecific anti-C5 antibody constructs comprising an anti-C5 antibody moiety fused to a fusion protein complex (complement factor H (FH) or a functional fragment thereof). Nucleic acids and vectors (e.g., viral vectors, such as AAV) encoding said antibody constructs are also provided. Methods for treating complement-mediated diseases in individuals (e.g., humans) are also provided, said methods comprising administering to said individual an effective amount of the isolated anti-C5 antibody construct described herein, or a nucleic acid or vector encoding said construct.

[0058] I. Definitions

[0059] Unless otherwise defined, 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 pertains. Although any methods and materials similar to or equivalent to those described and used herein may be used in the practice or testing of this invention, exemplary methods and materials are described.

[0060] The term "antibody" is used in its broadest sense herein and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and their antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or its antigen-binding fragment.

[0061] An "antibody" can refer to an immunoglobulin molecule or fragment thereof (including the basic 4-chain antibody unit) that can specifically bind to a specific epitope of an antigen. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, and can be the immunoreactive portion of a complete immunoglobulin. The antibodies used in this invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intramolecular antibodies”), 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 camel 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).

[0062] Full-length antibodies consist of two heavy chains and two light chains. Variable regions of the light and heavy chains are responsible for antigen binding. These variable domains of the heavy and light chains may be referred to as “VH” and “VL”, respectively. The variable regions of the two chains typically contain three highly variable loops called 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. The CDR boundaries of the antibody and antigen-binding fragments disclosed herein can be defined or identified according to the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three core-residue junctions (CDRs) of either the heavy or light chain are interspersed between flanking segments called framework regions (FRs), which are more conserved than the CDRs and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified according to the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further subdivided 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).

[0063] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, bifunctional antibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain Fv (scFv), scFv dimers (bivalent bifunctional antibodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, single-domain antibodies (sdAbs) (e.g., camelified single-domain antibodies), nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen with a parent antibody or a fragment of a parent antibody (e.g., a parental scFv). In some embodiments, the antigen-binding fragment may contain one or more CDRs from a specific human antibody that have been grafted into a frame region from one or more different human antibodies.

[0064] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in tight, non-covalent association. The folding of these two domains generates six hypervariable rings (three from each of the heavy and light chains), which contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, although the affinity is lower than the entire binding site, even a single variable domain (or half of the Fv containing only three antigen-specific CDRs) can recognize and bind to the antigen.

[0065] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing VH and VL antibody domains linked together to form a single polypeptide chain. In some embodiments, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the scFv to form the structure required for antigen binding. For a review of scFv, see Plückthun. The Pharmacology of Monoclonal Antibodies Volume 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0066] A basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units plus an additional polypeptide called the J chain and contain 10 antigen-binding sites, while IgA antibodies contain 2–5 basic 4-chain units that can polymerize to combine with the J chain to form a polyvalent assembly. In the case of IgG, the 4-chain unit is typically about 150,000 Daltons. Each L chain is linked to the H chain by a covalent disulfide bond, and two H chains are linked to each other by one or more disulfide bonds depending on the H chain isoform. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for both the α and γ chains, and four CH domains for the μ and ε isoforms. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain at its other end. VL is aligned with VH, and CL is aligned with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form the interface between the variable domains of the light chain and the variable domains of the heavy chain. VH and VL pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th ed., Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and Chapter 6. The L chain from any vertebrate species can be designated as one of two distinct types, called κ and λ, based on the amino acid sequence of its constant domain. Immunoglobulins can be designated as different classes or isotypes based on the amino acid sequence of the heavy chain (CH) constant domain. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain designated as α, δ, ε, γ, and μ, respectively. Based on relatively minor differences in CH sequence and function, the γ and α classes are further subdivided into subclasses, such as the following subclasses expressed in humans: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0067] The Fc fragment comprises the carboxyl-terminal portions of two H chains held together by disulfide bonds. The effector function of an antibody is determined by the sequence in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0068] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domains of the heavy and light chains are referred to as "VH" and "VL," respectively. These domains are typically the largest variable portion of the antibody (relative to other antibodies of the same class) and contain the antigen-binding site. Antibodies from camel species that consist only of a heavy chain have a single heavy-chain variable region, which is called a "VHH." Therefore, VHH is a special type of VH.

[0069] The term "variability" refers to the fact that the sequences of certain segments of a variable domain differ widely between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the entire span of the variable domain. Rather, in both the light and heavy chain variable domains, variability is concentrated in three segments called hypervariable regions (HVRs). The more conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions, predominantly employing a β-sheet configuration, linked by three HVRs that form loops connecting β-sheet structures and, in some cases, forming part of a β-sheet structure. The HVRs in each chain are held together closely adjacent to each other by the FR regions and, together with HVRs from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, Md. (1991)). Constant domains do not directly participate in the binding of antibodies to antigens, but they exhibit various effector functions, such as antibody participation in antibody-dependent cytotoxicity.

[0070] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., the individual antibodies constituting said group are identical except for possible small amounts of possibly naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). Monoclonal antibodies are highly specific (targeting a single antigenic site). Unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous because they are synthesized via hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the production of said antibody by any particular method. For example, the monoclonal antibody intended for use according to this application can be prepared by a variety of techniques, including, for example, hybridoma methods (e.g., Kohler and Milstein). Nature256:495-97 (1975); Hongo et al., Hybridoma 14 (3): 253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); Hammerling et al. Monoclonal Antibodies and T - Cell Hybridomas 563-681 (Elsevier, NY, 1981), recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567), phage display technology (see, for example, 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 techniques for generating human antibodies or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, for example, 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. Patent 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)).

[0071] The terms "full-length antibody," "intact antibody," or "complete antibody" are used interchangeably to refer to an antibody in essentially its complete form, rather than an antibody fragment. Specifically, full-length 4-chain antibodies include antibodies having both a heavy chain and a light chain and including an Fc region. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or a variant of its amino acid sequence. In some cases, an intact antibody may have one or more effector functions.

[0072] The term "bifunctional antibody" refers to an antibody constructed by combining the VH domain with the V... L Small antibody fragments are prepared from sFv fragments (see previous paragraph) with short linkers (approximately 5-10 residues) between their domains. These linkers enable interchain rather than intrachain pairing of the V domain, resulting in bivalent fragments—fragments with two antigen-binding sites. Bispecific bifunctional antibodies are heterodimers of two “cross-linked” sFv fragments, where the VH and V domains of both antibodies are linked. L The domains are located on different polypeptide chains. Bifunctional antibodies are described in detail, for example, EP 404,097; WO 93 / 11161; Hollinger et al. Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)

[0073] Monoclonal antibodies as used in this article explicitly include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chains is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of one or more chains is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; Morrison et al.). Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest in this paper include PRIMATTZFD® antibodies, wherein the antigen-binding region of the antibody is derived from antibodies produced by immunizing macaques, for example, with the antigen of interest. As used herein, “humanized antibody” is used as a subset of “chimeric antibody”.

[0074] As used herein, the term “CDR” or “complementarity-determining region” is intended to refer to a non-continuous antigenic combination site found within the variable region of both the heavy and light chain polypeptides. These specific regions have been described in the following literature: Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US 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 MP et al., Dev. Comp. Immunol. , 27: 55-77 (2003); and Honegger and Plückthun, J. Mol. Biol. , 309:657-670 (2001), where the definition includes overlap or subset of amino acid residues when compared with each other. However, the application of any definition to refer to the CDR of an antibody or its grafted antibody or variant is intended to fall within the scope of the terminology as defined and used herein. The amino acid residues covering the CDR defined by each of the foregoing references are listed in Table 1 below for comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, those by 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. References cited in this paragraph are incorporated herein by reference in their entirety for use in this application and may be included in one or more of the claims herein.

[0075] Table 1: CDR Definitions

[0076] 1 Residue numbering follows the nomenclature of Kabat et al., as above.

[0077] 2 Residue numbering follows the nomenclature of Chothia et al., as above.

[0078] 3 Residue numbering follows the nomenclature of MacCallum et al., as above.

[0079] 4 Residue numbering follows the nomenclature of Lefranc et al., as above.

[0080] 5 Residue numbering follows the Honegger and Plückthun nomenclature, as described above.

[0081] The expressions “as in Kabat variable domain residue numbering” or “as in Kabat amino acid position numbering” and their variations refer to the numbering system used by Kabat et al. (ibid.) for antibody editing of heavy chain or light chain variable domains. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, corresponding to shortening or insertion into the FR or hypervariable region (HVR) of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (according to Kabat residue 52a) and inserted residues after heavy chain FR residue 82 (e.g., according to Kabat residues 82a, 82b, and 82c, etc.). For a given antibody, the Kabat number of the residues can be determined by comparing homologous regions of the antibody sequence with a “standard” Kabat numbered sequence.

[0082] Unless otherwise indicated herein, the residue numbers in the immunoglobulin heavy chain are derived from the EU index, as described by Kabat et al. (ibid.), with slight modifications. "EU index, as described by Kabat," refers to the residue numbers of human IgG1 EU antibodies.

[0083] “Frame” or “FR” residues are those variable domain residues other than the CDR residues defined herein.

[0084] As used in this article, “immunoassay” refers to any binding assay that uses antibodies capable of specifically binding to target molecules to detect and quantify the target molecules.

[0085] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies containing a minimal sequence derived from a non-human antibody. To a large extent, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the hypervariable region (HVR) of the receptor are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mice, rats, rabbits, or non-human primates possessing the desired antibody specificity, affinity, and capability. In some cases, the frame region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not present in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally, humanized antibodies will contain substantially all at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. Humanized antibodies will also optionally contain at least a portion of the immunoglobulin constant region (Fc) (typically the constant region of human immunoglobulins). Suitable human receptor antibodies can be selected from conventional databases, such as the KABAT database, the Los Alamos database, the AbM database, and the Swiss protein database, based on homology with the nucleotide and amino acid sequences of the donor antibody. Human antibodies characterized by homology (based on amino acids) with the frame regions of the donor antibody may be suitable for providing heavy chain constant regions and / or heavy chain variable frame regions for insertion into the donor CDR. Suitable receptor antibodies capable of providing light chain constant regions or variable frame regions can be selected in a similar manner. It should be noted that the heavy and light chains of the receptor antibody do not need to originate from the same receptor antibody. Several methods for generating such humanized antibodies are described in the prior art (see, for example, EP-A-0239400 and EP-A-054951). For further details, see, for example, 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. Patent Nos. 6,982,321 and 7,087,409.

[0086] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of human-produced antibodies, and / or an antibody prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Various techniques known in the art, including phage display libraries, can be used to generate human antibodies. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Cole et al. also used this method to prepare human monoclonal antibodies. Monoclonal Antibodies and Cancer Therapy Alan R. Liss, 77 (1985); Boerner et al., J. Immunol., The method described in 147(1):86-95 (1991). See also van Dijk and van deWinkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen stimulation, but whose endogenous loci have been disabled, for example, by immunizing xenogeneic mice (see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). Also see, for example, Li et al., for human antibodies produced via human B-cell hybridoma technology. Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0087] The term "donor antibody" refers to an antibody (monoclonal antibody and / or recombinant antibody) that contributes its variable region, CDR or other functional fragment or analogue of amino acid sequence to a first immunoglobulin conjugate, thereby giving the altered immunoglobulin coding region and the altered antibody expressed therefrom the antigen specificity and neutralizing activity characteristic of the donor antibody.

[0088] The term "receptor antibody" refers to an antibody (monoclonal antibody and / or recombinant antibody) that is heterologous to a donor antibody, which contributes all (or any portion, but in some embodiments all) of its amino acid sequence encoding its heavy chain and / or light chain framework region and / or its heavy chain and / or light chain constant region to a first immunoglobulin conjugate. In some embodiments, the human antibody is a receptor antibody.

[0089] As used herein, the terms “attach,” “attached,” “fuse,” or “fused” refer to the connection or union of two or more components by bonds, linkages, forces, or ligations, including direct or indirect connections, thereby, for example, directly binding a first polypeptide to a second polypeptide or material, and, for example, placing one or more intermediate compounds (e.g., amino acids, peptides, polypeptides, etc.) between the first polypeptide and the second polypeptide or material.

[0090] The "percentage of amino acid sequence identity (%)" or "homology" for the peptide and antibody sequences identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the compared peptide, after sequence alignment and taking into account any conserved substitutions as part of sequence identity. The alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the capabilities of those skilled in the art, for example, 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 the alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences. However, for the purposes of this document, the amino acid sequence identity value % (Edgar, RC, ...) is generated using the sequence comparison computer program MUSCLE. Nucleic Acids Research 32 (5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0091] "Homologous" refers to the sequence similarity or identity between two polypeptides or two nucleic acid molecules. Two compared sequences are homologous when positions at those positions are occupied by the same base or amino acid monomer subunit. For example, if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage 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 being compared, multiplied by 100. For example, if six out of ten positions in two sequences are matching or homologous, then the two sequences are 60% homologous. For instance, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparisons are made when two sequences are aligned to give the maximum homology.

[0092] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to another part of the immunoglobulin (the variable domain, which contains the antigen-binding site). The constant domain contains the C16 heavy chain. H1. C H 2 and C H 3. Structural domains (collectively referred to as C) H ) and light chain CHL (or C L ) structural domain.

[0093] The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two distinct types (called kappa (“κ”) and lamuda (“λ”)) based on the amino acid sequence of their constant domains.

[0094] The “CH1 domain” (also known as the “C1” of the “H1” domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0095] The "hinge region" is generally defined as the region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). The hinge region of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain SS bond in the same position.

[0096] The "CH2 domain" (also known as the "C2 domain") of the human IgG Fc region typically extends from about amino acid 231 to about amino acid 340. The unique feature of the CH2 domain is that it does not pair tightly with another domain. Instead, two N-linked branched carbohydrate chains are intercalated between the two CH2 domains of the intact native IgG molecule. It is speculated that the carbohydrates provide an alternative to domain-domain pairing and contribute to the stability of the CH2 domain. (Burton) Molec Immunol. 22:161-206 (1985).

[0097] The “CH3 domain” (also known as the “C3 domain”) contains a chain segment from the C-terminus of the Fc domain to the CH2 domain (i.e., from approximately amino acid residue 341 of the antibody sequence to the C-terminus, typically at amino acid residue 446 or 447 of IgG).

[0098] The term "Fc domain" or "fragment crystallizable region" is used herein to define the C-terminal region of the immunoglobulin heavy chain, including both native sequence Fc domains and variant Fc domains. Although the boundaries of immunoglobulin heavy chain Fc domains may vary, the human IgG heavy chain Fc domain is generally defined as the segment extending from an amino acid residue at position Cys226 or from position Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc structure (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Therefore, compositions of complete antibodies may comprise antibody populations with all K447 residues removed, antibody populations without K447 residues removed, and antibody populations having mixtures with or without K447 residues. Suitable native sequence Fc domains for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0099] The term "Fc receptor" or "FcR" describes a receptor that binds to the Fc domain of an antibody. Preferred FcRs are naturally occurring human FcRs. Furthermore, preferred FcRs are receptors that bind IgG antibodies (γ receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences and are distinguished primarily by their cytoplasmic domains. The activating receptor FcγRIIA contains an activation motif (ITAM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an inhibitory motif (ITIM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. (See M. Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcR is a comprehensive review by 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 covered by the term "FcR" in this paper.

[0100] As used herein, the term "epitope" refers to a specific set of atoms or amino acids bound to an antigen by an antibody or antibody moiety. If two antibodies or antibody moiety exhibit competitive binding to an antigen, they can bind to the same epitope within the antigen.

[0101] As used herein, when a first antibody or fragment thereof inhibits the binding of a second antibody or fragment thereof to the target antigen by at least about 50% (e.g., at least about 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, the first antibody or fragment thereof “competes” with the second antibody or fragment thereof for binding to the target antigen, or vice versa. A high-throughput method for “binding” antibodies based on antibody cross-competition is described in PCT Publication No. WO 03 / 48731.

[0102] As used herein, the terms “specifically bind,” “specifically recognize,” and “specific to” refer to measurable and reproducible interactions, such as the binding between a target and an antibody or antibody moiety, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody or antibody moiety that specifically recognizes a target (which may be an epitope) binds to that target with greater affinity, cohesion, ease of binding, and / or longer duration of binding than it does to other targets. In some embodiments, the degree to which an antibody binds to an irrelevant target is less than about 10% of the antibody's binding to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, antibodies that specifically bind to a target have ≤10% binding. -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤10 -12 The dissociation constant of M (K) D In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved across proteins from different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding. The binding specificity of the antibody or antigen-binding domain can be determined experimentally using methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA assays, RIA assays, ECL assays, IRMA assays, EIA assays, BIACORE™ assays, and peptide scanning.

[0103] The term "specificity" refers to the selective recognition of a specific epitope of an antigen by an antigen-binding protein or antibody. For example, natural antibodies are monospecific. As used herein, the term "multispecific" means that an antigen-binding protein or antibody has two or more antigen-binding sites, wherein at least two antigen-binding sites bind to different antigens or different epitopes of the same antigen. As used herein, "bispecific" means that an antigen-binding protein or antibody has two different antigen-binding specificities. As used herein, the term "monospecific" antibody means an antibody having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.

[0104] Effector cells are leukocytes that express one or more FcRs and perform effector functions. Specifically, effector cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be derived from natural sources, such as blood. Effector cells are typically lymphocytes associated with the effector phase and are used to produce cytokines (helper T cells), kill infected pathogens (cytotoxic T cells), or secrete antibodies (differentiated B cells).

[0105] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is triggered by the binding of the first component of the complement system (C1q) to an antibody (an antibody of the appropriate subclass) that binds to a homologous antigen. To assess complement activation, one can, for example, use antibodies such as those from Gazzano-Santoro et al. J. Immunol. Methods The CDC assay is described in 202:163 (1996). Peptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capacity are described in U.S. Patent Nos. 6,194,551B1 and WO99 / 51642. The contents of those patents are expressly incorporated herein by reference. See also Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0106] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its bound collateral (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the inherent binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its collateral Y is generally expressed as a dissociation constant (Kd). Affinity can be measured by commonly used methods known in the art, including those described herein. Low-affinity antibodies typically bind antigens slowly and tend to dissociate readily, while high-affinity antibodies typically bind antigens more quickly and tend to maintain binding for longer periods. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this application. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0107] As used in this article, "on-rate", "rate of association", "association rate" or "k" on "It can also be determined using methods such as biolayer interferometry and surface plasmon resonance (SPR), as mentioned above."

[0108] "Isolated" antibodies are antibodies (e.g., natural or recombinant antibodies) that have been identified, isolated, and / or recovered from components of their production environment. Preferably, the isolated polypeptide does not associate with any other components from its production environment. Contaminant components of its production environment, such as those produced by recombinantly transfected cells, are substances that typically interfere with the research, diagnostic, or therapeutic use of the antibody and may include enzymes, hormones, and other proteins or non-protein solutes. In a preferred embodiment, the polypeptide will: (1) be purified to a greater than 95% by weight of antibody, and in some embodiments, greater than 99% by weight of antibody, as determined by, for example, the Lowry method; (2) be purified to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a twist-cup sequencer; or (3) be purified to a degree determined to be homogeneous by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. Isolated antibodies include in situ antibodies from recombinant cells, since at least one component of the antibody's native environment will be absent. However, isolated peptides or antibodies are typically prepared through at least one purification step.

[0109] An "isolated" nucleic acid molecule encoding the constructs, antibodies, or antigen-binding fragments described herein is a nucleic acid molecule that is identified and isolated from at least one contaminant nucleic acid molecule typically associated with it in the environment in which it is produced. Preferably, the isolated nucleic acid is not associated with any components related to the environment in which it is produced. The isolated nucleic acid molecule encoding the polypeptides and antibodies described herein is in a form different from the form or environment in which it is found in nature. Therefore, the isolated nucleic acid molecule is different from the nucleic acids naturally present in cells that encode the polypeptides and antibodies described herein. Isolated nucleic acids include nucleic acid molecules typically found in cells containing nucleic acid molecules, but which are located extrachromosomally or at chromosomal locations other than their natural chromosomal locations.

[0110] Nucleic acids are "operably linked" when they are placed in a functional relationship with another nucleic acid sequence. For example, if the DNA of a pre-sequence or secretory leader sequence is expressed as a pre-protein involved in polypeptide secretion, then the DNA of the pre-sequence or secretory leader sequence is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to that sequence; or, if a ribosome binding site is positioned to facilitate translation, then the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, they are contiguous and located within the reading frame. However, enhancers do not necessarily have to be contiguous. Ligation is accomplished by connecting at a convenient restriction site. If these sites are not present, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

[0111] "Separated" refers to a change from or separation from the native state. For example, nucleic acids or peptides that are naturally present in the native state in a living subject are not "separated," but the same nucleic acids or peptides that are partially or completely separated from their native coexisting substances are "separated." Separated nucleic acids or proteins can exist in a substantially purified form or in non-native environments, such as host cells.

[0112] As used herein, the term "hybridoma" refers to a cell resulting from the fusion of B lymphocytes with a fusion pair (such as myeloma cells). Hybridoma cells can be cloned and maintained indefinitely in cell cultures and are capable of producing monoclonal antibodies. Hybridoma cells can also be considered hybrid cells.

[0113] The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably and refer to polymers of nucleotides. Such nucleotide polymers may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to a linear sequence of nucleotides containing a nucleic acid molecule or polynucleotide. “Isolated nucleic acid” refers to a nucleic acid segment or fragment that is isolated from the sequence sidebanded therewith in its natural state, i.e., a DNA fragment removed from a sequence typically adjacent to the segment (i.e., a sequence adjacent to a segment in its naturally occurring genome). The term also applies to nucleic acids that have been substantially purified from other components naturally accompanying the nucleic acid (i.e., RNA, DNA, or protein) that naturally accompany the nucleic acid in cells. Therefore, the term includes, for example, recombinant DNA incorporated into vectors, autonomously replicating plasmids or viruses, or into the genomic DNA of prokaryotes or eukaryotes, or recombinant DNA existing as a separate molecule independent of other sequences (i.e., cDNA or genomic or cDNA fragments produced by PCR or restriction enzyme digestion). It also includes recombinant DNA, which is part of a hybrid gene encoding an additional polypeptide sequence.

[0114] As used herein, “complementarity” refers to nucleic acids, and is a 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 adenine residues in a first nucleic acid region can form specific hydrogen bonds (“base pairing”) with residues in a second nucleic acid region, and if said residues are thymine or uracil, then the second nucleic acid region is antiparallel to the first region. Similarly, it is known that cytosine residues in the first nucleic acid strand can base pair with residues on a second nucleic acid strand antiparallel to the first strand, provided that said residues are guanine. If a first region of a nucleic acid is arranged antiparallel to a second region of the same or different nucleic acid, and at least one nucleotide residue in the first region can base pair with a residue in the second region, then the two regions are complementary. In some embodiments, the first region comprises a first portion, and the second region comprises a second portion, such that when the first and second portions are arranged antiparallel, at least about 50% and / or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues in the first portion can base pair with nucleotide residues in the second portion. In some implementations, all nucleotide residues in the first part can be base-paired with nucleotide residues in the second part.

[0115] As used herein, the term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid to which it is linked. This term includes vectors in the form of self-replicating nucleic acid structures as well as vectors incorporated into the genome of a host cell. Vectors can be plasmids, bacteriophages, bacterial artificial chromosomes, or yeast artificial chromosomes. Vectors can be DNA vectors or RNA vectors. Vectors can be autonomously replicating extrachromosomal vectors or vectors integrated into the host genome. Some vectors are capable of guiding the expression of the nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0116] "Encoding" refers to the inherent characteristic of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence in biological processes, and the resulting biological characteristics. Therefore, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then the gene encodes that protein. The coding strand, whose nucleotide sequence is identical to the mRNA sequence and is generally provided in the sequence listing, and the non-coding strand, which serves as a transcription template for a gene or cDNA, can both be referred to as encoding the protein or other product of that gene or cDNA.

[0117] The terms "polypeptide" and "peptide" are used interchangeably and refer to a polymer of amino acid residues, and are not limited to a minimum length. Such a polymer of amino acid residues can contain native or non-native amino acid residues. Full-length proteins and fragments thereof are included in this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, "polypeptide" includes modifications to the native sequence, such as deletions, additions, and substitutions (often conserved), provided that the polypeptide retains the desired activity. These modifications can be intentional, such as by site-directed mutagenesis, or can be accidental, such as by mutations in the host producing the protein or errors caused by PCR amplification.

[0118] As used in this article, “combination” refers to the covalent connection between one molecule and another.

[0119] As used herein, the term "variant" is a nucleic acid or peptide sequence that differs from a reference nucleic acid or peptide sequence, respectively, but retains the essential biological characteristics of the reference molecule. Sequence changes in nucleic acid variants may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in substitutions, additions, deletions, fusions, or truncations of amino acids. Sequence changes in peptide variants are generally limited or conserved, so the sequences of the reference peptide and the variant are very similar overall and identical in many regions. The amino acid sequences of the variant and the reference peptide may differ due to any combination of one or more substitutions, additions, or deletions. Variants of nucleic acids or peptides can be naturally occurring variants, such as allelic variants, or can be variants known not to exist naturally. Non-naturally occurring variants of nucleic acids and peptides can be prepared by mutagenesis or 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%, or at least 85% identical to the reference sequence.

[0120] As described in this article by Spree USA. The term "regulation" can refer to any method that alters the level or activity of a substrate. Non-limiting examples of protein regulation include affecting expression (including transcription and / or translation), affecting folding, affecting degradation or protein turnover, and affecting protein localization. Non-limiting examples of enzyme regulation include affecting enzyme activity. A "regulator" is a molecule whose activity includes affecting the level or activity of a substrate. Regulators can be direct or indirect. Regulators can be used to activate, inhibit, or otherwise regulate their substrates.

[0121] The term "control sequence" refers to the DNA sequence required to express an operable, linked coding sequence in a specific host organism. Control sequences suitable for prokaryotes include, for example, promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0122] "Pharmaceutically acceptable carriers" refer to conventional, non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, formulation aids, or carriers used in conjunction with therapeutic agents to form a "pharmaceutical composition" for administration to a subject. Pharmaceutically acceptable carriers are non-toxic to the recipient at the doses and concentrations used and are compatible with other components in the formulation. Pharmaceutically acceptable carriers are suitable for the formulations used.

[0123] The “diluent” of interest herein is pharmaceutically acceptable (safe and non-toxic for human administration) and can be used to prepare liquid formulations, such as lyophilized and reconstituted formulations. Exemplary diluents include sterile water, water for sterile injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, or dextran solution. In an alternative embodiment, the diluent may comprise an aqueous solution of salt and / or buffer.

[0124] A "preservative" is a compound that can be added to the formulations described herein to reduce bacterial activity. Adding preservatives can, for example, facilitate the manufacture of multi-purpose (multi-dosage) formulations. Examples of potential preservatives include octadecyl dimethylbenzyl ammonium chloride, hexamethyl diammonium chloride, benzalkonium chloride (a mixture of alkylbenzyl dimethyl ammonium chlorides, where the alkyl group is a long-chain compound), and benzyl chloride. Other types of preservatives include aromatic alcohols such as phenol, butanol, and benzyl alcohol; alkyl hydroxybenzoates such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol. The most commonly used preservative in this article is benzyl alcohol.

[0125] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to formulations that are in a form that enables the biological activity of one or more active ingredients and do not contain any additional components that would cause unacceptable toxicity to a subject to whom the formulation will be administered. Such formulations may be sterile.

[0126] "Sterile" preparations are sterile or substantially free of live microorganisms and their spores.

[0127] "Stable" formulations are those in which proteins substantially retain their physical and chemical stability and integrity during storage. Various analytical techniques exist in the art for measuring protein stability, and a review of these techniques can be found in [link to relevant documentation]. Peptide and Protein Drug Delivery, 247-301, Vincent Lee (ed.), Marcel Dekker, Inc., New York, NY, Pubs. (1991); and Jones, A. Adv. Drug Delivery Rev.10: 29-90 (1993). Stability can be measured at a selected temperature and over a selected time period. For rapid screening, formulations can be kept at 40°C for 2 weeks to 1 month and then their stability measured. If a formulation is to be stored at 2-8°C, it should generally be stable at 30°C or 40°C for at least 1 month, and / or stable at 2-8°C for at least 2 years. If a formulation is to be stored at 30°C, it should generally be stable at 30°C for at least 2 years, and / or stable at 40°C for at least 6 months. For example, the degree of aggregation during storage can be used as an indicator of protein stability. Thus, a “stable” formulation can be one in which the amount of protein present in aggregate form is less than about 10%, and preferably less than about 5%. In other embodiments, any increase in aggregate formation during formulation storage can be identified.

[0128] A "reconstituted" formulation is a formulation prepared by dissolving a lyophilized protein or antibody formulation in a diluent, thereby dispersing the protein bulk. The reconstituted formulation is suitable for administration (e.g., subcutaneous injection) to a patient treated with the protein of interest, and in some embodiments may be suitable for parenteral or intravenous administration.

[0129] An "isotonic" formulation is one whose osmotic pressure is substantially the same as that of human blood. The osmotic pressure of an isotonic formulation is generally between approximately 250 and 350 mOsm. The term "hypotonic" describes a formulation with an osmotic pressure lower than that of human blood. Correspondingly, the term "hypertonic" is used to describe a formulation with an osmotic pressure higher than that of human blood. For example, isotonicity can be measured using a vapor pressure osmoremeter or a cryo-osmoremeter. The formulations in this application can be made hypertonic by adding salts and / or buffer solutions.

[0130] “ Recombinant AAV Vector (rAAV Vector)"rAAV" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., non-AAV-derived nucleic acid sequences), which are side-joined with at least one and, in some embodiments, two AAV inverted terminal repeat (ITR) sequences. Such an rAAV vector, when present in a host cell infected with a helper virus (or expressing a helper function) and expressing the AAV rep and cap gene products, can replicate and package into infectious viral particles (i.e., AAV receptors). When the rAAV vector is incorporated into a host cell (containing Rep and Cap proteins), it can replicate and be packaged into infectious viral particles. When the rAAV vector is incorporated into a larger polynucleotide (e.g., into a chromosome or another vector, such as a plasmid for cloning or transfection), the rAAV vector can be referred to as a "pre-vector," which is "rescued" through replication and encapsulation in the presence of AAV packaging and suitable helper functions. The rAAV vector can take many forms, including but not limited to plasmids, linear artificial chromosomes, lipid complexes, encapsulation in liposomes, and capsidation within viral particles, particularly AAV particles. The rAAV vector can be packaged into the AAV viral capsid to produce "recombinant adeno-associated virus particles (rAAV particles)."

[0131] “ AAV Inverted Terminal Repeat (ITR) "Sequence" is a well-known term in the art; it is a sequence of approximately 145 nucleotides located at both ends of a natural single-stranded AAV genome. The outermost 125 nucleotides of the ITR can be oriented in two different ways, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter self-complementary regions (called A, A', B, B', C, C', and D regions) that allow intrastrand base pairing to occur within this portion of the ITR.

[0132] As used herein, the terms “transfection” or “transformation” or “transduction” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected” or “transformed” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. The cells include primary subject cells and their progeny.

[0133] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of the number of passages. The nucleic acid content of progeny cells cannot be exactly the same as that of parent cells and may contain mutations. This article includes screened or selected mutant progeny that have the same function or biological activity as the initially transformed cells.

[0134] As used herein, “treatment” or “treating” is a method for obtaining a beneficial or desired outcome (including clinical outcomes). For the purposes of this application, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: relief of one or more symptoms caused by a disease, reduction of the severity of the disease, stabilization of the disease (e.g., prevention or delay of disease exacerbation), prevention or delay of disease spread (e.g., metastasis), prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of disease status, provision of disease remission (partial or complete remission), reduction of the dosage of one or more other medications required to treat the disease, delay of disease progression, increase or improvement of quality of life, increase in weight gain, and / or prolongation of survival. The methods of this application cover any one or more of these treatment aspects.

[0135] As used herein, the terms “effective amount” and “pharmaceutically effective amount” refer to an amount of a pharmaceutical agent sufficient to provide the desired biological outcome. This outcome may be a reduction (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and / or an alleviation of the signs, symptoms, or cause of a disease or condition, or any other desired alteration of a biological system.

[0136] The terms "inhibition" or "inhibit" refer to a reduction or cessation of any phenotypic trait, or a reduction or cessation of the occurrence, extent, or likelihood of that trait. "Reduction" or "inhibit" means a decrease, reduction, or cessation of activity, function, and / or quantity compared to a reference. In some embodiments, "reduction" or "inhibit" means an overall reduction of 20% or greater (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%). In another embodiment, "reduction" or "inhibit" means an overall reduction of 50% or greater. In yet another embodiment, "reduction" or "inhibit" means an overall reduction of 75%, 85%, 90%, 95%, or greater.

[0137] As used herein, “reference” refers to any sample, standard, or level used for comparative purposes. References may be obtained from healthy and / or disease-free samples. In some instances, references may be obtained from untreated samples. In some instances, references may be obtained from disease-free or untreated samples of individuals. In some instances, references may be obtained from one or more healthy individuals who are not individuals or patients.

[0138] As used herein, “delayed disease development” means postponing, hindering, slowing, delaying, stabilizing, suppressing, and / or postponing the development of a disease. This delay can vary in length depending on the individual’s medical history and / or the treatment received. As will be apparent to those skilled in the art, adequate or significant delay can effectively encompass prevention, as the individual does not develop the disease.

[0139] As used in this article, “prevention” includes providing protection against the onset or recurrence of disease in individuals who may be susceptible to the disease but have not yet been diagnosed with it.

[0140] As used herein, "suppression" function or activity is a decrease in function or activity when compared to conditions that are otherwise identical except for the condition or parameter of interest, or alternatively, when compared to another condition. For example, an antibody that suppresses tumor growth reduces the rate of tumor growth compared to the rate of tumor growth in the absence of that antibody.

[0141] As used herein, the terms “patient,” “subject,” “individual,” etc., are used interchangeably and refer to any animal with a complement system, which in some embodiments is a mammal and in some embodiments is a human, including those who require treatment or are susceptible to a disease or its sequelae. An individual may include, for example, dogs, cats, pigs, cattle, sheep, goats, horses, rats, monkeys, mice, and humans. In some embodiments, the individual is a human.

[0142] It should be understood that the embodiments of this application described herein include “consisting of embodiments” and / or “substantially consisting of embodiments”.

[0143] In this article, the reference to "about" a value or parameter includes (and describes) the variation with respect to that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0144] As used herein, referring to "not" a value or parameter generally means and describes something "different from" a value or parameter. For example, "The method is not used to treat type X disease" means that the method is used to treat types of diseases other than X.

[0145] The term “about XY” as used in this article has the same meaning as “about X to about Y”.

[0146] Unless the context clearly specifies otherwise, as used herein and in the appended claims, the singular forms “a,” “or,” and “this” include a plurality of indicators.

[0147] II. Multispecific constructs

[0148] In one aspect, this document provides a multispecific construct comprising: a first portion (e.g., a first antibody portion) that specifically recognizes complement factor D (“FD”); and a second portion (hereinafter also referred to as a “complement protein”) that specifically recognizes a component of the complement pathway. In some embodiments, the second portion specifically recognizes complement component 2 (“C2”). In some embodiments, the second portion specifically recognizes complement component 5 (“C5”). In some embodiments, the multispecific construct comprises two or more first portions (e.g., scFv) that are, for example, fused in tandem with each other or located on different polypeptide chains. In some embodiments, the multispecific construct comprises two or more second portions (e.g., scFv) that are, for example, fused in tandem with each other or located on different polypeptide chains. In some embodiments, the multispecific construct comprises one polypeptide chain (or consists substantially of one polypeptide chain, or consists of one polypeptide chain). In some implementations, the multispecific construct comprises two or more (e.g., 2) polypeptide chains (or consists substantially of two or more polypeptide chains, or is composed of two or more polypeptide chains), such as heterodimers or homodimers formed in the presence of an Fc domain.

[0149] In some embodiments, the first antibody portion specifically recognizing FD comprises an immunoglobulin heavy chain variable domain (“VH1”) and an immunoglobulin light chain domain (“VL1”). In some embodiments, the first antibody portion is selected from the group consisting of: full-length antibodies, Fab, Fab', F(ab')2, scFv, and sdAb. In some embodiments, the first antibody portion is an scFv. This scFv may contain, for example, a VH1-optional linker-VL1 from its N-terminus to its C-terminus. In some embodiments, the scFv may contain a VL1-optional linker-VH1 from its N-terminus to its C-terminus. In some embodiments, the first antibody portion comprises a plurality (e.g., 2, 3, or 4) anti-FD scFvs, which are optionally arranged in tandem. In some embodiments, the linker between VH1 and VL1 of the anti-FD scFv is a GS linker. In some embodiments, the linker between VH1 and VL1 of the anti-FD scFv comprises the amino acid sequence of any one of SEQ ID NO: 76, 89-95, and 110. In some embodiments, the linker between VH1 and VL1 of the anti-FD scFv comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 93) or GGGGSGGGGSGGGGSSGGGS (SEQ ID NO: 95).

[0150] In some embodiments, the second antibody portion that specifically recognizes the complement protein comprises an immunoglobulin heavy chain variable domain (“VH2”) and an immunoglobulin light chain domain (“VL2”). In some embodiments, the second antibody portion is selected from the group consisting of: full-length antibodies, Fab, Fab', F(ab')2, scFv, and sdAb. In some embodiments, the second antibody portion is an scFv. This scFv may contain, for example, a VH2-optional linker-VL2 from its N-terminus to its C-terminus. In some embodiments, the scFv may contain a VL2-optional linker-VH2 from its N-terminus to its C-terminus. In some embodiments, the second antibody portion comprises multiple (e.g., 2, 3, or 4) scFvs that recognize the complement protein, these scFvs optionally arranged in tandem. In some embodiments, the complement protein is C2. In some embodiments, the complement protein is C5. In some embodiments, the linker between VH2 and VL2 of the anti-complement protein scFv is a GS linker. In some embodiments, the linker between VH2 and VL2 of the anticomplement protein scFv comprises the amino acid sequence of any one of SEQ ID NO: 76, 89-95, and 110. In some embodiments, the linker between VH2 and VL2 of the anticomplement protein scFv comprises the amino acid sequence of SEQ ID NO: 93 or 95.

[0151] In some embodiments, the first portion (e.g., the first antibody portion) is directly linked to the second portion (e.g., the second antibody portion). In some embodiments, the first portion is linked to the second portion via a linker (e.g., a peptide linker). In some embodiments, the first portion is fused to the second portion via a peptide linker. In some embodiments, the linker has a length of about four to about fifty amino acids. In some embodiments, the linker connecting the first and second portions is a GS linker. In some embodiments, the linker is selected from the group consisting of (G)n (SEQ ID NO: 110), (GS)n (SEQ ID NO: 89), (GGGS)n (SEQ ID NO: 90), (GGGGS)n (SEQ ID NO: 91), and (GSGGS)n (SEQ ID NO: 92), where n is an integer of at least 1. In some embodiments, n is 1-8. In some embodiments, the linker connecting the first and second portions contains the amino acid sequence of GGGGS (SEQ ID NO: 76). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 93). In some embodiments, the linker comprises the amino acid sequence SEQ ID NO: 95. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 94).

[0152] In some embodiments, the first portion is fused to the second portion via an Fc domain. In some embodiments, the Fc domain is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, or IgM Fc domains. In some embodiments, the Fc region is IgG4 Fc. In some embodiments, the Fc domain contains an LALA mutation. In some embodiments, the Fc region is a human Fc region. In some embodiments, the human IgG4 Fc contains the amino acid sequence of SEQ ID NO: 96. In some embodiments, the Fc region is capable of mediating antibody effector functions, such as ADCC (antibody-dependent cell-mediated cytotoxicity) and / or CDC (complement-dependent cytotoxicity). In some embodiments, the Fc domain contains the carboxyl-terminal portions of two heavy chains held together by disulfide bonds. In some embodiments, the effector function of the antibody is determined by the sequence of the Fc domain. In some embodiments, the Fc domain is recognized by Fc receptors found on certain cell types. In some embodiments, the IgG4 Fc fragment contains an S228P mutation. In some embodiments, the IgG4 Fc fragment comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, the Fc region includes modifications that reduce the binding affinity of the Fc region to the Fc receptor. In some embodiments, the Fc domain is side-joined with GGGGS (SEQ ID NO: 76) at each end of its sequence. In some embodiments, the Fc domain is side-joined with GGGGSGGGGS (SEQ ID NO: 94) at each end of its sequence (hereinafter also referred to as the “GS-Fc-GS” linker or the “side-joined Fc domain linker”). In some embodiments, the IgG4 Fc fragment contains a PLA mutation (S228P / M428L / N434A). In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the side-joined Fc domain linker comprises the amino acid sequence of SEQ ID NO: 105.

[0153] In some embodiments, a multispecific construct is provided, comprising: a first antibody portion (e.g., scFv) specifically recognizing FD and a second antibody portion (e.g., scFv) specifically recognizing complement proteins (e.g., C2, C5), wherein the first antibody portion comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, H-CDR3 containing the amino acid sequence of SEQ ID NO: 3, L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first antibody portion comprises VH1 containing the amino acid sequence of SEQ ID NO: 7 and VL1 containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first antibody portion is scFv, for example, scFv containing the amino acid sequence of SEQ ID NO: 9 or 10. In some embodiments, the first antibody portion is fused to the N-terminus of the second antibody portion. In some embodiments, the first antibody portion is fused to the C-terminus of the second antibody portion. In some embodiments, a peptide linker and / or an Fc domain are present to link the first and second antibody portions. In some embodiments, the peptide linker is selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41.

[0154] In some embodiments, a multispecific construct is provided comprising a first antibody portion specifically recognizing FD and a second antibody portion specifically recognizing C2 (hereinafter also referred to as an "anti-FD / anti-C2 multispecific construct"). In some embodiments, a multispecific construct is provided comprising: a first antibody portion specifically recognizing FD (e.g., scFv) and a second antibody portion specifically recognizing C2 (e.g., scFv); wherein the first antibody portion comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, H-CDR3 containing the amino acid sequence of SEQ ID NO: 3, L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, a multispecific construct is provided, comprising: a first antibody portion (e.g., scFv) specifically recognizing FD and a second antibody portion (e.g., scFv) specifically recognizing C2; wherein the second antibody portion comprises: i) H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, H-CDR3 containing the amino acid sequence of SEQ ID NO: 13, L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16; or ii) H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, H-CDR3 containing the amino acid sequence of SEQ ID NO: 23, L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 25. L-CDR2 containing the amino acid sequence of SEQ ID NO: 25 and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26.In some embodiments, a multispecific construct is provided, the multispecific construct comprising: a first antibody portion (e.g., scFv) specifically recognizing FD and a second antibody portion (e.g., scFv) specifically recognizing C2; wherein the first antibody portion comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, H-CDR3 containing the amino acid sequence of SEQ ID NO: 3, L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6; and wherein the second antibody portion comprises: i) H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, H-CDR3 containing the amino acid sequence of SEQ ID NO: 13, L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 15. L-CDR2 containing the amino acid sequence of SEQ ID NO: 15 and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16; or ii) H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, H-CDR3 containing the amino acid sequence of SEQ ID NO: 23, L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25 and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, the multispecific construct comprises: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2. In some embodiments, the first antibody portion comprises VH1 containing the amino acid sequence of SEQ ID NO: 7 and VL1 containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first antibody portion is an scFv, such as an scFv containing the amino acid sequence of SEQ ID NO: 9 or 10. In some embodiments, the second antibody portion comprises: i) a VH2 containing the amino acid sequence of SEQ ID NO: 17 and a VL2 containing the amino acid sequence of SEQ ID NO: 18; or ii) a VH2 containing the amino acid sequence of SEQ ID NO: 27 and a VL2 containing the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second antibody portion is an scFv, such as an scFv containing the amino acid sequence of any one of SEQ ID NO: 19, 20, 29, and 30.In some embodiments, the first antibody portion is located at the N-terminus of the second antibody portion. In some embodiments, the first antibody portion is located at the C-terminus of the second antibody portion. In some embodiments, the first antibody portion is directly linked to the second antibody portion. In some embodiments, the first antibody portion is linked to the second antibody portion via a peptide linker (e.g., SEQ ID NO: 76). In some embodiments, the first antibody portion is linked to the second antibody portion via an Fc domain (e.g., SEQ ID NO: 41 or 105).

[0155] Therefore, in some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VH1-optional linker-VL1-optional linker-VH2-optional linker-VL2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, and VL2 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28.In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 19 or 29. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 63.

[0156] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VL1-optional linker-VH1-optional linker-VL2-optional linker-VH2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, and VL2 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28.In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 20 or 30. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 106.

[0157] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VH1-optional linker-VL1-optional linker-VL2-optional linker-VH2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first antibody portion comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, and VL2 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28.In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 20 or 30. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 67.

[0158] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VL1-optional linker-VH1-optional linker-VH2-optional linker-VL2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, and VL2 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28.In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 19 or 29. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 108.

[0159] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus: VH1-optional linker-VL1-optional linker-Fc-optional linker-VH2-optional linker-VL2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 contains the amino acid sequence of SEQ ID NO: 17, and VL2 contains the amino acid sequence of SEQ ID NO: 18.In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 19 or 29. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, the multispecific construct comprises two polypeptide chains, each comprising the amino acid sequence of SEQ ID NO: 65.

[0160] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus: VL1-optional linker-VH1-optional linker-Fc-optional linker-VL2-optional linker-VH2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 contains the amino acid sequence of SEQ ID NO: 17, and VL2 contains the amino acid sequence of SEQ ID NO: 18.In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 20 or 30. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 53. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 53.

[0161] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, the following sequence: VH1-optional linker-VL1-optional linker-Fc-optional linker-VL2-optional linker-VH2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 contains the amino acid sequence of SEQ ID NO: 17, and VL2 contains the amino acid sequence of SEQ ID NO: 18.In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 20 or 30. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 69.

[0162] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, the following sequence: VL1-optional linker-VH1-optional linker-Fc-optional linker-VH2-optional linker-VL2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 contains the amino acid sequence of SEQ ID NO: 17, and VL2 contains the amino acid sequence of SEQ ID NO: 18.In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 19 or 29. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 52.

[0163] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing an FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing a C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VH2-optional linker-VL2-optional linker-VH1-optional linker-VL1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, and VL2 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 19 or 29. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8.In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 77.

[0164] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VL2-optional linker-VH2-optional linker-VH1-optional linker-VL1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, and VL2 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 20 or 30. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8.In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 81.

[0165] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VH2-optional linker-VL2-optional linker-VL1-optional linker-VH1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, and VL2 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 19 or 29. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8.In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 43 or 45.

[0166] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VL2-optional linker-VH2-optional linker-VL1-optional linker-VH1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, and VL2 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 20 or 30. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8.In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 42 or 44.

[0167] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus: VH2-optional linker-VL2-optional linker-Fc-optional linker-VH1-optional linker-VL1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, and VL2 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 19 or 29. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6.In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 78.

[0168] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, the following sequence: VL2-optional linker-VH2-optional linker-Fc-optional linker-VH1-optional linker-VL1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, and VL2 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 20 or 30. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6.In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 82. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 82.

[0169] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, the following sequence: VH2-optional linker-VL2-optional linker-Fc-optional linker-VL1-optional linker-VH1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, and VL2 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 19 or 29. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6.In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 54.

[0170] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C2, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, the following sequence: VL2-optional linker-VH2-optional linker-Fc-optional linker-VL1-optional linker-VH1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, and VL2 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, and VL2 comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 20 or 30. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6.In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 51 or 55. In some embodiments, the multispecific construct comprises two polypeptide chains, each comprising the amino acid sequence of SEQ ID NO: 51 or 55.

[0171] In some embodiments, a multispecific construct is provided comprising a first antibody portion specifically recognizing FD and a second antibody portion specifically recognizing C5 (hereinafter also referred to as an "anti-FD / anti-C5 multispecific construct"). In some embodiments, a multispecific construct is provided comprising: a first antibody portion specifically recognizing FD (e.g., scFv) and a second antibody portion specifically recognizing C5 (e.g., scFv); wherein the first antibody portion comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, H-CDR3 containing the amino acid sequence of SEQ ID NO: 3, L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, a multispecific construct is provided, the multispecific construct comprising: a first antibody portion (e.g., scFv) specifically recognizing FD and a second antibody portion (e.g., scFv) specifically recognizing C5; wherein the second antibody portion comprises: H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, H-CDR3 containing the amino acid sequence of SEQ ID NO: 33, L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36.In some embodiments, a multispecific construct is provided, the multispecific construct comprising: a first antibody portion (e.g., scFv) specifically recognizing FD and a second antibody portion (e.g., scFv) specifically recognizing C5; wherein the first antibody portion comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, H-CDR3 containing the amino acid sequence of SEQ ID NO: 3, L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6; and wherein the second antibody portion comprises: H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, H-CDR3 containing the amino acid sequence of SEQ ID NO: 33, L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. The L-CDR2 containing the amino acid sequence of SEQ ID NO: 35 and the L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, the multispecific construct comprises: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2. In some embodiments, the first antibody portion comprises VH1 containing the amino acid sequence of SEQ ID NO: 7 and VL1 containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first antibody portion is an scFv, for example, an scFv containing the amino acid sequence of SEQ ID NO: 9 or 10. In some embodiments, the second antibody portion comprises VH2 containing the amino acid sequence of SEQ ID NO: 37 and VL2 containing the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second antibody portion is an scFv, for example, an scFv containing the amino acid sequences of SEQ ID NO: 39 and 40. In some embodiments, the first antibody portion is at the N-terminus of the second antibody portion. In some embodiments, the first antibody portion is located at the C-terminus of the second antibody portion. In some embodiments, the first antibody portion is directly linked to the second antibody portion. In some embodiments, the first antibody portion is linked to the second antibody portion via a peptide linker (e.g., SEQ ID NO: 76). In some embodiments, the first antibody portion is linked to the second antibody portion via an Fc domain (e.g., SEQ ID NO: 41 or 105).

[0172] Therefore, in some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VH1-optional linker-VL1-optional linker-VH2-optional linker-VL2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 71.

[0173] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VL1-optional linker-VH1-optional linker-VL2-optional linker-VH2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 49.

[0174] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VH1-optional linker-VL1-optional linker-VL2-optional linker-VH2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 73.

[0175] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VL1-optional linker-VH1-optional linker-VH2-optional linker-VL2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 48.

[0176] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus: VH1-optional linker-VL1-optional linker-Fc-optional linker-VH2-optional linker-VL2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 72.

[0177] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus: VL1-optional linker-VH1-optional linker-Fc-optional linker-VL2-optional linker-VH2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 61.

[0178] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, the following sequence: VH1-optional linker-VL1-optional linker-Fc-optional linker-VL2-optional linker-VH2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 74.

[0179] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, the following sequence: VL1-optional linker-VH1-optional linker-Fc-optional linker-VH2-optional linker-VL2. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 75.

[0180] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VH2-optional linker-VL2-optional linker-VH1-optional linker-VL1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 85.

[0181] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VL2-optional linker-VH2-optional linker-VH1-optional linker-VL1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 87.

[0182] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VH2-optional linker-VL2-optional linker-VL1-optional linker-VH1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 50.

[0183] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, VL2-optional linker-VH2-optional linker-VL1-optional linker-VH1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76, 93, and 95. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, a multispecific construct is provided comprising the amino acid sequence of SEQ ID NO: 46 or 47.

[0184] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus: VH2-optional linker-VL2-optional linker-Fc-optional linker-VH1-optional linker-VL1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 86. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 86.

[0185] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, the following sequence: VL2-optional linker-VH2-optional linker-Fc-optional linker-VH1-optional linker-VL1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 88. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 88.

[0186] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, the following sequence: VH2-optional linker-VL2-optional linker-Fc-optional linker-VL1-optional linker-VH1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and said VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 62.

[0187] In some embodiments, a multispecific construct is provided comprising: a first scFv specifically recognizing FD, the first scFv comprising VH1 and VL1; and a second scFv specifically recognizing C5, the second scFv comprising VH2 and VL2, wherein the multispecific construct comprises, from the N-terminus to the C-terminus, the following sequence: VL2-optional linker-VH2-optional linker-Fc-optional linker-VL1-optional linker-VH1. In some embodiments, the one or more linkers are independently selected from the group consisting of SEQ ID NO: 76 and 93-95. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, VH2 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and VL2 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, and VL2 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the second scFv comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, VH1 comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and VL1 comprises L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, and VL1 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, a multispecific construct is provided, the multispecific construct comprising the amino acid sequence of SEQ ID NO: 59. In some embodiments, the multispecific construct comprises two polypeptide chains, each of which comprises the amino acid sequence of SEQ ID NO: 59.

[0188] Biological characteristics of multispecific constructs

[0189] In some embodiments, the multispecific construct inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) AP. In some embodiments, the multispecific construct or its use preserves an individual's ability to resist infection through CP and LP.

[0190] Because C3 spontaneously hydrolyzes to form C3(H2O), AP is considered to have constitutive activity at low levels. C3(H2O) behaves similarly to C3b because it can associate with fB, making fB readily cleaved and activated by fD. The resulting C3(H2O)Bb then cleaves C3 to produce C3b and C3a, initiating the AP cascade by forming the C3 convertase C3bBb of AP. As the initial C3 convertase produces increasing amounts of C3b, an amplification loop is established. It should be noted that since CP and LP also produce C3b, which can bind to factor B and conjugate AP, the AP amplification loop also participates in CP and LP once these pathways are activated. Therefore, AP consists of two functional entities: an independent complement activation pathway independent of CP or LP, and an amplification process that participates in and facilitates the full expression of CP and LP. In some embodiments, the multispecific construct suppresses (e.g., suppresses at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the amplification process or amplification loop.

[0191] In some embodiments, the AP activity inhibited using the method of the present invention or the multispecific construct described herein is AP activation induced by one or more of the following: lipopolysaccharide (LPS), lipooligosaccharide (LOS), pathogen-associated molecular patterns (PAMPs), and danger-associated molecular patterns (DAMPs). In some embodiments, the AP activity inhibited using the method of the present invention or the multispecific construct described herein is the generation of the C3bBb protein complex. In some embodiments, the AP activity inhibited using the method of the present invention or the multispecific construct described herein is FD-dependent.

[0192] In some embodiments, the binding of the multispecific construct described herein to FD is associated with a reduction in the production of C3bBb in the complement activation pathway of intact organisms (e.g., humans) (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the multispecific construct inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) LP-induced C3b and / or C5b-9 deposition.

[0193] In some embodiments, the complement pathway component recognized by the second antibody moiety is also located within or regulates the AP, such as C3b, fB, fD, fH, or P. In some embodiments, the multispecific construct recognizing such AP components exhibits at least about 20% greater inhibitory activity against AP than the same anti-FD antibody moiety in inhibiting AP (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, or higher). In some embodiments, the multispecific construct recognizing such AP components exhibits at least about 20% greater inhibitory activity against LPS-induced C3b and / or C5b-9 deposition than the same anti-FD antibody moiety in inhibiting LPS-induced C3b and / or C5b-9 deposition (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, or higher).

[0194] In some embodiments, the anti-FD / anti-C2 multispecific construct i) inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) CP and LP, for example, inhibiting about 50% to about 80% of CP and LP, and ii) inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) AP. In some embodiments, the anti-FD / anti-C2 multispecific construct inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) all of CP, LP, AP, and TP. In some embodiments, the anti-FD / anti-C2 multispecific construct inhibits (e.g., inhibits at least about 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-FD / anti-C2 multispecific construct inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) sheep RBC lysis and rabbit RBC lysis (e.g., induced by human serum).

[0195] In some embodiments, the anti-FD / anti-C2 multispecific construct inhibits CP and / or LP (e.g., inhibits sheep RBC lysis in 20% human serum), wherein IC 50The value is at most about 50 nM, for example, at most about 40 nM, 35 nM, 30 nM, 20 nM, 10 nM, 1 nM or lower. In some embodiments, the anti-FD / anti-C2 multispecific construct inhibits CP and / or LP (e.g., inhibits sheep RBC lysis in 20% human serum), wherein IC50 50 The value is from about 1 nM to about 50 nM, for example, any one of about 10 nM to about 50 nM, about 20 nM to about 30 nM, about 20 nM to about 40 nM, about 20 nM to about 35 nM, or about 24 nM to about 32 nM.

[0196] In some embodiments, the anti-FD / anti-C2 multispecific construct inhibits CP and / or LP (e.g., inhibits sheep RBC lysis in 50% human serum), wherein IC 50 The value is at most about 150 nM, for example, at most about 120 nM, 110 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 35 nM, 30 nM, 20 nM, 10 nM or lower. In some embodiments, the anti-FD / anti-C2 multispecific construct inhibits CP and / or LP (e.g., inhibits sheep RBC lysis in 50% human serum), wherein IC50 50 The value is about 10 nM to about 150 nM, for example, about 100 nM to about 150 nM, about 40 nM to about 120 nM, about 50 nM to about 100 nM, about 60 nM to about 100 nM, about 60 nM to about 70 nM, about 65 nM to about 95 nM, about 80 nM to about 100 nM, or about 80 nM to about 90 nM.

[0197] In some embodiments, the anti-FD and / or anti-C2 multispecific constructs inhibit AP (e.g., inhibit rabbit RBC lysis in 20% human serum), wherein IC50 50 The value is at most about 20 nM, for example, at most about 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 1 nM or lower. In some embodiments, the anti-FD / anti-C2 multispecific construct inhibits AP (e.g., inhibits rabbit RBC lysis in 20% human serum), wherein IC50... 50 The value is from about 0.5 nM to about 20 nM, for example any of about 1 nM to about 20 nM, about 10 nM to about 20 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, or about 5 nM to about 10 nM.

[0198] In some implementations, the anti-FD / anti-C2 multispecific construct inhibits AP (e.g., inhibits rabbit RBC lysis in 50% human serum), where IC50... 50 The value is at most about 60 nM, for example, at most about 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 1 nM or lower. In some embodiments, the anti-FD / anti-C2 multispecific construct inhibits AP (e.g., inhibits rabbit RBC lysis in 50% human serum), wherein IC50... 50 The value is about 1 nM to about 60 nM, for example, about 1 nM to about 20 nM, about 10 nM to about 60 nM, about 10 nM to about 40 nM, about 15 nM to about 30 nM, or about 15 nM to about 25 nM.

[0199] In some embodiments, the anti-FD / anti-C5 multispecific construct i) inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) TP, and ii) inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) AP. In some embodiments, the anti-FD / anti-C5 multispecific construct inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) IgM, mannan, and LPS-induced C5b-9 deposition. In some embodiments, the anti-FD / anti-C5 multispecific construct inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) LPS-induced C3b deposition. In some embodiments, the anti-FD / anti-C5 multispecific construct does not inhibit (or inhibits at most about 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 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) sheep RBC lysis and rabbit RBC lysis (e.g., induced by human serum).

[0200] In some implementations, the anti-FD / anti-C5 multispecific construct inhibits CP (e.g., inhibits sheep RBC lysis in 20% human serum), where IC50... 50The value is at most about 150 nM, for example, at most about 120 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 35 nM, 30 nM, 20 nM, 10 nM or lower. In some embodiments, the anti-FD / anti-C5 multispecific construct inhibits CP (e.g., inhibits sheep RBC lysis in 20% human serum), wherein IC50 50 The value is from about 10 nM to about 150 nM, for example, any one of about 10 nM to about 100 nM, about 40 nM to about 120 nM, about 50 nM to about 100 nM, about 60 nM to about 100 nM, about 70 nM to about 90 nM, about 80 nM to about 100 nM, or about 80 nM to about 90 nM.

[0201] In some implementations, the anti-FD / anti-C5 multispecific construct inhibits CP (e.g., inhibits sheep RBC lysis in 50% human serum), where IC50... 50 The value is at most about 450 nM, for example, any or lower of about 400 nM, 300 nM, 250 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, or 10 nM. In some embodiments, the anti-FD / anti-C5 multispecific construct inhibits CP (e.g., inhibits sheep RBC lysis in 50% human serum), wherein IC50... 50 The value is about 50 nM to about 450 nM, for example, about 100 nM to about 300 nM, about 50 nM to about 250 nM, about 150 nM to about 400 nM, about 200 nM to about 300 nM, or about 200 nM to about 250 nM.

[0202] In some implementations, the anti-FD / anti-C5 multispecific construct inhibits AP (e.g., inhibits rabbit RBC lysis in 20% human serum), where IC50... 50 The value is at most about 20 nM, for example, at most about 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 1 nM or lower. In some embodiments, the anti-FD / anti-C5 multispecific construct inhibits AP (e.g., inhibits rabbit RBC lysis in 20% human serum), wherein IC50 50 The value is about 0.5 nM to about 20 nM, for example, about 1 nM to about 20 nM, about 10 nM to about 20 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, or about 5 nM to about 10 nM.

[0203] In some implementations, the anti-FD / anti-C5 multispecific construct inhibits AP (e.g., inhibits rabbit RBC lysis in 50% human serum), where IC50... 50 The value is at most about 60 nM, for example, at most about 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 1 nM or lower. In some embodiments, the anti-FD / anti-C5 multispecific construct inhibits AP (e.g., inhibits rabbit RBC lysis in 50% human serum), wherein IC50... 50 The value is about 1 nM to about 60 nM, for example, any one of about 1 nM to about 20 nM, about 10 nM to about 60 nM, about 10 nM to about 40 nM, about 15 nM to about 30 nM, or about 20 nM to about 25 nM.

[0204] In some embodiments, the complement pathway activity inhibited by the anti-FD / anti-C5 multispecific construct described herein is complement pathway activation induced by one or more of LPS, LOS, PAMP, and DAMP. In some embodiments, the complement signaling activity inhibited by the anti-FD / anti-C5 multispecific construct is the production of C5a protein, the production of C5b protein, and / or the formation of MAC. In some embodiments, the complement pathway activity inhibited by the anti-FD / anti-C5 multispecific construct is C5-dependent.

[0205] In some implementations, the binding of the anti-FD / anti-C5 multispecific construct to C5 (e.g., human C5) is associated with a reduction in the production of C5a or C5b and the formation of MAC in the complement activation pathway of intact organisms (e.g., humans) (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).

[0206] The activity assays for the multispecific constructs described herein are well known in the art, including, for example, inhibition of one or more of AP, CP, LP, and TP, including but not limited to, rabbit RBC lysis assays (e.g., induced by human serum), sheep RBC lysis assays (e.g., induced by human serum), IgM-induced C3b and / or C5b-9 deposition assays, mannan-induced C3b and / or C5b-9 deposition assays, or LPS-induced C3b and / or C5b-9 deposition assays. See, for example, the assays in US11434279, PCT / CN2023 / 073730, US20220204602, US20220177556, US11578137, WO2022134047, and PCT / US2023 / 063305, the contents of which are incorporated herein by reference in their entirety. See also Examples 2-3 herein.

[0207] Anti-FD antibody portion (primary antibody portion)

[0208] The anti-FD antibody portion of the multispecific construct described in this application includes any antibody portion that specifically binds to FD. For example, any anti-FD antibody or its antigen-binding fragment, such as 11-8A1 or 1F10-5, described in US11434279 (the contents of which are incorporated herein by reference in their entirety) may be used in the anti-FD antibody portion described herein.

[0209] Factor D, also known as adipsin, is a 24 kDa serine protease containing 228 amino acids. The structure of factor D contains two antiparallel β-barrel domains, each containing six β-chains; all enzymes have the same type of β-chains. Unlike most complement system proteins synthesized by the liver and immune cells, factor D is primarily produced and secreted into the bloodstream by adipocytes. However, macrophages and monocytes can also synthesize this substance, and small amounts are synthesized by astrocytes in the brain. Serum levels of factor D may vary, but under normal conditions, they are found in the range of 1–2 µg / mL. In a healthy state, factor D, like other low-molecular-weight proteins, is filtered by the glomerulus and almost completely reabsorbed in the renal tubules, then rapidly metabolized intracellularly.

[0210] Factor D is produced as a precursor or prozymogen (pre-factor D), requiring subsequent cleavage of a 6-amino acid peptide for maturation. The conversion of pre-factor D to its mature form appears to be rapid, occurring either during or immediately after secretion in the secretory pathway. Despite some controversy, the maturation of pre-factor D to mature factor D is believed to occur primarily through the activation of mannose-binding lectin-associated serine protease-3 (MASP-3), a MASP believed to function in the lectin pathway. Although enzyme-mediated maturation is required, factor D is primarily present in its mature form in resting blood, likely because MASP-3 lacks physiological inhibitors.

[0211] Because mature factor D is the predominant form in resting blood and because it has no known endogenous inhibitors, it must be highly controlled to prevent inappropriate cleavage of endogenous proteins other than its substrate. Therefore, mature factor D is locked in an inactive state through a self-inhibitory loop. This loop determines the enzyme's low reactivity and extremely high specificity for its substrate, factor B. Importantly, factor B can only be cleaved by factor D when it binds to C3b or C3(H2O). After binding and cleaving factor B, factor D is not permanently incorporated into the complex but is recycled through a reversible reaction. Therefore, glomerular filtration by the kidneys plays a crucial role in regulating factor D concentration. Besides host defense against pathogens, the bypass pathway and factor D are also involved in various other physiological processes. For example, C3b-mediated opsonization is known to be responsible for labeling damaged hepatocytes for clearance by phagocytes following acute liver injury. This process provides a scaffold for the development of newly formed cells and helps prevent persistent inflammation. The bypass pathway and factor D have been shown to be essential in this process. However, the activation level of the alternative pathway needs to be kept in good balance, because an overactive complement cascade is known to lead to widespread hepatocellular death, persistent inflammation, and liver damage.

[0212] Recent studies have shown that factor D is also involved in the skin aging process. With age, the extracellular matrix of the dermis is known to degenerate with an increase in the number of senescent cells. In human skin samples, expression levels in older subjects were higher than in younger subjects. Another tissue in which factor D plays a role is adipose tissue. Adipocytes are energy reservoirs and play a crucial role in energy balance. They are responsible not only for lipolysis but also for glucose uptake and triglyceride synthesis. Interestingly, factor D is not the only complement system component produced in adipose tissue. Other factors, including C3 and factor B, are also expressed to some extent in this tissue, and in the absence of pathogens, they have been found to activate the proximal portion of the alternative pathway (i.e., upstream of C5 cleavage). In fact, studies have shown that factor D plays an important role in adipocyte differentiation and lipid accumulation through C3a signaling. In contrast, activation of the terminal or cleaved portion of this pathway has not been observed in adipose tissue because proteins such as C5 are not expressed. As part of their role in energy homeostasis, adipocytes also have endocrine functions. In response to certain stimuli, they secrete regulatory molecules that play a role in the metabolic functions of other tissues. These regulatory molecules include fatty acids and adipokines, such as factor D. Studies have shown that when blood glucose levels are elevated, factor D indirectly induces insulin secretion from pancreatic β-cells by controlling the production of C3a via the alternative pathway. Furthermore, research has found that factor D / C3a signaling can protect pancreatic β-cells by preventing cell dedifferentiation and death. Studies have even indicated that higher levels of circulating factor D are associated with a lower risk of developing diabetes in middle-aged individuals.

[0213] A deficiency in components of the proximal portion of the alternative pathway (such as factor D) leads to an inability to opsonize invading pathogens and insufficient MAC formation. This ultimately limits phagocytosis and lysis of invaders. Therefore, it is not surprising that complete factor D deficiency has been identified as a risk factor for severe bacterial infections. For example, complete factor D deficiency was observed in a Dutch patient with meningitis due to a Ser42Stop mutation in both alleles of the gene. Complete factor D deficiency is associated with a decreased ability to opsonize and phagocytose bacteria, and this has also been observed in other family members. Other case studies have also found that factor D deficiency is associated with Neisseria gonorrhoeae infection and various respiratory infections. Although the underlying factor D mutations are not always the same, they can all lead to protein instability or abnormal protein folding, resulting in a failure to secrete. Complete factor D deficiency requires mutations in both alleles, therefore, its inheritance pattern is autosomal recessive.

[0214] The anti-FD antibody moiety or its functional fragment can specifically bind to FD from any source, such as any organism with a complement system, including but not limited to dogs, cats, pigs, cattle, 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.

[0215] In some embodiments, the anti-FD antibody portion binds to human FD. In some embodiments, the anti-FD antibody portion is cross-species reactive to FDs other than human FD. Exemplary non-human FDs 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 portion cross-reacts with cynomolgus monkey FD. In some embodiments, the anti-FD antibody portion does not cross-react with mouse FD.

[0216] The anti-FD antibody moiety can be any suitable form known in the art. In some embodiments, the anti-FD antibody moiety is selected from the group consisting of: full-length antibodies, Fab, Fab', F(ab')2, scFv, sdAb, and combinations thereof. In some embodiments, the anti-FD antibody moiety comprises an sdAb bound to FD. In some embodiments, the anti-FD 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 containing a PLA mutation, such as SEQ ID NO:41). In some embodiments, the anti-FD antibody moiety comprises anti-FD scFv (or consists essentially of anti-FD scFv, or consists of anti-FD scFv). In some embodiments, the anti-FD antibody moiety is a mouse antibody, a chimeric antibody, a humanized antibody, or a human 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.

[0217] In some embodiments, the binding of the anti-FD antibody moiety to FD (e.g., human FD) is pH-dependent, and the anti-FD antibody moiety binds to FD more strongly at neutral pH (e.g., about pH 7.4; e.g., the pH found in blood) than it binds to FD at acidic pH (e.g., about pH 5.8; e.g., the pH found in endocrine systems). In some embodiments, the pH-dependent anti-FD antibody moiety has a binding affinity for FD (e.g., human FD) at about pH 7.4 that is at least about 3 times (e.g., at least about 4, 5, 6, 7, 8, 9, 10 times or higher) than the pH-dependent anti-FD antibody moiety has a binding affinity for FD (e.g., human FD) at about pH 5.8.

[0218] In some embodiments, the anti-FD antibody portion (also referred to as the first antibody portion) comprises: i) VH1, which comprises H-CDR1, H-CDR2, and H-CDR3, wherein H-CDR1, H-CDR2, and H-CDR3 each comprise the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 in a reference VH having the amino acid sequences shown in SEQ ID NO: 7; and ii) VL1, which comprises L-CDR1, L-CDR2, and L-CDR3, wherein L-CDR1, L-CDR2, and L-CDR3 each comprise the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 in a reference VL having the amino acid sequences shown in SEQ ID NO: 8. In some embodiments, the anti-FD antibody portion has an affinity for FD (e.g., human FD) comparable to that of a reference antibody for FD (e.g., the same, or with a difference of 8, 7, 6, 5, 4, 3, 2, or 1 times), the reference antibody comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 8.

[0219] In some embodiments, the anti-FD antibody portion or the first antibody portion comprises: (i) VH1, said VH1 comprising an H-CDR1 containing the amino acid sequence of SEQ ID NO: 1 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); an H-CDR2 containing the amino acid sequence of SEQ ID NO: 2 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and an H-CDR3 containing the amino acid sequence of SEQ ID NO: 3 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (ii) VL1, said VL1 comprising an H-CDR1 containing the amino acid sequence of SEQ ID NO: 1 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (ii) VL1 containing the amino acid sequence of SEQ ID NO: 1 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (iii) VL1 containing the amino acid sequence of SEQ ID NO: 1 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (ii ... L-CDR1 containing the amino acid sequence of SEQ ID NO: 4 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); L-CDR2 containing the amino acid sequence of SEQ ID NO: 5 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution). In some embodiments, the anti-FD antibody portion or the first antibody portion comprises (i) VH1, which includes H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 3; and (ii) VL1, which includes L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0220] In some embodiments, the anti-FD antibody portion or the first antibody portion comprises: VH1, said VH1 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid homology with SEQ ID NO: 7; and VL1, said VL1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid homology with SEQ ID NO: 8. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions), and VL1 comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions).

[0221] In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertion, deletion, and / or substitution, such as conservative substitution) in H-CDR1 and / or H-CDR2 and / or H-CDR3; and VL1 comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertion, deletion, and / or substitution, such as conservative substitution) in L-CDR1 and / or L-CDR2 and / or L-CDR3. In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in H-CDR1 and / or H-CDR2; and VL1 comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in L-CDR1 and / or L-CDR2.

[0222] In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to 10 amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in the frame region; and VL1 comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to 10 amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in the frame region.

[0223] In some embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof containing amino acid changes (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in CDR and FR; and VL1 comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof containing amino acid changes (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in CDR and FR.

[0224] In some embodiments, the anti-FD antibody portion or the first antibody portion includes VH1 containing the amino acid sequence of SEQ ID NO: 7 and VL1 containing the amino acid sequence of SEQ ID NO: 8.

[0225] In some embodiments, the anti-FD antibody portion or the first antibody portion is scFv. In some embodiments, the anti-FD antibody portion or the first antibody portion includes VH1-optional linker-VL1 from the N-terminus to the C-terminus. In some embodiments, the anti-FD antibody portion or the first antibody portion includes the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-FD antibody portion or the first antibody portion includes VL1-optional linker-VH1 from the N-terminus to the C-terminus. In some embodiments, the anti-FD antibody portion or the first antibody portion includes the amino acid sequence of SEQ ID NO: 10.

[0226] Biological characteristics of anti-FD antibody

[0227] In some embodiments, the anti-FD antibody described herein partially inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) AP, but does not inhibit (or inhibits at most about 10%, 5%, 2%, 1%, or less) CP or LP activation. In some embodiments, the portion of the anti-FD antibody described herein, or its use, preserves an individual's ability to fight infection through CP and LP.

[0228] In some embodiments, the anti-FD antibody described herein partially inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the amplification process or amplification loop.

[0229] In some implementations, the anti-FD antibody partially inhibits the IC50 of CP and / or LP (e.g., inhibiting the lysis of sheep RBCs in 20% human serum). 50 The value could not be detected.

[0230] In some implementations, the anti-FD antibody partially inhibits AP (e.g., inhibits rabbit RBC lysis in 20% human serum), where IC50... 50 The value is at most about 50 nM, for example, at most about 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 1 nM or lower. In some embodiments, the anti-FD antibody partially inhibits AP (e.g., inhibits rabbit RBC lysis in 20% human serum), wherein IC50... 50 The value is from about 0.5 nM to about 50 nM, for example, any one of about 1 nM to about 20 nM, about 10 nM to about 20 nM, about 5 nM to about 15 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, or about 5 nM to about 10 nM.

[0231] In some embodiments, the AP activity partially inhibited by the method of the present invention or the anti-FD antibody is AP activation induced by one or more of LPS, LOS, PAMP, and DAMP. In some embodiments, the AP activity partially inhibited by the method of the present invention or the anti-FD antibody is the generation of the C3bBb protein complex. In some embodiments, the AP activity partially inhibited by the method of the present invention or the anti-FD antibody is FD-dependent.

[0232] In some embodiments, the binding of the anti-FD antibody portion to FD is associated with a reduction in the production of C3bBb in the complement activation pathway of intact organisms (e.g., humans) (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the anti-FD antibody portion inhibits (e.g., inhibits at least about 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 portion does not inhibit (or inhibits at most about 10%, 5%, 2%, 1%, or less) IgM-induced or mannan-induced C3b and / or C5b-9 deposition.

[0233] Methods for testing the activity of anti-FD antibodies or their antigen-binding fragments (e.g., AP inhibition) are well known in the art, including but not limited to rabbit RBC lysis assays (e.g., induced by human serum) or LPS-induced C3b and / or C5b-9 deposition assays. See, for example, the assay in US11434279. See also Examples 2-3 herein.

[0234] Anti-complement protein portion (second antibody portion)

[0235] The second antibody described in this article partially and specifically recognizes complement proteins, such as complement proteins other than FD.

[0236] The complement system plays a crucial role in the pathogenesis of many autoimmune, inflammatory, and ischemic diseases. Improper complement activation and complement deposition on host cells can lead to complement-mediated cell and target tissue lysis and / or damage, as well as tissue destruction due to the production of potent inflammatory mediators. The complement system, also known as the complement cascade, is part of the immune system. It enhances the ability of (complementary) antibodies and phagocytes to clear microorganisms and damaged cells from the body, promote inflammation, and attack pathogen cell membranes. It is part of the innate immune system, lacks adaptive response, and does not change throughout an individual's life. However, the complement system can be recruited and function through antibodies produced by the adaptive immune system.

[0237] Unbound by any theory or assumption, three complement pathways are known: the alternative complement pathway (AP), the classical pathway (CP), and the lectin pathway (LP). Generally, CP is initiated by antigen-antibody complexes, LP is activated by lectin binding to sugar molecules on microbial surfaces, and AP, while constitutively active at low levels, can rapidly proliferate on bacterial, viral, and parasitic cell surfaces due to a lack of regulatory proteins. Host cells are usually protected from AP complement activation by regulatory proteins. However, in certain situations, such as when regulatory proteins are defective or absent, AP can also be uncontrolled activated on host cells, leading to complement-mediated diseases or symptoms. CP consists of components C1, C2, and C4, and merges with AP in the C3 activation step. LP consists of mannose-binding lectin (MBL) and MBL-associated serine protease (MASP), and shares components C4 and C2 with CP. AP consists of component C3 and several factors, such as factor B, factor D, propertin, and fluid phase regulator H. Complement activation consists of three phases: (a) recognition, (b) enzymatic activation, and (c) membrane attack leading to cell death. The first phase of CP complement activation begins at C1. C1 is composed of three distinct proteins: the recognition subunit C1q and the serine protease subfractions C1r and C1s, which bind together to form the calcium-dependent tetrameric complex C1r2s2. The intact C1 complex is essential for the physiological activation of C1. Activation occurs when the intact C1 complex binds to an immunoglobulin complex conjugated to an antigen. This binding activates C1s, which then cleaves C4 and C2 proteins to produce 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. LP activation is initiated by the binding of MBL to certain sugars on the target surface, which triggers the activation of MBL-associated serine proteases (MASP). MASP then cleaves C4 and C2 in a manner similar to the activity of C1s in CP, resulting in the C3 convertase C4b2a. Therefore, CP and LP are activated through different mechanisms, but they share the same components C4 and C2, and both pathways lead to the production of the same C3 convertase C4b2a. The cleavage of C3 into C3b and C3a by C4b2a is a central event in the complement pathway for two reasons. It initiates the AP amplification loop because surface-deposited C3b is a key intermediate for the AP C3 convertase C3bBb. Both C3a and C3b have important biological significance. C3a has pro-inflammatory effects and, along with C5a, is known as an allergen. C3b and its further cleavage products also bind to complement receptors present on neutrophils, eosinophils, monocytes, and macrophages, thereby promoting the phagocytosis and clearance of C3b opsonization granules.Finally, C3b can associate with C4b2a or C3bBb to form C5 convertases of CP and LP and AP, respectively, activating the terminal complement sequence, leading to the production of the potent pro-inflammatory mediator C5a and the assembly of the soluble membrane attack complex (MAC) C5-C9.

[0238] Complement dysfunction is a cause of several human glomerular diseases, including atypical hemolytic uremic syndrome (aHUS), antineutrophil cytoplasmic antibody-mediated vasculitis (ANCA), C3 glomerulonephropathy, IgA nephropathy, immune complex-induced membranoproliferative glomerulonephritis, renal ischemia-reperfusion injury, lupus nephritis, membranous nephropathy, and chronic transplant-mediated glomerulonephropathy. Abnormal complement component activation has been proposed as a biomarker for various types of cancer and their clinical outcomes. Lung cancer patients show significantly higher plasma complement protein and activation fragment levels than control donors, and elevated complement levels correlate with lung tumor size. Complement-related proteins are also elevated in biofluids 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 the treatment of glomerular diseases and cancer.

[0239] Complement component 2 (C2)

[0240] In some embodiments, the complement protein bound to the second antibody portion is complement component 2 (C2). Any anti-C2 antibody or its antigen-binding fragment described in PCT / CN2023 / 073730 (the contents of which are incorporated herein by reference in their entirety) may be used in the anti-C2 antibody portion described herein.

[0241] The anti-C2 antibody portion can specifically bind to C2 from any source, such as any organism having a complement system, including but not limited to dogs, cats, pigs, cattle, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans. In some embodiments, C2 is mouse C2. In some embodiments, C2 is human C2.

[0242] In some embodiments, the anti-C2 antibody portion binds to C2. In some embodiments, the anti-C2 antibody portion binds to C2a. In some embodiments, the anti-C2 antibody portion exhibits cross-species reactivity with C2 other than human C2 and / or with C2a other than human C2a. Exemplary non-human C2s and C2a include, but are not limited to, mouse C2 and C2a, rat C2 and C2a, rabbit C2 and C2a, sheep C2 and C2a, and cynomolgus monkey C2 and C2a. In some embodiments, the anti-C2 antibody portion cross-reacts with cynomolgus monkey C2 and / or C2a.

[0243] Human C2 is an 83 kDa protein (100 kDa after glycosylation) produced as an inactive, highly glycosylated zymogen, consisting of five domains: three N-terminal complement control protein (CCP) domains, one von Willebrand factor A (VWA) domain, and one C-terminal trypsin-like serine protease (SP) domain. The last two domains, VWA and SP, form the C2a fragment (residues 224–732, 70 kDa), which is generated in the canonical and lectin-binding proteolytic activation cascade. The larger C2a fragment is 57.4 kDa (70 kDa after glycosylation) and provides the catalytic center for the invertase complex of the complement-activated canonical and lectin-binding pathways. C2, bound to C4b, is cleaved by classical proteases (C1s) or lectin proteases (MASP2) to produce C4bC2a. C4bC2a is a short-lived C3 convertase that further cleaves C3 into C3a and C3b, ultimately leading to the formation of the membrane attack complex (MAC) and the lysis of bacteria and damaged cells. C2 shares the same serine protease domain as C4bC2a, but exhibits an inactive, zymogeneous conformation, requiring a cofactor-induced conformational change to exert its activity.

[0244] The anti-C2 antibody moiety can be any suitable form known in the art. In some embodiments, the anti-C2 antibody moiety is selected from the group consisting of: full-length antibodies, Fab, Fab', F(ab')2, scFv, sdAb, and combinations 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 containing a PLA mutation, such as SEQ ID NO:41). In some embodiments, the anti-C2 antibody moiety comprises (or is substantially composed of) an anti-C2 scFv. In some embodiments, the anti-C2 antibody moiety is a mouse antibody, a chimeric antibody, a humanized antibody, or a 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.

[0245] In some embodiments, the binding of the anti-C2 antibody moiety to C2 (e.g., human C2) is pH-dependent, and the binding of the anti-C2 antibody moiety to C2 at neutral pH (e.g., about pH 7.4; e.g., the pH found in blood) is stronger than its binding at acidic pH (e.g., about pH 5.8; e.g., the pH found in endocrine systems). 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 (e.g., at least about 4, 5, 6, 7, 8, 9, 10 times or higher) than the binding affinity of the pH-dependent anti-C2 antibody moiety to C2 (e.g., human C2) at about pH 5.8.

[0246] In some embodiments, the anti-C2 antibody portion (also referred to as the second antibody portion) comprises: i) VH2, which comprises H-CDR1, H-CDR2, and H-CDR3, wherein H-CDR1, H-CDR2, and H-CDR3 each comprise the amino acid sequences of reference VH having the amino acid sequences shown in SEQ ID NO: 17; and ii) VL2, which comprises L-CDR1, L-CDR2, and L-CDR3, wherein L-CDR1, L-CDR2, and L-CDR3 each comprise the amino acid sequences of reference VL having the amino acid sequences shown in SEQ ID NO: 18. In some embodiments, the affinity of this anti-C2 antibody portion for C2 (e.g., human C2) is comparable to that of a reference antibody (e.g., the same, or differing by about 8, 7, 6, 5, 4, 3, 2, or 1 times), said reference antibody comprising VH containing the amino acid sequence of SEQ ID NO: 17 and VL containing the amino acid sequence of SEQ ID NO: 18.

[0247] In some embodiments, the anti-C2 antibody portion comprises: (i) VH2, said VH2 comprising H-CDR1 containing the amino acid sequence of SEQ ID NO: 11 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); H-CDR2 containing the amino acid sequence of SEQ ID NO: 12 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (ii) VL2, said VL2 comprising H-CDR1 containing the amino acid sequence of SEQ ID NO: 12 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (ii) VL2 containing the amino acid sequence of SEQ ID NO: 12 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (iii) VL2 containing the amino acid sequence of SEQ ID NO: 12 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (iv ... L-CDR1 containing the amino acid sequence of SEQ ID NO: 14 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); L-CDR2 containing the amino acid sequence of SEQ ID NO: 15 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution). In some embodiments, the anti-C2 antibody portion comprises: (i) VH2, which includes H-CDR1 containing the amino acid sequence of SEQ ID NO: 11, H-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and (ii) VL2, which includes L-CDR1 containing the amino acid sequence of SEQ ID NO: 14, L-CDR2 containing the amino acid sequence of SEQ ID NO: 15, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 16.

[0248] In some embodiments, the anti-C2 antibody portion comprises: VH2, said VH2 comprising the amino acid sequence of SEQ ID NO: 17 or a variant thereof having at least about 80% (including, for example, at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid homology with SEQ ID NO: 17; and VL2, said VL2 comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least about 80% (for example, at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid homology with SEQ ID NO: 18. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to 10 amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in VH2; and VL2 comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to 10 amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in VL2.

[0249] In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in H-CDR1 and / or H-CDR2 and / or H-CDR3; and VL2 comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in L-CDR1 and / or L-CDR2 and / or L-CDR3. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in H-CDR1 and / or H-CDR2; and VL2 comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in L-CDR1 and / or L-CDR2.

[0250] In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to 10 amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in the frame region; and VL2 comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to 10 amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in the frame region.

[0251] In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof containing amino acid changes (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in CDR and FR; and VL2 comprises the amino acid sequence of SEQ ID NO: 18, or a variant thereof containing amino acid changes (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in CDR and FR.

[0252] In some embodiments, the anti-C2 antibody portion comprises VH2 containing the amino acid sequence of SEQ ID NO: 17 and VL2 containing the amino acid sequence of SEQ ID NO: 18.

[0253] In some embodiments, the anti-C2 antibody moiety is scFv. In some embodiments, the anti-C2 antibody moiety comprises, from the N-terminus to the C-terminus: VH2-optional linker-VL2. In some embodiments, the anti-C2 antibody moiety comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-C2 antibody moiety comprises, from the N-terminus to the C-terminus: VL2-optional linker-VH2. In some embodiments, the anti-C2 antibody moiety comprises the amino acid sequence of SEQ ID NO: 20.

[0254] In some embodiments, the anti-C2 antibody portion (also referred to as the second antibody portion) comprises: i) VH2, which comprises H-CDR1, H-CDR2, and H-CDR3, wherein each of the H-CDR1, H-CDR2, and H-CDR3 comprises an amino acid sequence of reference VH having the amino acid sequence shown in SEQ ID NO: 27; and ii) VL2, which comprises L-CDR1, L-CDR2, and L-CDR3, wherein each of the L-CDR1, L-CDR2, and L-CDR3 comprises an amino acid sequence of reference VL having the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the affinity of this anti-C2 antibody portion for C2 (e.g., human C2) is comparable to that of a reference antibody (e.g., the same, or differing by about 8, 7, 6, 5, 4, 3, 2, or 1 times), said reference antibody comprising VH containing the amino acid sequence of SEQ ID NO: 27 and VL containing the amino acid sequence of SEQ ID NO: 28.

[0255] In some embodiments, the anti-C2 antibody portion comprises: (i) VH2, said VH2 comprising H-CDR1 containing the amino acid sequence of SEQ ID NO: 21 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); H-CDR2 containing the amino acid sequence of SEQ ID NO: 22 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (ii) VL2, said VL2 comprising H-CDR1 containing the amino acid sequence of SEQ ID NO: 22 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (ii) VL2 containing the amino acid sequence of SEQ ID NO: 23 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (iii) VL2 containing the amino acid sequence of SEQ ID NO: 23 ...iv) VL2 containing the amino acid sequence of SEQ ID NO: 23 or a variant thereof containing up to 3 amino acid L-CDR1 containing the amino acid sequence of SEQ ID NO: 24 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); L-CDR2 containing the amino acid sequence of SEQ ID NO: 25 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution). In some embodiments, the anti-C2 antibody portion comprises: (i) VH2, which includes H-CDR1 containing the amino acid sequence of SEQ ID NO: 21, H-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and (ii) VL2, which includes L-CDR1 containing the amino acid sequence of SEQ ID NO: 24, L-CDR2 containing the amino acid sequence of SEQ ID NO: 25, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 26.

[0256] In some embodiments, the anti-C2 antibody portion comprises: VH2, said VH2 comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% (including, for example, at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid homology with SEQ ID NO: 27; and VL2, said VL2 comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid homology with SEQ ID NO: 28. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to 10 amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in VH2; and VL2 comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof comprising up to 10 amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in VL2.

[0257] In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in H-CDR1 and / or H-CDR2 and / or H-CDR3; and VL2 comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in L-CDR1 and / or L-CDR2 and / or L-CDR3. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in H-CDR1 and / or H-CDR2; and VL2 comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in L-CDR1 and / or L-CDR2.

[0258] In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to 10 amino acid variations (e.g., any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) in the frame region (e.g., insertion, deletion, and / or substitution, such as conservative substitution); and VL2 comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof comprising up to 3 amino acid variations (e.g., any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) in the frame region (e.g., insertion, deletion, and / or substitution, such as conservative substitution).

[0259] In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 27, or a variant thereof containing amino acid changes (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in CDR and FR; and VL2 comprises the amino acid sequence of SEQ ID NO: 28, or a variant thereof containing amino acid changes (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in CDR and FR.

[0260] In some embodiments, the anti-C2 antibody portion comprises VH2 containing the amino acid sequence of SEQ ID NO: 27 and VL2 containing the amino acid sequence of SEQ ID NO: 28.

[0261] In some embodiments, the anti-C2 antibody moiety is scFv. In some embodiments, the anti-C2 antibody moiety comprises, from the N-terminus to the C-terminus: VH2-optional linker-VL2. In some embodiments, the anti-C2 antibody moiety comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the second antibody moiety comprises, from the N-terminus to the C-terminus: VL2-optional linker-VH2. In some embodiments, the second antibody moiety comprises the amino acid sequence of SEQ ID NO: 30.

[0262] Biological characteristics of the anti-C2 antibody portion

[0263] In some embodiments, the anti-C2 antibody described herein partially inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher) CP and / or LP. In some embodiments, the anti-C2 antibody described herein partially inhibits no more than about 70% (e.g., no more than about 60%, 50%, 40%, 30%, 20%, 10% or lower) CP and / or LP. In some embodiments, the anti-C2 antibody partially does not inhibit (or inhibits at most about 10%, 5%, 2%, 1% or lower) AP activation. In some embodiments, the anti-C2 antibody partially inhibits about 50% to about 80% of CP and / or LP.

[0264] In some implementations, the anti-C2 antibody partially inhibits CP and / or LP (e.g., inhibits the lysis of sheep RBCs in 20% human serum), wherein IC 50 The value is at most about 100 nM, for example, at most about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 35 nM, 30 nM, 20 nM, 10 nM, 1 nM or lower. In some embodiments, the anti-C2 antibody partially inhibits the CP and / or LP pathways (e.g., inhibits sheep RBC lysis in 20% human serum), wherein IC50... 50 The value is from about 1 nM to about 100 nM, for example, from about 1 nM to about 50 nM, from about 10 nM to about 40 nM, from about 20 nM to about 80 nM, from about 20 nM to about 50 nM, or from about 20 nM to about 35 nM.

[0265] In some implementations, the anti-C2 antibody partially inhibits the IC50 of AP (e.g., inhibits the lysis of rabbit RBCs in 20% human serum). 50 The value could not be detected.

[0266] In some embodiments, the anti-C2 antibody partially inhibits (e.g., inhibits at least about 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 partially does not inhibit (or inhibits at most about 10%, 5%, 2%, 1%, or less) LPS-induced C3b and / or C5b-9 deposition.

[0267] Methods for testing the activity of anti-C2 antibodies or their antigen-binding fragments (e.g., CP and / or LP inhibition) are well known in the art, including but not limited to sheep RBC lysis assays (e.g., induced by human serum), IgM-induced C3b or C5b-9 deposition assays, or mannan-induced C3b or C5b-9 deposition assays. See, for example, the assays in PCT / CN2023 / 073730. See also Examples 2-3 herein.

[0268] Complement component 5 (C5)

[0269] In some embodiments, the complement protein bound to the second antibody portion is complement component 5 (C5). Any anti-C5 antibody or antigen-binding fragment thereof described in US20220204602, US20220177556, US11578137, WO2022134047, and PCT / US2023 / 063305 may be used in the anti-C5 antibody portion described herein, the contents of which are incorporated herein by reference in their entirety.

[0270] The anti-C5 antibody moiety can specifically bind to C5 from any source, such as any organism having a complement system, including but not limited to dogs, cats, pigs, cattle, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans. In some embodiments, C5 is mouse C5. In some embodiments, C5 is human C5.

[0271] In some embodiments, the anti-C5 antibody portion binds to C5. In some embodiments, the anti-C5 antibody portion binds to C5b. In some embodiments, the anti-C5 antibody portion exhibits cross-species reactivity with C5s other than human C5 and / or with C5b other than human C5b. Exemplary non-human C5s and C5b include, but are not limited to, mouse C5 and C5b, rat C5 and C5b, rabbit C5 and C5b, sheep C5 and C5b, and cynomolgus monkey C5 and C5b. In some embodiments, the anti-C5 antibody portion cross-reacts with cynomolgus monkey C5 and / or C5b. In some embodiments, the anti-C5 antibody portion does not bind to C5a.

[0272] The C5 gene encodes a protoprotein that is proteased to produce various protein products, including the C5 α chain, C5 β chain, C5a anaphylatoxin, and C5b. Human C5 is a 188 kDa protein containing a C5 α chain and a β chain linked by disulfide bonds. Invertase cleaves the α chain, forming the potent anaphylatoxin C5a and the large C5b cleavage product, which possess strong convulsant and chemotactic activities. The C5b large cleavage product can form a complex with 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.

[0273] The anti-C5 antibody moiety can be any suitable form known in the art. In some embodiments, the anti-C5 antibody moiety is selected from the group consisting of: full-length antibodies, Fab, Fab', F(ab')2, scFv, sdAb, and combinations 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 containing a PLA mutation, such as SEQ ID NO:41). In some embodiments, the anti-C5 antibody moiety comprises (or is substantially composed of) an anti-C5 scFv, or is composed of. In some embodiments, the anti-C5 antibody moiety is a mouse antibody, a chimeric antibody, a humanized antibody, or a 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.

[0274] In some embodiments, the binding of the anti-C5 antibody moiety to C5 (e.g., human C5) is pH-dependent, and the binding of the anti-C5 antibody moiety to C5 at neutral pH (e.g., about pH 7.4; e.g., the pH found in blood) is stronger than its binding at acidic pH (e.g., about pH 5.8; e.g., the pH found in endocrine systems). 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 (e.g., at least about 4, 5, 6, 7, 8, 9, 10 times or more) than the binding affinity of the pH-dependent anti-C5 antibody moiety to C5 (e.g., human C5) at about pH 5.8.

[0275] In some implementations, the anti-C5 antibody portion binds to an epitope in the α chain of C5 and / or an epitope in the β chain of C5.

[0276] In some embodiments, the anti-C5 antibody portion (also referred to as the second antibody portion) comprises: VH2, which comprises H-CDR1, H-CDR2, and H-CDR3, wherein H-CDR1, H-CDR2, and H-CDR3 each comprise the amino acid sequences of reference VH having the amino acid sequences shown in SEQ ID NO: 37; and VL2, which comprises L-CDR1, L-CDR2, and L-CDR3, wherein L-CDR1, L-CDR2, and L-CDR3 each comprise the amino acid sequences of reference VL having the amino acid sequences shown in SEQ ID NO: 38. In some embodiments, the affinity of this anti-C5 antibody portion for C5 (e.g., human C5) is comparable to that of a reference antibody (e.g., the same, or differing by about 8, 7, 6, 5, 4, 3, 2, or 1 times), said reference antibody comprising VH containing the amino acid sequence of SEQ ID NO: 37 and VL containing the amino acid sequence of SEQ ID NO: 38.

[0277] In some embodiments, the anti-C5 antibody portion comprises: (i) VH2, said VH2 comprising H-CDR1 containing the amino acid sequence of SEQ ID NO: 31 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); H-CDR2 containing the amino acid sequence of SEQ ID NO: 32 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (ii) VL2, said VL2 comprising H-CDR1 containing the amino acid sequence of SEQ ID NO: 32 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (ii) VL2 containing the amino acid sequence of SEQ ID NO: 33 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (iii) VL2 containing the amino acid sequence of SEQ ID NO: 32 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (ii) VL2 containing the amino acid sequence of SEQ ID NO: 32 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (iii) VL2 containing the amino acid sequence of SEQ ID NO: 32 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and (iv) VL2 containing the amino acid sequence of SEQ ID NO: 32 or a variant thereof containing up to 3 amino acid L-CDR1 containing the amino acid sequence of SEQ ID NO: 34 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); L-CDR2 containing the amino acid sequence of SEQ ID NO: 35 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution); and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36 or a variant thereof containing up to 3 amino acid variations (e.g., insertion, deletion, and / or substitution, e.g., conservative substitution). In some embodiments, the anti-C5 antibody portion comprises: (i) VH2, which includes H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and H-CDR3 containing the amino acid sequence of SEQ ID NO: 33; and (ii) VL2, which includes L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36.

[0278] In some embodiments, the anti-C5 antibody portion comprises: VH2, said VH2 comprising the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least about 80% (e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid homology with SEQ ID NO: 37; and VL2, said VL2 comprising the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least about 80% (e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid homology with SEQ ID NO: 38. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to 10 amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in VH2; and VL2 comprises the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to 10 amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in VL2.

[0279] In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in H-CDR1 and / or H-CDR2 and / or H-CDR3; and VL2 comprises the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in L-CDR1 and / or L-CDR2 and / or L-CDR3. In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in H-CDR1 and / or H-CDR2; and VL2 comprises the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to three (e.g., any one of 1, 2, or 3) amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in L-CDR1 and / or L-CDR2.

[0280] In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, or a variant thereof comprising up to 10 amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in the frame region; and VL2 comprises the amino acid sequence of SEQ ID NO: 38, or a variant thereof comprising up to 10 amino acid variations (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in the frame region.

[0281] In some embodiments, VH2 comprises the amino acid sequence of SEQ ID NO: 37, or a variant thereof containing amino acid changes (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in CDR and FR; and VL2 comprises the amino acid sequence of SEQ ID NO: 38, or a variant thereof containing amino acid changes (e.g., insertions, deletions, and / or substitutions, such as conservative substitutions) in CDR and FR.

[0282] In some embodiments, the anti-C5 antibody portion comprises VH2 containing the amino acid sequence of SEQ ID NO: 37 and VL2 containing the amino acid sequence of SEQ ID NO: 38.

[0283] In some embodiments, the anti-C5 antibody moiety is scFv. In some embodiments, the anti-C5 antibody moiety comprises, from the N-terminus to the C-terminus: VH2-optional linker-VL2. In some embodiments, the anti-C5 antibody moiety comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the anti-C5 antibody moiety comprises, from the N-terminus to the C-terminus: VL2-optional linker-VH2. In some embodiments, the anti-C5 antibody moiety comprises the amino acid sequence of SEQ ID NO: 40.

[0284] Biological characteristics of the anti-C5 antibody portion

[0285] In some embodiments, the anti-C5 antibody described herein partially inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) downstream effects of AP, CP, and / or LP activation.

[0286] In some embodiments, the complement pathway activity inhibited by the anti-C5 antibody portion described herein is complement pathway activation induced by one or more of LPS, LOS, PAMP, and DAMP. In some embodiments, the complement signaling activity inhibited by the anti-C5 antibody portion is the production of C5a protein, the production of C5b protein, and / or the formation of MAC. In some embodiments, the complement pathway activity inhibited by the anti-C5 antibody portion is C5-dependent.

[0287] In some implementations, the binding of the anti-C5 antibody portion to C5 (e.g., human C5) is associated with a reduction in the production of C5a or C5b and the formation of MAC in the complement activation pathway of an intact organism (e.g., human) (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).

[0288] In some implementations, the anti-C5 antibody partially inhibits CP and / or LP and / or AP and / or TP (terminal pathways) (e.g., inhibiting sheep RBC lysis in 20% human serum), wherein IC50 50 The value is at most about 150 nM, for example, at most about 120 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM or lower. In some embodiments, the anti-C5 antibody partially inhibits CP and / or LP and / or AP and / or TP (e.g., inhibiting sheep RBC lysis in 20% human serum), wherein IC50 50 The value is from about 1 nM to about 150 nM, for example, any one of about 100 nM to about 150 nM, about 1 nM to about 100 nM, about 50 nM to about 100 nM, about 40 nM to about 120 nM, about 60 nM to about 120 nM, about 70 nM to about 100 nM, about 80 nM to about 100 nM, or about 80 nM to about 90 nM. In some embodiments, the anti-C5 antibody partially inhibits about 100% of sheep RBC lysis (e.g., in 20% human serum).

[0289] In some implementations, the anti-C5 antibody partially inhibits AP (e.g., inhibits rabbit RBC lysis in 20% human serum), where IC50... 50The value is at most about 150 nM, for example, any or lower of about 120 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM. In some embodiments, the anti-C5 antibody partially inhibits AP (e.g., inhibits rabbit RBC lysis in 20% human serum), wherein IC50... 50 The value is from about 1 nM to about 150 nM, for example, about 100 nM to about 150 nM, about 1 nM to about 100 nM, about 50 nM to about 100 nM, about 40 nM to about 120 nM, about 60 nM to about 120 nM, about 70 nM to about 100 nM, about 70 nM to about 90 nM, about 80 nM to about 100 nM, or about 80 nM to about 90 nM. In some embodiments, the anti-C5 antibody partially inhibits up to about 60% (e.g., up to about 50%, 40%, 30%, 20% or less) of rabbit RBC lysis (e.g., in 20% human serum).

[0290] In some implementations, the anti-C5 antibody partially inhibits (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) one or more of IgM-induced, mannan-induced, and LPS-induced C5b-9 deposition.

[0291] Methods for testing the activity of anti-C5 antibodies or their antigen-binding fragments (e.g., inhibition of terminal pathways) are well known in the art, including but not limited to sheep RBC lysis assays (e.g., induced by human serum), IgM-induced, mannan-induced, or LPS-induced C5b-9 deposition assays. See, for example, the assays in US20220204602, US20220177556, US11578137, WO2022134047, and PCT / US2023 / 063305. See also Examples 2-3 herein.

[0292] connector

[0293] The multispecific constructs and isolated anti-C5 antibody constructs described herein may include one or more optional linkers. For example, in some embodiments, the first antibody portion specifically recognizing FD is an scFv, and one or more linkers are included between VH and VL. In some embodiments, the second antibody portion specifically recognizing complement proteins is an scFv, and one or more linkers are included between VH and VL. In some embodiments, the linkers within the anti-FD scFv and / or the complement protein-targeting scFv independently comprise the amino acid sequence of any one of SEQ ID NO: 76, 89-95, and 110. In some embodiments, the multispecific construct includes one or more linkers between the first antibody portion (e.g., scFv) specifically recognizing FD and the second antibody portion (e.g., scFv) specifically recognizing complement proteins. The length, flexibility, and / or other characteristics of one or more linkers used in the multispecific construct or isolated anti-C5 antibody construct may affect the properties of the multispecific construct or isolated anti-C5 antibody construct, including but not limited to affecting the affinity, specificity, or affinity for one or more specific antigens or epitopes. For example, a longer linker can be chosen to ensure that two adjacent domains do not interfere with each other spatially. In some embodiments, the linker (e.g., a peptide linker) contains flexible residues (e.g., glycine (G) and serine (S)) such that a first antibody portion specifically recognizing FD and a second antibody portion specifically recognizing complement proteins can move freely 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.

[0294] Other linker considerations include the impact on the physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (various degrees of stability and planned degradation), rigidity, flexibility, immunogenicity, regulation of antibody binding, and ability to be incorporated into micelles or liposomes.

[0295] Peptide linkers can have naturally occurring or non-natural sequences. For example, sequences derived from the hinge region of heavy-chain-only antibodies can be used as linkers. See, for example, WO1996 / 034103.

[0296] The peptide linker can have any suitable length. In some embodiments, the length of the peptide linker is at least about 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 amino acids or more. In some embodiments, the length of the peptide linker is no more than about 200, 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 amino acids or fewer. In some embodiments, the length of the peptide linker is any of the following: 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, 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.

[0297] In some embodiments, the peptide linker does not contain any polymerization activity. Peptide linkers are known in the art to contain no promotion of secondary structure, and their characteristics are described, for example, by Dall'Acqua 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 structure. The bonding of the domains to each other can be provided, for example, by genetic engineering. Methods for preparing fused and operatively linked bispecific single-stranded constructs and expressing these constructs 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, NY 1989 and 1994; or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0298] Peptide linkers can be stable linkers that cannot be cleaved by proteases, especially matrix metalloproteinases (MMPs).

[0299] In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G)n, where n is at least one integer; and glycine-serine polymers (including, for example, (GS)n, where n is at least one integer); glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, the linker is a GS linker.

[0300] In some embodiments, a first antibody portion that specifically recognizes FD and a second antibody portion that specifically recognizes complement protein are linked together by a sufficiently long linker such that the antigen-binding domains of the first and second antibody portions can fold in a manner that allows binding to FD and complement protein. In some embodiments, the linker comprises the amino acid sequence shown in any one of SEQ ID NO: 76, 89-95, and 110.

[0301] Fc structural domain

[0302] In some embodiments, the multispecific construct or isolated anti-C5 antibody construct includes an Fc domain. In some embodiments, the multispecific construct or isolated anti-C5 antibody construct comprises two polypeptide chains, each containing a subunit of the Fc domain, and the two polypeptide chains are dimerized by the Fc domain (see, for example, [link to relevant documentation]). Figure 3B In some embodiments, the first portion (e.g., the first antibody portion) or the second portion (e.g., the second antibody portion) described herein may also (or additionally) include an Fc domain. For example, the first antibody portion may be a full-length anti-FD antibody, and / or the second antibody portion may be a full-length anti-C2 or anti-C5 antibody.

[0303] In some embodiments, the multispecific construct containing the Fc domain or the isolated anti-C5 antibody construct has one or more of the following properties: i) an in vivo half-life extended by at least about 20% (e.g., an extension of at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold or more); and ii) an activity at least about 20% stronger (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold or more) than the same multispecific construct or the same isolated anti-C5 antibody construct without the Fc domain (e.g., different antibody moieties linked by a G4S linker).

[0304] In some embodiments, the Fc domain is an Fc effector domain, i.e., an Fc domain with some or all of the effector functions, including, for example, complement and ADCC functions. In some embodiments, the Fc effector domain is the Fc region of IgG1 or IgG3.

[0305] In some implementations, one or more amino acid modifications may be introduced into the Fc domain of the antibody moiety, thereby creating an Fc domain variant. The Fc domain variant may contain a human Fc domain sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) with amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0306] In some implementations, the Fc domain has some, but not all, effector functions that make the Fc domain a desirable candidate for applications where the in vivo half-life of the antibody moiety is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental.

[0307] In vitro and / or in vivo cytotoxicity assays can be performed to analyze the CDC and / or ADCC activity of the Fc domain. For example, an Fc receptor (FcR) binding assay can be performed to determine whether the antibody has FcγR binding (and therefore may have ADCC activity), and / or retains FcRn binding ability. NK cells, the main cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet. Annu. Rev. Immunol. Table 2 on page 464 of 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al.). Proc. Nat'l Acad. Sci. USA83:7059-7063 (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 may be used (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); and CytoTox 96). ® Non-radioactive cytotoxicity assays (Promega, Madison, WI). Effector cells that can be used for this type of assay include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, assays can be performed in vivo, such as in animal models, for example, Clynes et al. Proc. Nat'l Acad. Sci. USA The ADCC activity of the molecule of interest was assessed in animal models disclosed in 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB, et al.). , Int'l. Immunol. 18(12):1759-1769 (2006)).

[0308] Antibodies with reduced effector function include those with substitutions at one or more of the Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with residues 265 and 297 substituted for alanine (US Patent No. 7,332,581). In some embodiments, the Fc domain of the multispecific construct or isolated anti-C5 antibody construct does not contain mutations that reduce its effector function, such as one or more mutations described herein. In some embodiments, the Fc domain of the multispecific construct or isolated anti-C5 antibody construct contains one or more of these mutations.

[0309] Certain antibody variants that improve or reduce binding to FcR have been described. See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312; and Shields et al. , J. Biol. Chem. 9(2): 6591-6604 (2001).

[0310] In some embodiments, the Fc domain is an IgG1 Fc domain. In some embodiments, the IgG1 Fc domain does not contain the L234A mutation and / or the L235A mutation. In some embodiments, the IgG1 Fc domain contains the L234A mutation and / or the 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 containing the amino acid sequence of SEQ ID NO: 96. In some embodiments, the Fc domain is an IgG4 Fc domain containing the S228P, F234A, and / or L235A mutation. In some embodiments, the Fc domain is an IgG4 Fc fragment containing the S228P mutation, for example, containing the amino acid sequence of SEQ ID NO: 97. In some implementations, the Fc domain is an IgG4 Fc fragment containing the S228P / M428L / N434A (“PLA”) triple mutation, such as the amino acid sequence of SEQ ID NO: 41.

[0311] In some embodiments, the antibody moiety (or multispecific construct or isolated anti-C5 antibody construct) comprises an Fc domain with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333 and / or 334 (EU numbers of residues) in the Fc domain.

[0312] In some embodiments, the Fc domain is modified to alter (i.e., improve or reduce) C1q binding and / or complement-dependent cytotoxicity (CDC), as described in, for example, U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al. , J. Immunol. As described in 164: 4178-4184 (2000).

[0313] In some embodiments, the antibody moiety (or multispecific construct or isolated anti-C5 antibody construct) includes a variant Fc domain comprising one or more amino acid substitutions that alter the half-life and / or change the binding to the neonatal Fc receptor (FcRn). The increased half-life and binding to the neonatal Fc receptor (FcRn, which is responsible for transferring maternal IgG to the fetus) (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. Antibodies that improve binding to FcRn (24:249 (1994)) are described in US2005 / 0014934A1 (Hinton et al.). Those antibodies contain an Fc domain having one or more substitutions that alter the binding of the Fc domain to FcRn. Such Fc variants include those with substitutions at one or more Fc domain residues (e.g., substitution at Fc domain residue 434 (US Patent No. 7,371,826)).

[0314] For other examples of variants of the Fc domain, see also Duncan and Winter. Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0315] In some embodiments, the Fc domain comprises the amino acid sequence of any one of SEQ ID NO: 41, 96, and 97. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 41.

[0316] In some embodiments, the first antibody portion is linked to the second antibody portion via an Fc domain, such as an Fc domain comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the Fc domain is side-linked with linkers (e.g., SEQ ID NO: 94) at one or both ends of its sequence and then located between the first and second antibody portions. In some embodiments, the Fc domain is side-linked with linkers (e.g., SEQ ID NO: 94) at both ends of its sequence (“side-linked Fc domain linkers”) and then located between the first and second antibody portions. In some embodiments, the side-linked Fc domain linkers comprise the amino acid sequence of SEQ ID NO: 105.

[0317] Antibody binding affinity

[0318] The binding specificity of the multispecific constructs described herein and the antibody moiety of the isolated anti-C5 antibody construct can be determined experimentally using methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, EIA, BIACORE™, and peptide scanning.

[0319] In some implementations, the anti-FD antibody partially and specifically binds to FD (e.g., human FD), where K D It is about 10 -7 M to approximately 10 -12 M, for example, any of the following: approximately 10 -7 M to approximately 10 -10 M, approximately 10 -8 M to approximately 10 -11 M, approximately 10 -9 M to approximately 10 -11 M, approximately 10 -8 M to approximately 10 -10 M, approximately 10 -8 M to approximately 10 -9 M, approximately 10 -9 M to approximately 10 -10 M, or about 10 -10 M to approximately 10 -11 M. In some embodiments, the anti-FD antibody partially and specifically binds to FD (e.g., human FD), where K D It is about 10 -8 M to approximately 10 -12 M, or approximately 1×10 -10 M to approximately 5 × 10 -10 M.

[0320] In some implementations, the anti-C2 antibody partially and specifically binds to C2 (e.g., human C2), where K D It is about 10 -7 M to approximately 10 -12 M, for example, any of the following: approximately 10 -7 M to approximately 10 -10 M, approximately 10 -8 M to approximately 10 -11 M, approximately 10 -9 M to approximately 10 -11 M, approximately 10 -8 M to approximately 10 -10 M, approximately 10 -8 M to approximately 10 -9 M, approximately 10 -9 M to approximately 10 -10 M or approximately 10 -10 M to approximately 10 -11 M. In some embodiments, the anti-C2 antibody partially and specifically binds to C2 (e.g., human C2), where K D It is approximately 1×10 -10 M to approximately 5 × 10 -10 M.

[0321] In some implementations, the anti-C5 antibody partially and specifically binds to C5 (e.g., human C5), where K D It is about 10 -7 M to approximately 10 -12 M, for example, any of the following: approximately 10 -7 M to approximately 10 -10 M, approximately 10 -8 M to approximately 10 -11 M, approximately 10 -9 M to approximately 10 -11 M, approximately 10 -8 M to approximately 10 -10 M, approximately 10 -8 M to approximately 10 -9 M, approximately 10 -9 M to approximately 10 -10 M or approximately 10 -10 M to approximately 10 -11 M. In some implementations, the anti-C5 antibody partially and specifically binds to C5 (e.g., human C5), where K D It is approximately 1×10 -9 M to approximately 5 × 10 -9 M.

[0322] Chimeric antibodies or humanized antibodies

[0323] In some embodiments, one or more antibody portions of the multispecific construct or isolated anti-C5 antibody construct of this application are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and in Morrison et al. Proc. Natl. Acad. Sci. USA In 81:6851-6855 (1984), chimeric antibodies comprise a non-human variable region (e.g., a variable region derived from a mouse) and a human constant region. In some embodiments, chimeric antibodies are "class-switched" antibodies, where the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include their antigen-binding fragments.

[0324] In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains, wherein the HVR, such as the CDR (or a portion thereof), is derived from the non-human antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Optionally, the humanized antibody will also contain at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the HVR residues are derived from the antibody therein) to, for example, restore or enhance antibody specificity or affinity.

[0325] Humanized antibodies and their preparation methods have been reviewed, for example, by Almagro and Fransson. Front. Biosci. 13:1619-1633 (2008), and further described in, for example, Riechmann et al. Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (Description of SDR (a-CDR) porting); Padlan, Mol. Immunol. 28:489-498 (1991) (Description of “Surface Remodeling”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing the “FR reorganization”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka, Br. J. Cancer, 83:252-260 (2000) (Describes the “guided selection” approach to FR reorganization).

[0326] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using the "best-fit" method (see, for example, Sims et al.) , J. Immunol. 15 1 :2296 (1993)); Frame regions derived from common sequences of specific subgroups of the variable regions of human antibody light or heavy chains (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA , 8 9 :4285 (1992); and Presta et al., J. Immunol. , 151:2623 (1993)); human maturation (somatic mutation) framework region or human germline framework region (see, for example, Almagro and Fransson, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and the frame regions obtained by screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0327] Human antibodies

[0328] In some embodiments, one or more antibody portions of the multispecific construct and the isolated anti-C5 antibody construct of this application are human antibodies (referred to as human domain antibodies, or human dAbs). Human antibodies can be produced using various techniques known in the art. Human antibodies are broadly described 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. In 47(4):912-21 (2010). Transgenic mice or rats capable of producing fully human single-domain antibodies (or dAbs) are known in the art. See, for example, US20090307787A1, US Patent No. 8,754,287, US20150289489A1, US20100122358A1 and WO2004049794.

[0329] Human antibodies (e.g., human dAbs) can be prepared by administering an immunogen to transgenic animals modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen stimulation. Such animals typically contain all or part of the human immunoglobulin loci, which replace endogenous immunoglobulin loci or are located extrachromosomally or randomly integrated into the animal's chromosome. In such transgenic mice, endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg. Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; and U.S. Patent No. 5,770,429, which describes... Technology; US Patent No. 7,041,870 describes the KM MOUSE ® The technology; and U.S. Patent Application Publication No. US 2007 / 0061900, which describes Technology. The human variable region derived from complete antibodies produced by such animals can be further modified, for example, by combining it with different human constant regions.

[0330] Human antibodies (e.g., human dAbs) can also be prepared using hybridoma-based methods. Human myeloma and mouse-human xenograft cell lines for generating human monoclonal antibodies have been described (see, for example, Kozbor). J. Immunol. ,133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications Pages 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al. J. Immunol ., 147: 86 (1991)). Human antibodies produced via human B-cell hybridoma technology were also described by 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 (description of generating monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue , 26(4):265-268 (2006) (Description of those methods for human-human hybridomas). Human hybridoma technology (tri-source hybridoma technology) is also described by 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)

[0331] Human antibodies (e.g., human dAbs) can also be generated by isolating variable domain sequences from Fv clones selected from a human phage display library. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0332] Replacement, insertion and missing variants

[0333] In some embodiments, the multispecific constructs or isolated anti-C5 antibody constructs described herein include antibody variants containing one or more amino acid substitutions. Sites of interest for substitution mutagenesis include HVR (or CDR) and FR. Conserved substitutions are shown in... Table 2 Under the heading "Preferred Substitutions," the most significant changes are provided. Table 2 Under the heading “Exemplary Substitutions”, and further described below with reference to the amino acid side chain category, amino acid substitutions can be introduced into antibodies of interest, and products can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0334] Table 2. Amino acid substitutions

[0335] Amino acids can be grouped according to the properties of their common side chains: (1) hydrophobic: leucine, 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 affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0336] Non-conservative replacement would require replacing members of one of these categories with members of another category.

[0337] One type of substitution variant involves replacing one or more hypervariable residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further research will have modifications (e.g., improvements) to certain biological properties relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will retain some biological properties of the parent antibody substantially. Exemplary substitution variants are affinity-matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. In short, one or more HVR residues are mutated, the variant antibody is displayed on a phage, and it is screened for specific biological activities (e.g., binding affinity).

[0338] HVR can be modified (e.g., substituted), for example, to improve antibody affinity. HVR "hotspots," which are residues encoded by codons that undergo mutations at a high frequency during somatic cell maturation (see, for example, Chowdhury), can also be modified. Methods Mol. Biol . 207:179-196 (2008)), and / or SDR (a-CDR) make such changes, wherein the resulting variant VH or VL is tested for binding affinity. Affinity maturation via the construction and reselection of secondary libraries has been described, for example, by Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene for maturation selection by any of a variety of methods (e.g., error-prone PCR, strand 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 approach to introducing diversity involves an HVR-directed pathway, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. Specifically, CDR-H3 and CDR-L3 are often targeted.

[0339] In some implementations, substitutions, insertions, or deletions can occur within one or more HVRs, as long as such changes do not materially reduce the antibody's ability to bind to the antigen. For example, conserved changes to the HVR that do not materially reduce binding affinity can be made (e.g., conserved substitutions as described herein). Such changes can be made outside the HVR "hotspot" or CDR. In some implementations of the variant VHH sequences provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0340] One method that can be used to identify residues or regions in antibodies that can serve as mutagenic targets is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989). Science As described in 244:1081-1085. In this method, groups of residues or target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and antigen. Such contact residues and adjacent residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0341] Amino acid sequence insertions include N-terminal and / or C-terminal fusions of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a peptide that extends the serum half-life of the antibody.

[0342] Glycosylation variants

[0343] In some embodiments, one or more antibody portions of the multispecific construct or isolated anti-C5 antibody construct of this application are modified to increase or decrease the degree of glycosylation of said construct. The addition or deletion of glycosylation sites on the antibody can be conveniently achieved by altering the amino acid sequence to generate or remove one or more glycosylation sites.

[0344] When an antibody moiety (or multispecific construct, or isolated anti-C5 antibody construct) contains an Fc domain, the carbohydrate linked to it can be altered. Native antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, generally linked to the C-terminal of the Fc domain via N-bonds. H 2. Asn297 of the structural domain. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibody moiety can be modified to produce antibody variants with certain improved properties.

[0345] In some implementations, the antibody moiety (or multispecific construct, or isolated anti-C5 antibody construct) has a carbohydrate structure lacking fucose linked (directly or indirectly) to the Fc domain. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 within the glycan chain relative to the sum of all glycan structures linked to Asn 297 (e.g., complex, heterogeneous, and high-mannose structures), measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to the asparagine residue located approximately at position 297 (EU number of the Fc domain residue) within the Fc domain; however, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297 due to minor sequence variations in the antibody, i.e., between positions 294 and 300. Such fucosylation variants can have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Publicly disclosed examples involving "defucosylated" or "fucosylated" antibody variants include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US2004 / 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 defucosylation antibodies include Lec13 CHO cells with protein fucosylation defects (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312A1, Adams et al., especially Example 11); and gene knockout cell lines, such as α-1,6-fucosyltransferase gene. FUT8 Gene knockout CHO cells (see, for example, Yamane-Ohnuki et al.) Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al. Biotechnol. Bioeng ., 94(4):680-688 (2006); and WO2003 / 085107).

[0346] In some embodiments, the antibody moiety (or multispecific construct, or isolated anti-C5 antibody construct) has a bisected oligosaccharide, for example, wherein the biantennary oligosaccharide linked to the antibody Fc domain is bisected via GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc domain are also provided. Such antibody variants may have improved CDC function. This antibody variant is described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0347] Cysteine-engineered antibody variants

[0348] In some embodiments, it may be desirable to produce cysteine-engineered antibody moieties, such as "thioMAb," wherein one or more residues of one or more antibodies in the multispecific construct or isolated anti-C5 antibody construct described herein are replaced by cysteine ​​residues. In a particular embodiment, the substituted residues are located at an accessible site on the antibody. By replacing those residues with cysteine, a reactive thiol group is thereby positioned at an accessible site on the antibody and can be used to conjugate the antibody to other moieties (e.g., a pharmaceutical moieties or a linker-pharmaceutical moieties) to produce immunoconjugates, as further described herein. In some embodiments, cysteine ​​may be used to replace any or more of the following residues: A118 (EU number) of the heavy chain; and S400 (EU number) of the Fc domain of the heavy chain. Cysteine-engineered antibody moieties can be produced, for example, as described in U.S. Patent No. 7,521,541.

[0349] III. Isolated anti-C5 antibody construct

[0350] Isolated antibody constructs (anti-C5 antibody constructs) are also provided, which contain any of the aforementioned anti-C5 antibody moieties. Any properties of the aforementioned anti-C5 antibody moieties (e.g., binding affinity, biological properties) also apply to the isolated anti-C5 antibody constructs described herein.

[0351] In some embodiments, an isolated anti-C5 antibody construct is provided, the antibody construct comprising an antibody moiety specifically recognizing C5 (anti-C5 antibody moiety), wherein the anti-C5 antibody moiety comprises: H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, H-CDR3 containing the amino acid sequence of SEQ ID NO: 33, L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36; and wherein the anti-C5 antibody moiety comprises: i) VH, the VH comprising the amino acid sequence of SEQ ID NO: 37, or thereof combined with SEQ ID NO: 37. 37 has a variant having at least about 80% (e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid homology; and ii) VL, said VL comprising the amino acid sequence of SEQ ID NO: 38, or a variant thereof having at least about 80% (e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid homology with SEQ ID NO: 38. In some embodiments, an anti-C5 antibody portion, or an anti-C5 antibody construct comprising an anti-C5 antibody portion comprising a VH containing the amino acid sequence of SEQ ID NO: 37 and a VL containing the amino acid sequence of SEQ ID NO: 38. In some implementations, the anti-C5 antibody portion is selected from the group consisting of: full-length antibody, Fab, Fab', F(ab)2, F(ab')2, scFv, or combinations thereof.

[0352] In some embodiments, the anti-C5 antibody portion is a full-length antibody (“anti-C5 full-length antibody”). In some embodiments, the anti-C5 full-length antibody comprises an Fc fragment derived from human IgG4. In some embodiments, the Fc fragment comprises the amino acid sequence of any one of SEQ ID NO: 41, 96, and 97 (e.g., SEQ ID NO: 41). Therefore, in some embodiments, an anti-C5 full-length antibody, or an anti-C5 antibody construct comprising the anti-C5 full-length antibody, wherein the anti-C5 full-length antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 98 and a light chain containing the amino acid sequence of SEQ ID NO: 99.

[0353] In some embodiments, the anti-C5 antibody portion is an anti-C5 scFv. Therefore, in some embodiments, an anti-C5 scFv, or an anti-C5 antibody construct comprising anti-C5 scFv, is provided, wherein the anti-C5 scFv comprises the amino acid sequence of SEQ ID NO: 39 or 40.

[0354] In some embodiments, the isolated anti-C5 antibody construct is multispecific. In some embodiments, the isolated anti-C5 antibody construct also includes an antibody portion that specifically recognizes FD (anti-FD antibody portion) (hereinafter also referred to as "isolated anti-C5 / anti-FD antibody construct"). Any of the anti-FD antibody portions described above may be used herein. Any properties of the anti-FD antibody portions and the anti-FD / anti-C5 multispecific construct described above (e.g., binding affinity, biological properties) also apply to the isolated anti-C5 / anti-FD antibody construct described herein.

[0355] In some embodiments, an isolated anti-C5 antibody construct comprising an anti-C5 antibody moiety (e.g., a full-length antibody, Fab, or scFv) is provided, wherein the isolated anti-C5 antibody construct further comprises an anti-FD antibody moiety; and wherein the anti-C5 antibody moiety comprises: H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, H-CDR3 containing the amino acid sequence of SEQ ID NO: 33, L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, the anti-C5 antibody portion comprises: i) VH, said VH comprising the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid homology with SEQ ID NO: 37; and ii) VL, said VL comprising the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least about 80% (e.g., at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) amino acid homology with SEQ ID NO: 38. In some embodiments, the anti-C5 antibody portion comprises a VH containing the amino acid sequence of SEQ ID NO: 37 and a VL containing the amino acid sequence of SEQ ID NO: 38. In some embodiments, the anti-C5 antibody portion is an anti-C5 scFv. In some embodiments, the anti-C5 scFv comprises the amino acid sequence of SEQ ID NO: 39 or 40. In some embodiments, the anti-FD antibody portion comprises: H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, H-CDR3 containing the amino acid sequence of SEQ ID NO: 3, L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-FD antibody portion comprises a VH containing the amino acid sequence of SEQ ID NO: 7 and a VL containing the amino acid sequence of SEQ ID NO: 8. In some implementations, the anti-FD antibody portion is scFv (anti-FDscFv).In some embodiments, the anti-FD scFv comprises the amino acid sequence of SEQ ID NO: 9 or 10. In some embodiments, the anti-C5 antibody moiety is the anti-C5 scFv, and the anti-C5 scFv is directly linked to the anti-FD scFv to form a fusion peptide. In some embodiments, the anti-C5 antibody moiety is the anti-C5 scFv, and the anti-C5 scFv is linked to the anti-FD scFv via a peptide linker and / or an Fc domain to form a fusion peptide. Any linker and Fc domain described in Part II may be used herein. In some embodiments, the linker within the anti-FD scFv and / or the anti-C5 scFv, and the linker between the anti-C5 scFv and the anti-FD scFv, independently comprises the amino acid sequence of any one of SEQ ID NO: 76, 93, and 95. In some embodiments, the anti-C5 scFv and the anti-FD scFv are linked via a linker comprising the amino a...

Claims

1. A multispecific construct comprising a first antibody portion that specifically recognizes complement factor D ("FD") and a second antibody portion that specifically recognizes components of the complement pathway.

2. The multispecific construct of claim 1, wherein the first antibody portion that specifically recognizes the FD comprises an immunoglobulin heavy chain variable domain ("VH1") and an immunoglobulin light chain variable domain ("VL1").

3. The multispecific construct of claim 2, wherein: (i) The VH1 comprises: heavy chain CDR1 ("H-CDR1"), the H-CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising up to 3 amino acid variations; heavy chain CDR2 ("H-CDR2"), the H-CDR2 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof comprising up to 3 amino acid variations; and heavy chain CDR3 ("H-CDR3"), the 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 VL1 comprises: a light chain CDR1 ("L-CDR1"), the 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"), the 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"), the L-CDR3 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising up to 3 amino acid variations.

4. The multispecific construct of claim 2 or 3, wherein the VH1 comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 7; and the VL1 comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 80% amino acid homology with SEQ ID NO:

8.

5. The multispecific construct according to any one of claims 1 to 4, wherein the first antibody portion is scFv.

6. The multispecific construct of claim 5, wherein the first antibody portion is an scFv comprising the amino acid sequence of SEQ ID NO: 9 or 10.

7. The multispecific construct according to any one of claims 1 to 6, wherein the second antibody portion that specifically recognizes the complement pathway component comprises an immunoglobulin heavy chain variable domain ("VH2") and an immunoglobulin light chain variable domain ("VL2").

8. The multispecific construct of claim 7, wherein the second antibody portion is scFv.

9. The multispecific construct according to any one of claims 1 to 8, wherein the complement pathway component is complement component 2 ("C2").

10. The multispecific construct of claim 9, wherein: (i) The VH2 comprises: H-CDR1, which comprises the amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising up to 3 amino acid variations; H-CDR2, which comprises the amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising up to 3 amino acid variations; and H-CDR3, which comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to 3 amino acid variations; and The VL2 comprises: L-CDR1, which comprises the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising up to 3 amino acid variations; L-CDR2, which comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof comprising up to 3 amino acid variations; and L-CDR3, which comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof comprising up to 3 amino acid variations; or (ii) The VH2 comprises: H-CDR1, which comprises the amino acid sequence of SEQ ID NO: 21 or a variant thereof comprising up to 3 amino acid variations; H-CDR2, which comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof comprising up to 3 amino acid variations; and H-CDR3, which comprises the amino acid sequence of SEQ ID NO: 23 or a variant thereof comprising up to 3 amino acid variations; and The VL2 comprises: L-CDR1, which comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof comprising up to 3 amino acid variations; L-CDR2, which comprises the amino acid sequence of SEQ ID NO: 25 or a variant thereof comprising up to 3 amino acid variations; and L-CDR3, which comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof comprising up to 3 amino acid variations.

11. The multispecific construct of claim 9 or 10, wherein: (i) The VH2 comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 17; and the VL2 comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 18; or (ii) The VH2 comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 27; and the VL2 comprises the amino acid sequence of SEQ ID NO: 28 or a variant thereof having at least about 80% amino acid homology with SEQ ID NO:

28.

12. The multispecific construct according to any one of claims 9 to 11, wherein the second antibody portion is an scFv comprising the amino acid sequence of any one of SEQ ID NO: 19, 20, 29 and 30.

13. The multispecific construct according to any one of claims 1 to 8, wherein the complement pathway component is complement component 5 ("C5").

14. The multispecific construct of claim 13, wherein: (i) The VH2 comprises: H-CDR1, which comprises the amino acid sequence of SEQ ID NO: 31 or a variant thereof comprising up to 3 amino acid variations; H-CDR2, which comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof comprising up to 3 amino acid variations; and H-CDR3, which comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof comprising up to 3 amino acid variations; and (ii) The VL2 comprises: L-CDR1, which comprises the amino acid sequence of SEQ ID NO: 34 or a variant thereof comprising up to 3 amino acid variations; L-CDR2, which comprises the amino acid sequence of SEQ ID NO: 35 or a variant thereof comprising up to 3 amino acid variations; and L-CDR3, which comprises the amino acid sequence of SEQ ID NO: 36 or a variant thereof comprising up to 3 amino acid variations.

15. The multispecific construct of claim 13 or 14, wherein: (i) The VH2 comprises the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 37; and (ii) The VL2 comprises the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least about 80% amino acid homology with SEQ ID NO:

38.

16. The multispecific construct according to any one of claims 13 to 15, wherein the second antibody portion is an scFv comprising the amino acid sequence of SEQ ID NO: 39 or 40.

17. The multispecific construct according to any one of claims 1 to 16, wherein: (i) The first antibody portion is directly connected to the second antibody portion; or (ii) The first antibody portion is linked to the second antibody portion via a peptide linker.

18. The multispecific construct according to any one of claims 1 to 17, wherein the multispecific construct comprises the amino acid sequence of any one of the following: SEQ ID NO: 42-50, 63, 67, 71, 73, 77, 81, 85, 87, 106, and 108.

19. The multispecific construct of any one of claims 1 to 16, wherein the first antibody portion is linked to the second antibody portion via an Fc domain.

20. The multispecific construct of claim 19, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:

41.

21. The multispecific construct of claim 19 or 20, wherein the multispecific construct comprises the amino acid sequence of any of the following: SEQ ID NO: 51-55, 59-62, 65, 69, 72, 74, 75, 78, 82, 86 and 88.

22. An isolated nucleic acid or a vector containing the same, said isolated nucleic acid or vector encoding the multispecific construct according to any one of claims 1 to 21.

23. The isolated nucleic acid or vector as described in claim 22, wherein: (i) The vector is a viral vector; or (ii) The isolated nucleic acid is mRNA.

24. A pharmaceutical composition comprising any one of claims 1 to 21, an isolated nucleic acid as described in claim 22 or 23, or a vector as described in claim 22 or 23, and optionally a pharmaceutically acceptable vector.

25. A method of treating an individual with a complement-mediated disease, the method comprising administering to the individual an effective amount of the pharmaceutical composition of claim 24.

26. The method of claim 25, wherein the complement-mediated disease is an ocular disease.

27. An isolated antibody construct (anti-C5 antibody construct), said isolated antibody construct comprising an antibody moiety specifically recognizing C5 (anti-C5 antibody moiety), wherein said anti-C5 antibody moiety comprises: H-CDR1 containing the amino acid sequence of SEQ ID NO: 31, H-CDR2 containing the amino acid sequence of SEQ ID NO: 32, H-CDR3 containing the amino acid sequence of SEQ ID NO: 33, L-CDR1 containing the amino acid sequence of SEQ ID NO: 34, L-CDR2 containing the amino acid sequence of SEQ ID NO: 35, and L-CDR3 containing the amino acid sequence of SEQ ID NO: 36; and wherein said anti-C5 antibody moiety comprises: i) VH, said VH containing the amino acid sequence of SEQ ID NO: 37, or a variant thereof having at least about 80% amino acid homology with SEQ ID NO: 37; and ii) VL, said VL containing the amino acid sequence of SEQ ID NO: 38, or a variant thereof having at least about 80% amino acid homology with SEQ ID NO:

37. NO:38 is a variant with at least approximately 80% amino acid homology.

28. The isolated anti-C5 antibody construct of claim 27, wherein the anti-C5 antibody portion is selected from the group consisting of: full-length antibody, Fab, Fab', F(ab)2, F(ab')2, scFv, and combinations thereof.

29. The isolated anti-C5 antibody construct of claim 27 or 28, wherein the anti-C5 antibody portion is a full-length antibody ("anti-C5 full-length antibody").

30. The isolated anti-C5 antibody construct of claim 29, wherein the full-length anti-C5 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 98 and a light chain containing the amino acid sequence of SEQ ID NO:

99.

31. The isolated anti-C5 antibody construct according to any one of claims 27 to 29, wherein the anti-C5 antibody portion is anti-C5 scFv.

32. The isolated anti-C5 antibody construct of claim 31, wherein the anti-C5 scFv comprises the amino acid sequence of SEQ ID NO: 39 or 40.

33. The isolated anti-C5 antibody construct according to any one of claims 27 to 32, wherein the isolated anti-C5 antibody construct is multispecific.

34. The isolated anti-C5 antibody construct of claim 33, wherein the isolated anti-C5 antibody construct further comprises an antibody portion that specifically recognizes FD (anti-FD antibody portion).

35. The isolated anti-C5 antibody construct of claim 34, wherein the anti-FD antibody portion comprises H-CDR1 containing the amino acid sequence of SEQ ID NO: 1, H-CDR2 containing the amino acid sequence of SEQ ID NO: 2, H-CDR3 containing the amino acid sequence of SEQ ID NO: 3, L-CDR1 containing the amino acid sequence of SEQ ID NO: 4, L-CDR2 containing the amino acid sequence of SEQ ID NO: 5, and L-CDR3 containing the amino acid sequence of SEQ ID NO:

6.

36. The isolated anti-C5 antibody construct of claim 34 or 35, wherein the anti-FD antibody portion comprises VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO:

8.

37. The isolated anti-C5 antibody construct according to any one of claims 34 to 36, wherein the anti-FD antibody portion is scFv (anti-FD scFv).

38. The isolated anti-C5 antibody construct of claim 37, wherein the anti-FD scFv comprises the amino acid sequence of SEQ ID NO: 9 or 10.

39. The isolated anti-C5 antibody construct of claim 37 or 38, wherein the anti-C5 antibody portion is anti-C5scFv, and wherein: (i) The anti-C5 scFv is directly linked to the anti-FD scFv to form a fusion polypeptide; or (ii) The anti-C5 scFv is linked to the anti-FD scFv via a peptide linker and / or an Fc domain to form a fusion polypeptide.

40. The isolated anti-C5 antibody construct of claim 37 or 38, wherein the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 46-50, 59-62, 71-75, and 85-88.

41. The isolated anti-C5 antibody construct according to any one of claims 27 to 33, wherein the isolated anti-C5 antibody construct further comprises complement factor H (FH) or a functional fragment thereof.

42. The isolated anti-C5 antibody construct of claim 41, wherein the anti-C5 antibody construct inhibits C3 activation.

43. The isolated anti-C5 antibody construct of claim 41 or 42, wherein the functional fragment of FH comprises short common repeat (SCR) domains 1-4 of FH.

44. The isolated anti-C5 antibody construct of claim 43, wherein the functional fragment of FH comprises the amino acid sequence of SEQ ID NO:

101.

45. The isolated anti-C5 antibody construct according to any one of claims 41 to 44, wherein the FH or a functional fragment thereof is fused to the N-terminus or C-terminus of the anti-C5 antibody moiety.

46. ​​The isolated anti-C5 antibody construct of any one of claims 41 to 44, wherein the anti-C5 antibody portion is a full-length anti-C5 antibody, wherein the isolated anti-C5 antibody construct comprises a first FH or a functional fragment thereof and a second FH or a functional fragment thereof, wherein the first FH or a functional fragment thereof is fused to the C-terminus of a first heavy chain of the full-length anti-C5 antibody, and the second FH or a functional fragment thereof is fused to the C-terminus of a second heavy chain of the full-length anti-C5 antibody.

47. The isolated anti-C5 antibody construct of claim 46, wherein each heavy chain fused with FH or a functional fragment thereof contains the amino acid sequence of SEQ ID NO: 100, and each light chain of the full-length anti-C5 antibody contains the amino acid sequence of SEQ ID NO:

99.

48. An isolated nucleic acid or a vector comprising the isolated nucleic acid or the vector encoding the isolated anti-C5 antibody construct of any one of claims 27 to 47.

49. The vector of claim 48, wherein the vector is a viral vector.

50. A host cell comprising the isolated nucleic acid of any one of claims 22, 23, and 488, or the vector of any one of claims 22, 23, 48, and 49.

51. A pharmaceutical composition comprising a multispecific construct according to any one of claims 1 to 21, an isolated nucleic acid according to any one of claims 22, 23 and 48, or a vector according to any one of claims 22, 23, 48 and 49, and optionally a pharmaceutically acceptable vector.

52. A method of treating an individual with a complement-mediated disease, the method comprising administering to the individual an effective amount of the pharmaceutical composition of claim 51.

53. The method of claim 52, 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, asthma, allergic asthma, lupus, ulcerative colitis, stroke, postoperative systemic inflammatory syndrome, chronic obstructive pulmonary disease (COPD), paroxysmal nocturnal hemoglobinuria (PNH) syndrome, myasthenia gravis, neuromyelitis optica (NMO), multiple sclerosis, delayed graft function, and antibodies. Mediated rejection, aHUS, central retinal vein occlusion (CRVO), central retinal artery occlusion (CRAO), bullous epidermolysis, sepsis, organ transplantation, inflammation, C3 glomerulonephropathy (C3G), membranous nephropathy, IgA nephropathy (IgAN), glomerulonephritis, thrombotic microangiopathy secondary to systemic lupus erythematosus (SLE-TMA), antineutrophil cytoplasmic antibody (ANCA) mediated vasculitis, Shiga toxin-induced HUS, antiphospholipid antibody-induced abortion, and any combination thereof.

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