Complement-inhibiting hybrid protein mutants and antibodies and their fusion proteins

Abstract

A hybrid protein containing a CFH functional unit and a DAF functional unit, a mutant thereof, a nucleic acid encoding the hybrid protein, a method for treating a disease or condition associated with the complement system, and a use thereof, as well as a fusion protein constructed based on the hybrid protein mutant and an antibody that specifically binds to complement C5. Further provided are a polynucleotide encoding the fusion protein, an expression vector, and a host cell, pharmaceutical compositions thereof, and a method for treating a disease associated with C5 protein, and a use thereof.

Description

[Technical Field]

[0001] The present invention relates to hybrid proteins and mutants thereof comprising a CFH functional unit and a DAF functional unit, nucleic acids encoding the hybrid proteins, and methods and uses thereof for treating diseases or disorders associated with the complement system; the present invention further relates to fusion proteins constructed based on the hybrid protein mutants and antibodies that specifically bind to complement C5. The present invention also relates to polynucleotides encoding the fusion proteins, expression vectors and host cells, and pharmaceutical compositions thereof, as well as methods and uses for treating C5 protein-related diseases. [Background technology]

[0002] The complement system is composed of over 30 soluble protein molecules and is part of the innate immune system. Its components include endogenous complement components, various regulatory factors, and complement receptors. The complement system is activated through three relatively independent but interrelated pathways, thereby exerting various biological effects, including opsonophagocytosis, cell lysis, mediation of inflammation, immunomodulation, and clearance of immune complexes, including enhanced phagocytosis, enhanced chemotaxis, increased vascular permeability, virus neutralization, cell lysis, and modulation of the immune response. While complement activation provides an important first line of defense against potential pathogens, complement activation, which promotes a protective inflammatory response, can also pose a potential threat to the host. Complement activation and its deposition on target structures can also indirectly cause cell or tissue destruction. Complement activation products that mediate tissue damage are generated at various points along the complement pathway. Inappropriate complement activation in host tissues plays an important role in the pathology of many autoimmune and inflammatory diseases.

[0003] Complement proteins, designated C1 through C9, are sequentially activated through three distinct pathways (classical, lectin, and alternative pathways) to elicit immune responses. Complement C5 is a key component of complement and plays an important role in inflammation and cell death. This protein consists of alpha and beta polypeptide chains linked by disulfide bonds. The activation peptide C5a is an anaphylatoxin that induces inflammatory responses in various cells via C5aR (CD88) and C5L2 (GPR77). The activation peptide C5a possesses potent contractile and chemotactic activities. The activation peptide C5a is derived from the alpha polypeptide by cleavage with C5 convertase. The C5b high molecular weight cleavage product can form a complex with the C6 complement component, which is the basis for the formation of the membrane attack complex (MAC), which contains additional complement components. The complement system can be highly cytotoxic to host cells if not properly regulated or overactivated.

[0004] Numerous studies have shown that complement C5 is associated with a variety of diseases, including those associated with the hemolytic activity of human complement. The anti-C5 monoclonal antibody eculizumab (Soliris®) exhibits high affinity for complement C5 and inhibits complement activation by inhibiting the cleavage of C5 into C5a / C5b and the associated formation of the membrane attack complex. Therefore, eculizumab exhibits an inhibitory effect on hemolysis and is therefore used as a therapeutic agent for paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Additionally, eculizumab is known as a therapeutic agent for generalized myasthenia gravis (gMG).

[0005] In vitro hemolysis inhibitory activity tests showed that eculizumab had a good hemolysis inhibitory effect on the classical pathway, but its activity in blocking the alternative complement pathway (AP) was insufficient, and therefore its efficacy did not adequately meet the needs of patients. For example, some PNH patients treated with eculizumab (Soliris) still cannot become transfusion-dependent due to extravascular hemolysis.

[0006] Additionally, because dysregulated or inappropriate activation of complement can lead to damage to host tissues, the complement system is also closely regulated by a series of proteins (complement regulatory proteins). Among them, the complement activation regulator (RCA) family of proteins is primarily responsible for complement regulation. RCA proteins include membrane proteins such as decay-accelerating factor (DAF; CD55), membrane cofactor protein (MCP; CD46), and complement receptor 1 (CR1; CD35), as well as fluid-phase proteins such as factor H (FH or CFH) and C4b-binding protein (C4BP). The structure of RCA proteins is composed of complement regulatory protein repeat (CCP) modules, of which two to four consecutive modules contribute to regulatory functions known as decay-accelerating activity (DAA) and cofactor activity (CFA). RCA proteins act by targeting C3 / C5 convertases, central enzymes in the complement pathway. RCA proteins bind to these convertases or their non-catalytic subunits, thereby inactivating them. DAA has been proposed as the irreversible dissociation of invertase into subunits after binding to the RCA protein, whereas CFA has been proposed as the cleavage and inactivation of invertase via recruitment of the serine protease factor I (FI) after RCA binds to the noncatalytic subunit (C3b / C4b), thereby preventing the formation of C3 convertase. Factor H is an opsonin and a ligand for complement receptors 2 and 3, and acts as a cofactor for factor I in the catalytic cleavage of C3b to iC3b, inhibiting C3b amplification. Factor H promotes the irreversible dissociation of the C3 convertase C3bBb in the alternative pathway and may also compete with factor B for C3b binding during the formation of the proconvertase. Factor H is a soluble complement regulator essential for protecting surfaces, including the extracellular matrix (ECM). FH can bind to the peptide hormone adrenomedullin and prevent its degradation. FH plays a role in managing cellular aging, stress or damage through its interactions with C-reactive protein, pentameric protein, DNA, histones, annexin II, malondialdehyde-acetaldehyde adducts of proteins, and oxidized lipids.FHL1 also has cofactor activity for factor I and C3bBb attenuation promoting activity (The Complement FactsBook. Edited by Scott Barnum and Theresa Schein. Copyright (C) 2018 Elsevier Ltd. All rights reserved. https: / / doi.org / 10.1016 / C2015-0-06595-9, Chapter 30).

[0007] DAF endogenously protects host cells from autologous complement attack by preventing the formation and accelerating the degradation of classical and alternative C3 convertases and C5 convertases, thereby inhibiting the cleavage of C3 and C5. When purified DAF is added to cells, it is incorporated into the cell membrane and can therefore exhibit functional activity. DAF has also been found to regulate T cell tolerance and thus negatively regulate several animal models of autoimmune disease (The Complement Facts Book, edited by Scott Barnum and Theresa Schein, Copyright (C) 2018 Elsevier Ltd. All rights reserved. https: / / doi.org / 10.1016 / C2015-0-06595-9, Chapter 25). Summary of the Invention

[0008] Therefore, there remains a need in the art to develop new proteins with stronger complement regulatory activity, as well as fusion proteins based on anti-C5 antibodies and new complement regulatory proteins, in particular to optimize complement regulatory proteins by mutation, and to optimize fusion proteins obtained based on preferred proteins with improved properties and developability, for example, better efficacy or improved solubility and purity while maintaining activity.

[0009] Description of the Invention The present invention relates to hybrid proteins and mutants thereof with better complement regulatory activity, which may have advantages such as better therapeutic effects.

[0010] In some embodiments, the hybrid proteins and mutants of the present invention contain CPP1 / SCR1 of CFH, but also contain less CCP of DAF, resulting in hybrid protein mutants with smaller molecular weights. In therapeutic applications, the hybrid proteins and mutants thereof can be administered at the same mass concentration as higher molar doses, and therefore have the potential for better therapeutic efficacy or longer dosing intervals compared to other commonly used complement regulatory proteins with larger molecular weights (especially compared to hybrid proteins that also contain other CCPs of CFH), for use in, for example, ophthalmic drugs and brain drugs. In other cases, a smaller molecular weight also greatly facilitates penetration into tissues such as tumor tissues and the blood-brain barrier.

[0011] In other embodiments, the hybrid proteins and mutants thereof of the present invention have better complement inhibitory activity, including activity in inhibiting the alternative pathway (AP), the classical pathway (CP), and / or C3b deposition. In some embodiments, the hybrid proteins and mutants thereof are effective in blocking human complement hemolytic activity. In some embodiments, mutants of the hybrid proteins of the present invention are effective in inhibiting human classical complement pathway hemolysis and / or human alternative complement pathway hemolysis. In some embodiments, mutants of the hybrid proteins of the present invention are effective in inhibiting C3b deposition.

[0012] Furthermore, the present invention relates to a hybrid protein of CFH and DAF, in which CCP1 / SCR1 of CFH hybridizes with a CCP of DAF (eg, CCP3-4 / SCR3-4 of DAF) to form the hybrid protein.

[0013] In some embodiments, the present invention relates to hybrid protein mutants that have improved purity, improved aggregation characteristics, or improved expression levels compared to the parent hybrid protein. For example, hybrid protein mutants of the present invention have a reduced proportion of (large) aggregates or improved purity compared to the parent hybrid protein. As another example, hybrid protein mutants of the present invention have increased expression levels compared to the parent hybrid protein. As another example, hybrid protein mutants of the present invention have a reduced proportion of (large) aggregates but improved expression levels compared to the parent hybrid protein. In some embodiments, the present invention relates to hybrid protein mutants that have a reduced proportion of aggregates, increased expression levels, and substantially equivalent activity (e.g., complement inhibitory activity) compared to the parent protein (e.g., the parent hybrid protein).

[0014] In some embodiments, the present invention relates to a fusion protein comprising an anti-C5 antibody, the fusion protein comprising an anti-C5 antibody and a complement regulatory protein optimized by mutation (e.g., a hybrid protein of a complement regulatory protein, e.g., a hybrid protein mutant of the present invention). In some embodiments, compared to an anti-C5 antibody, the fusion protein (1) further improves classical complement pathway (CP) blocking activity, enhancing efficacy; and (2) significantly improves alternative complement pathway (AP) blocking activity. In further embodiments, the fusion protein has more potent activity in inhibiting C3b deposition on cell surfaces than C5-antibody fusion proteins known in the art, where C3b deposition is believed to be the primary cause of extravascular hemolysis in PNH patients. In some embodiments, the fusion protein of the present invention has improved solubility while maintaining its activity.

[0015] In some embodiments, the present invention relates to the following specific embodiments. 1. A hybrid protein mutant, wherein the parent hybrid protein is: (a) CCP1 of human complement factor H (CFH); (b) CCP3 and CCP4 of human decay-accelerating factor (DAF), and Optionally, a signal peptide and / or tag comprising or consisting of Compared to the parent hybrid protein, the mutant: Mutations, e.g., substitutions, at positions 56, 59, 82, 86, 107, 109 and / or 110, e.g., substitutions with amino acids that are more hydrophilic than the original amino acid, and / or mutations at the C-terminus, e.g., addition of one or more amino acids at the C-terminus. and one or more mutations selected from the group consisting of: A hybrid protein mutant wherein the positions of the amino acid mutations are numbered corresponding to the amino acid positions set forth in SEQ ID NO:20.

[0016] 2. The hybrid protein mutant of embodiment 1, wherein the optimized hybrid protein comprises substitutions at positions 59, 82 and 110, numbered corresponding to the amino acid positions set forth in SEQ ID NO: 20, and / or the addition of one or more amino acids at the C-terminus.

[0017] 3. The hybrid protein mutant of embodiment 2, comprising substitutions at positions 56, 86, 107 and / or 109, numbered corresponding to the amino acid positions set forth in SEQ ID NO: 20.

[0018] 4. The hybrid protein mutant of embodiment 2, comprising substitutions at positions 56, 59, 82 and / or 110, numbered corresponding to the amino acid positions set forth in SEQ ID NO: 20, such as substitutions at 59, 82 and 110, or substitutions at 56, 59, 82 and 110.

[0019] 5. Mutations Substitution of the amino acid at positions 56, 59, 82, 86, 107, 109 and / or 110, numbered corresponding to the amino acid positions set forth in SEQ ID NO: 20, with an amino acid that is more hydrophilic than the original amino acid, for example, with D, E, S, Y or T; or Addition of 1 to 5 amino acids, e.g., 2 amino acids, at the C-terminus 5. The hybrid protein mutant of any one of embodiments 1 to 4, selected from the group consisting of:

[0020] 6. Mutations 56D / E, 59D / S, 82D, 86Y, 107E, 109D / E, 110T, or the addition of C-terminal amino acids KS, or combinations thereof 6. The hybrid protein mutant of any one of embodiments 1 to 5, selected from the group consisting of:

[0021] 7. 56E, 59D / S, 82D, 86Y, 107E, 109D / E, 110T, 59D-82D-110T, 59S-82D-110T, 59D-82D-110T-56E, 59S-82D-110T-56E, or Addition of amino acids KS at the C-terminus 7. The hybrid protein mutant of embodiment 6, comprising:

[0022] 8. A hybrid protein mutant according to any one of embodiments 1 to 7, wherein CCP3 and CCP4 of DAF are directly linked together to form CCP3-4.

[0023] 9. The hybrid protein mutant of any one of embodiments 1 to 8, wherein CCP1 of human CFH comprises or consists of the amino acid sequence of positions 19 to 82 of the human CFH protein, optionally wherein CCP1 comprises a V62I mutation, and wherein the amino acid positions are numbered corresponding to the amino acid positions set forth in SEQ ID NO:3.

[0024] 10. The hybrid protein mutant of any one of embodiments 1 to 9, wherein CCP1 of human CFH comprises or consists of the amino acid sequence of positions 19 to 84 of the human CFH protein, optionally wherein CCP1 comprises a V62I mutation, and wherein the amino acid positions are numbered corresponding to the amino acid positions set forth in SEQ ID NO:3.

[0025] 11. A hybrid protein mutant according to any one of embodiments 1 to 10, wherein CCP3 of human DAF comprises or consists of the amino acid sequence of positions 161 to 222 of the human DAF protein, and / or CCP4 of human DAF comprises or consists of the amino acid sequence of positions 223 to 285 of the human DAF protein, the amino acid positions being numbered corresponding to the amino acid positions set forth in SEQ ID NO: 1.

[0026] 12. A hybrid protein mutant according to any one of embodiments 1 to 11, wherein CCP3 of human DAF comprises or consists of the amino acid sequence of positions 163 to 222 of the human DAF protein, and / or CCP4 of human DAF comprises or consists of the amino acid sequence of positions 223 to 285 of the human DAF protein, the amino acid positions being numbered corresponding to the amino acid positions set forth in SEQ ID NO: 1.

[0027] 13. A hybrid protein mutant according to any one of embodiments 1 to 12, wherein CCP3-4 of human DAF comprises or consists of the amino acid sequence of positions 161 to 285 of the human DAF protein, the amino acid positions being numbered corresponding to the amino acid positions set forth in SEQ ID NO: 1.

[0028] 14. A hybrid protein mutant according to any one of embodiments 1 to 13, wherein CCP3-4 of human DAF comprises or consists of the amino acid sequence of positions 163 to 285 of the human DAF protein, the amino acid positions being numbered corresponding to the amino acid positions set forth in SEQ ID NO: 1.

[0029] 15. (1) CCP1 of human CFH comprises or consists of the amino acid sequence of positions 19 to 82 of the human CFH protein, CCP3 of human DAF comprises or consists of the amino acid sequence of positions 161 to 222 of the human DAF protein, and CCP4 of human DAF comprises or consists of the amino acid sequence of positions 223 to 285 of the human DAF protein; (2) CCP1 of human CFH comprises or consists of the amino acid sequence of positions 19 to 84 of the human CFH protein, CCP3 of human DAF comprises or consists of the amino acid sequence of positions 163 to 222 of the human DAF protein, and CCP4 of human DAF comprises or consists of the amino acid sequence of positions 223 to 285 of the human DAF protein; (3) CCP1 of human CFH comprises or consists of the amino acid sequence of positions 19 to 82 of the human CFH protein, and CCP3-4 of human DAF comprises or consists of the amino acid sequence of positions 161 to 285 of the human DAF protein; or (4) CCP1 of human CFH comprises or consists of the amino acid sequence of positions 19 to 84 of the human CFH protein, and CCP3-4 of human DAF comprises or consists of the amino acid sequence of positions 163 to 285 of the human DAF protein; A hybrid protein mutant according to any one of embodiments 1 to 14, wherein the amino acid positions of the human CFH protein are numbered corresponding to the amino acid positions set forth in SEQ ID NO: 3, and the amino acid positions of the human DAF protein are numbered corresponding to the amino acid positions set forth in SEQ ID NO: 1.

[0030] 16. The hybrid protein mutant of any one of embodiments 1 to 15, wherein CCP1 of human CFH has a V62I mutation.

[0031] 17. The human CFH protein is a human native CFH protein, or (i) the amino acid sequence set forth in SEQ ID NO: 3 or 5; (ii) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 4 or 6; (iii) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of (i) or (ii); Contains, or A hybrid protein mutant according to any one of embodiments 1 to 16, consisting of an amino acid sequence according to any one of (i) to (iii).

[0032] 18. The human DAF protein is a native human DAF protein, or (i) the amino acid sequence set forth in SEQ ID NO: 1; (ii) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:2; (iii) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of (i) or (ii); Contains, or A hybrid protein mutant according to any one of embodiments 1 to 17, consisting of an amino acid sequence according to any one of (i) to (iii).

[0033] 19. CCP1 of human CFH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, 13, 14 or 15, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 12, 13, 14 or 15; CCP3 of human DAF comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7 or 8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8; CCP4 of human DAF comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 9, and / or A hybrid protein mutant according to any one of embodiments 1 to 18, wherein CCP3-4 of human DAF comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11.

[0034] 20. The hybrid protein mutant of any one of embodiments 1 to 19, wherein the tag is a purification tag, such as a hexahistidine tag or a biotin marker, for example, comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0035] 21. The hybrid protein mutant of any one of embodiments 1 to 20, wherein the signal peptide is a secretory signal peptide comprising, for example, the amino acid sequence set forth in SEQ ID NO: 17.

[0036] 22. A hybrid protein mutant according to any one of embodiments 1 to 21, wherein the parent hybrid protein comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 18 to 33, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence.

[0037] 23. The hybrid protein mutant of embodiment 22, wherein the parent hybrid protein comprises or consists of the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence.

[0038] 24. A hybrid protein mutant according to any one of embodiments 1 to 23, comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 53 to 54, 56 to 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 to 96, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0039] 25. (1) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 60, and including a C-terminal KS addition; (2) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 62, and including a C-terminal KS addition and a 56E mutation; (3) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 64, and including a C-terminal KS addition and a 59D mutation; (4) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 66, and including a C-terminal KS addition and a 59S mutation; (5) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 68, and including a C-terminal KS addition and an 82D mutation; (6) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 70, and including a C-terminal KS addition and an 86Y mutation; (7) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 72, and including a C-terminal KS addition and a 107E mutation; (8) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 74, and including a C-terminal KS addition and a 109D mutation; (9) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 76, and including a C-terminal KS addition and a 109E mutation; (10) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 78, and including a C-terminal KS addition and a 110T mutation; (11) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 80, and including C-terminal KS and 59D-82D-110T mutations; (12) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 82, and including C-terminal KS and 59S-82D-110T mutations; (13) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 84, and including a C-terminal KS and 59D-82D-110T-56E mutation; (14) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 86, and C-terminal KS and 59S-82D-110T-56E mutations. 24. A hybrid protein mutant according to any one of embodiments 1 to 23, comprising:

[0040] 26. A fusion protein comprising an anti-C5 antibody or antigen-binding fragment thereof and a hybrid protein mutant according to any one of embodiments 1 to 25, wherein the antibody or antigen-binding fragment thereof is linked to the hybrid protein mutant with or without a linker.

[0041] 27. The fusion protein of embodiment 26, wherein one or more hybrid protein mutants are connected (at their N-terminus or at their C-terminus) to the N-terminus and / or C-terminus of the heavy chain and / or light chain, respectively, of the anti-C5 antibody, with or without a linker.

[0042] 28. The fusion protein of any one of embodiments 1 to 27, wherein the anti-C5 antibody is a humanized or chimeric antibody.

[0043] 29. The fusion protein of any one of embodiments 1 to 28, wherein the anti-C5 antibody is a monoclonal antibody.

[0044] 30. The fusion protein of any one of embodiments 1 to 29, wherein the antigen-binding fragment of the anti-C5 antibody is selected from Fab, Fab', Fab'-SH, Fv, a single-chain antibody (e.g., scFv), (Fab')2, a single-domain antibody, such as a VHH, a dAb (domain antibody), or a linear antibody.

[0045] 31. The fusion protein of any one of embodiments 1 to 30, wherein the anti-C5 antibody or antigen-binding fragment thereof comprises an Fc region, and preferably the Fc region is connected at its C-terminus to the N-terminus of the hybrid protein, with or without a linker.

[0046] 32. The linker contains one or more glycines (G). n , G.S., G. n S, G n S n , (G n S) n or (GSG) n or (G4S) nwherein n is an integer greater than or equal to 1, e.g., n is an integer of 2, 3, 4, 5, 6, or 7, for example, wherein the linker is G, GSG, or G4S.

[0047] 33. Includes full-length anti-C5 antibodies and hybrid proteins, an anti-C5 antibody joined at the C-terminus of its Fc region to the N-terminus of the hybrid protein to form the heavy chain of the fusion protein (with or without a linker); 33. The fusion protein of any one of embodiments 1 to 32, wherein the light chain of the anti-C5 antibody forms the light chain of the fusion protein.

[0048] 34. The anti-C5 antibody or antigen-binding fragment thereof i) an anti-C5 antibody or antigen-binding fragment thereof disclosed in CN113754763A; or ii) Eculizumab, ravulizumab, pozelimab, crovalimab, tesidolumab, or antigen-binding fragments thereof 34. The fusion protein of any one of embodiments 1 to 33, selected from:

[0049] 35. The anti-C5 antibody or antigen-binding fragment thereof is A fusion protein described in any one of embodiments 1 to 33, comprising three complementarity determining regions (CDRs) of the heavy chain variable region (VH), HCDR1, HCDR2 and HCDR3, and three CDRs of the light chain variable region (VL), LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2 and HCDR3 are the three CDRs contained in the VH set forth in SEQ ID NO: 39, and LCDR1, LCDR2 and LCDR3 are the three CDRs contained in the VL set forth in SEQ ID NO: 45.

[0050] 36. The anti-C5 antibody or antigen-binding fragment thereof is - an HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 40, - an HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 41, - an HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 42, - LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 46, - an LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 47, and - LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 48 34. The fusion protein of any one of embodiments 1 to 33, comprising:

[0051] 37. The anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH, wherein the heavy chain variable region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 39; or (ii) A fusion protein according to embodiment 35 or 36, comprising or consisting of the amino acid sequence of SEQ ID NO: 39.

[0052] 38. The anti-C5 antibody or antigen-binding fragment thereof comprises a light chain variable region VL, wherein the light chain variable region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 45; or (ii) A fusion protein according to any one of embodiments 35 to 37, comprising or consisting of the amino acid sequence of SEQ ID NO: 45.

[0053] 39. The anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL; - the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; - the fusion protein of embodiment 35 or 36, wherein the light chain variable region comprises or consists of an amino acid sequence of SEQ ID NO: 45, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0054] 40. The fusion protein of embodiment 35 or 36, wherein the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region, VH, and a light chain variable region, VL, wherein VH comprises or consists of the amino acid sequence of SEQ ID NO: 39, and VL comprises or consists of the amino acid sequence of SEQ ID NO: 45.

[0055] 41. The fusion protein of any one of embodiments 35 to 40, wherein the anti-C5 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region HC, e.g., the heavy chain constant region (HC) is an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, preferably an IgG2 or IgG4 heavy chain constant region, or an IgG2 / IgG4 hybrid form of the heavy chain constant region.

[0056] 42.The heavy chain constant region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 43; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 43; or (iii) The fusion protein of embodiment 41, comprising or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 43.

[0057] 43. The fusion protein of embodiment 41 or 42, wherein the heavy chain constant region comprises one or more mutations that enhance FcRn binding, such as YTE mutations (M252Y / S254T / T256E), LA mutations (M428L / N434A), or LS mutations (M428L / N434S), preferably LA mutations, and / or comprises a mutation that enhances stability, such as S228P.

[0058] 44. The fusion protein of any one of embodiments 35-43, wherein the anti-C5 antibody or antigen-binding fragment thereof comprises a light chain constant region, e.g., the light chain constant region is a lambda or kappa constant region.

[0059] 45. The light chain constant region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 49; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 49; or (iii) An antibody or antigen-binding fragment thereof according to embodiment 44, comprising or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 49.

[0060] 46. ​​The anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain; The heavy chain (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 44; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 44; or (iii) The fusion protein according to any one of embodiments 35 to 45, comprising or consisting of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2 or 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 44, preferably the amino acid changes occur outside the CDR regions, preferably the amino acid changes occur outside the heavy chain variable region.

[0061] 47. The anti-C5 antibody or antigen-binding fragment thereof comprises a light chain; The light chain (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 50; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 50; or (iii) The fusion protein according to any one of embodiments 35 to 46, comprising or consisting of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2 or 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 50, preferably the amino acid changes occur outside of the CDR regions, preferably the amino acid changes occur outside of the heavy chain variable region.

[0062] 48. The anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; - the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; - the fusion protein according to embodiment 46 or 47, wherein the light chain comprises or consists of an amino acid sequence of SEQ ID NO: 50, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0063] 49. The fusion protein of embodiment 48, wherein the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50.

[0064] 50. The fusion protein of embodiment 33, comprising two heavy chains and two light chains. 51. The fusion protein of embodiment 33 or 50, wherein the heavy chain of the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 or 87, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and / or the light chain of the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0065] 52. A nucleic acid molecule encoding a hybrid protein mutant according to any one of embodiments 1 to 25 or a fusion protein according to any one of embodiments 26 to 51.

[0066] 53. An expression vector comprising the nucleic acid molecule of embodiment 52, preferably pcDNA3.1.

[0067] 54. A host cell comprising a nucleic acid molecule according to embodiment 52 or an expression vector according to embodiment 53, which is preferably a prokaryote or a eukaryote, such as a CHO cell, a 293 cell, e.g., an Expi293 cell.

[0068] 55. A method for preparing a hybrid protein mutant according to any one of embodiments 1 to 25 or a fusion protein according to any one of embodiments 26 to 51, comprising culturing a host cell according to embodiment 54 under conditions suitable for expression of the hybrid protein mutant or fusion protein, and optionally further comprising isolating the hybrid protein mutant or fusion protein from the host cell or host cell culture medium and / or purifying the hybrid protein mutant or fusion protein.

[0069] 56. An immunoconjugate comprising a fusion protein according to any one of embodiments 26 to 51 and another agent, such as an antihemolytic agent or a label.

[0070] 57. A pharmaceutical composition or medicament or formulation comprising a hybrid protein mutant according to any one of embodiments 1 to 25, or a fusion protein according to any one of embodiments 26 to 51, and optionally a pharmaceutical excipient.

[0071] 58. A hybrid protein mutant according to any one of embodiments 1 to 25 or a fusion protein according to any one of embodiments 26 to 51, and Further therapeutic agents 1. A pharmaceutical combination product comprising:

[0072] 59. A method for preventing or treating a disease or condition associated with the complement system in a subject, comprising administering to the subject an effective amount of a hybrid protein mutant according to any one of embodiments 1 to 25, or a fusion protein according to any one of embodiments 26 to 51, or an immunoconjugate according to embodiment 56, or a pharmaceutical composition or formulation according to embodiment 57, or a pharmaceutical combination product according to embodiment 58.

[0073] 60. The method of embodiment 59, wherein the disease or condition is caused by abnormal activation of the complement system or dysregulation of the complement system, or is a complement C5-associated disease or condition.

[0074] 61. The method of embodiment 60, wherein the abnormal activation of the complement system or dysregulation of the complement system is due to, for example, a microbial infection or an increase in autoimmune antibodies, or due to a decrease, loss, impairment, or functional interference or disruption of a complement regulatory protein.

[0075] 62. The method of embodiment 60, wherein the complement C5-associated disease or condition comprises a disease phenotype caused by unregulated C5 function, such as due to dysregulated C5 activation, e.g., elevated C5 activation.

[0076] 63. The method of embodiment 60, wherein the complement C5-related disease or condition is a disease or condition in which the subject has an elevated level of complement C5 protein (e.g., at the nucleic acid or protein level) (e.g., compared to a healthy subject) or in which the subject has an elevated level of complement C5 protein (e.g., at the nucleic acid or protein level) in their blood or blood cells (e.g., compared to the blood or blood cells of a healthy subject).

[0077] 64. The method of embodiment 63, wherein the complement system-related disease or disorder is selected from diseases requiring inhibition of hemolysis, such as diseases requiring inhibition of hemolysis of the classical pathway of complement immunity and / or the alternative pathway of complement immunity, or diseases requiring inhibition of C3b deposition activity, such as dense deposition disease (DDD).

[0078] 65. A method for detecting the presence of complement C5 in a biological sample, comprising contacting the biological sample with the fusion protein of any one of embodiments 26 to 51 under conditions that allow binding to complement C5, and detecting whether a complex is formed between the antibody or antigen-binding fragment thereof or the fusion protein and complement C5, wherein the formation of a complex indicates the presence of complement C5. [Brief explanation of the drawings]

[0079] [Figure 1] FIG. 1 shows the crystal structures of CFH CCP1-4 and DAF CCP1-4 and a structural model of the hybrid proteins. [Figure 2] FIG. 1 shows the SDS-PAGE electrophoresis patterns of each protein tested. [Figure 3] FIG. 1 shows the results of SEC-HPLC purity analysis of hybrid proteins. [Figure 4-1] FIG. 1 shows the CP inhibitory activity of each protein tested. [Figure 4-2] Same as above. [Figure 5-1] FIG. 1 shows the AP inhibitory activity of each protein tested. [Figure 5-2] Same as above. [Figure 6-1] FIG. 1 shows the activity of each tested protein in inhibiting C3b deposition on the surface of red blood cells. [Figure 6-2] Same as above. [Figure 7] FIG. 1 shows the dissociation characteristics of anti-C5 antibodies and complement C5 in a ForteBio assay at different pH levels. [Figure 8] FIG. 1 shows the activity of anti-C5 antibodies to inhibit hemolysis of serum CP from human C5 transgenic mice. [Figure 9] FIG. 1 shows a schematic diagram of the fusion protein structure of an antibody and a hybrid protein. [Figure 10] FIG. 1 shows the prediction of potential amino acid sites mediating protein aggregation in hybrid proteins by computer-aided design software (DS2020). [Figure 11] FIG. 1 shows the AP inhibitory activity of fusion proteins comprising different mutant hybrid proteins (the fusion proteins are identified by the mutations contained in the mutant hybrid proteins). [Figure 12-1]FIG. 1 shows the CP inhibitory activity of fusion proteins comprising different mutant hybrid proteins (the fusion proteins are identified by the mutations contained in the mutant hybrid proteins). [Figure 12-2] Same as above. [Figure 13-1] FIG. 1 shows the AP and CP inhibitory activity of fusion proteins, including hybrid proteins containing different mutation combinations (fusion proteins are identified by the mutations contained in the mutant hybrid proteins). [Figure 13-2] Same as above. [Figure 14-1] FIG. 1 shows a comparison of the AP inhibitory activity of a fusion protein comprising a hybrid protein containing the 59D / 82D / 110T / 56E mutation combination with that of eculizumab and a fusion protein comprising CFH CCP1-5 (the fusion protein is identified by the mutations contained in the mutant hybrid protein). [Figure 14-2] Same as above. [Figure 15-1] FIG. 1 shows a comparison of the CP inhibitory activity of a fusion protein comprising a hybrid protein containing the 59D / 82D / 110T / 56E mutation combination with the CP inhibitory activity of eculizumab and a fusion protein comprising CFH CCP1-5 (the fusion proteins are identified by the mutations contained in the mutant hybrid protein). [Figure 15-2] Same as above. [Figure 16-1] FIG. 1 shows a comparison (rabbit erythrocyte method) of the C3b deposition inhibitory activity of a fusion protein comprising a hybrid protein containing the 59D / 82D / 110T / 56E mutation combination with the C3b deposition inhibitory activity of eculizumab and a fusion protein comprising CFH CCP1-5 (the fusion protein is identified by the mutations contained in the mutant hybrid protein). [Figure 16-2] Same as above. [Figure 17]FIG. 1 shows a comparison (Wieslab method) of the C3b deposition inhibitory activity of a fusion protein comprising a hybrid protein containing the 59D / 82D / 110T / 56E mutation combination with the C3b deposition inhibitory activity of eculizumab and a fusion protein comprising CFH CCP1-5 (fusion proteins are identified by the mutations contained in the mutant hybrid protein). [Figure 18] FIG. 1 shows a comparison (Wieslab method) of the LP inhibitory activity of a fusion protein comprising a hybrid protein containing the 59D / 82D / 110T / 56E mutation combination with that of eculizumab and a fusion protein comprising CFH CCP1-5 (fusion proteins are identified by the mutations contained in the mutant hybrid protein). [Figure 19-1] 19A-H show purity results and large aggregate characteristics of hybrid protein mutants detected by HPLC-SEC. [Figure 19-2] Same as above. [Figure 19-3] Same as above. [Figure 19-4] Same as above. [Figure 20] FIG. 1 shows a comparison of the percentage of large aggregates and the percentage of monomer purity of hybrid protein mutants. [Figure 21] FIG. 1 shows a comparison of the expression levels (relative expression levels) of hybrid protein mutants. [Figure 22-1] FIG. 1 shows a comparison of AP and CP inhibitory activities of hybrid protein mutants. [Figure 22-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0080] I. Definition It is understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. 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 belongs.

[0081] Decay-accelerating factor (DAF) Decay-accelerating factor (DAF, CD55) is a membrane-associated regulatory protein that protects autologous cells from the activation of autologous complement on their surface. DAF acts by rapidly dissociating the C3 and C5 convertases (the central enzymes of the cascade). DAF has the most potent attenuating-accelerating activity among proteins involved in complement regulation, acting on classical pathway enzymes (C4b2a and C4b2a3b) and alternative pathway enzymes (C3bBb and C3BbC3b). However, DAF does not have any cofactor function.

[0082] Structural analysis of DAF shows that, starting from its N-terminus, DAF consists of units of 4 to 60 amino acids in length, followed by a highly O-glycosylated serine (S)- and threonine (T)-rich fragment (STP), followed by a post-translationally added glycosylphosphatidylinositol (GPI) anchor.

[0083] In some embodiments, the DAF is a human DAF having the following accession numbers: Genbank accession numbers M31516, M15799, M64653, S72858, or M643567.

[0084] In some embodiments, the DAF is human DAF. In some embodiments, the human DAF is native human DAF. In some embodiments, the amino acid sequence of native human DAF is set forth in SEQ ID NO: 1, and the various modules are described in Table 1 below. Four repeating units, each 60 amino acids long, are referred to as complement control protein repeats (CCPs) or short consensus repeats (SCRs). CCP1 contains amino acids 35-96, CCP2 contains amino acids 96-160, CCP3 contains amino acids 161-222, and CCP4 contains amino acids 223-285. All of these contribute to the regulatory activity of DAF. The heavily O-glycosylated region functions as a cushion, positioning the CCP at an appropriate distance above the surface membrane in a suspended state. The GPI anchor allows DAF to move freely across the plasma membrane, thereby enabling it to inactivate the invertase complex wherever it is assembled. In this context, references to amino acid positions of modules of DAF are made to the numbering of amino acid positions corresponding to those set out in SEQ ID NO:1.

[0085] [Table 1]

[0086] In some embodiments, the nucleotide sequence of a cDNA encoding DAF is set forth in SEQ ID NO:2. Factor H (CFH or FH) "Complement factor H," "factor H," "FH," "CFH protein," or "CFH" are used interchangeably and refer to an approximately 150 kDa protein that is a member of the complement activation family of regulators and a complement control protein. CFH is a large soluble glycoprotein that circulates in human plasma and functions to regulate the alternative pathway of the complement system, ensuring that the complement system is directed against pathogens or other dangerous agents and does not damage host tissues.

[0087] Factor H is primarily a monomer, but can weakly self-associate (KD = 28 μM) and may oligomerize in the presence of glycosaminoglycans or high concentrations of metal ions. CFH is composed of 20 homologous units called complement regulatory protein repeats (CCPs) (SCRs or sushi domains), some of which function in cell adhesion, while others function to remove C3b from the cell surface. Each of the 20 SCRs is approximately 60 amino acids long, arranged head-to-tail, and contains four cysteine ​​residues, forming two disulfide bonds per module. SCRs 19 and 20 are involved in C3b binding.

[0088] The splice variant FHL-1 consists of the first seven CCPs followed by the C-terminal sequence Ser-Pro-Leu-Thr. I -Cys III , Cys II -Cys IV It contains approximately 60 residues, including four invariant cysteines that form disulfides. Adjacent modules are connected by sequences of 3 to 8 residues.

[0089] Images from negative stain electron microscopy showed that the FH molecule adopts diverse conformations but is primarily half-folded, and analytical ultracentrifugation, small-angle X-ray scattering (module entries in the PDB, e.g., 3GAV) and chemical cross-linking also showed that the modular chain itself is folded. The following high-resolution structures have been determined for several FH fragments, alone or in complex with other molecules (PDB identifiers are provided in parentheses below): CCP1-2 (2RLP), 2-3 (2RLQ), 1-4 (2WII), 5, 6-7 (e.g., 2W80, 2YBY), 7 (2JGW and 2JGX), 6-8 (2UWN and 2V8E), 9 (4K12), 10-11 (4B2R), 11-12 (4B2S), 12-13 (2KMS), 15 (1HFI), 16 (1HCC), 15-16 (1HFH), 18-20 (3SWO), 19-20 (e.g., 2BZM, 2G7I, and 4ONT). Each CCP resembles a prolate spheroid with a long axis of approximately 4 nm and a short axis of approximately 2 nm, containing beta strands in an antiparallel patch that roughly coincides with the long axis, with the N- and C-termini positioned at opposite ends of the long axis, thereby facilitating end-to-end arrangement of tandem CCPs with varying inter-module contacts, tilts, and twists.

[0090] In some embodiments, the CFH is human CFH, for example, native human CFH. In some embodiments, the amino acid sequence of CFH has the following accession number: HGNC:HGNC:4883, Ensembl:ENSG00000000971, HPRD:00601, MIM:134370, or Vega:OTTHUMG00000035607.

[0091] In some embodiments, the amino acid sequence of CFH is set forth in SEQ ID NO: 3. In some embodiments, the amino acid positions corresponding to each module of CFH are as set forth in Table 2 below.

[0092] [Table 2]

[0093] In some embodiments, the nucleotide sequence of the cDNA encoding CFH is set forth in SEQ ID NO:4. In some embodiments, the protein sequence encoding the splice variant FHL-1 of CFH is set forth in SEQ ID NO: 5. In some embodiments, the nucleotide sequence of the cDNA encoding the splice variant FHL-1 of CFH is set forth in SEQ ID NO: 6.

[0094] In this context, references to amino acid positions of modules of CFH are made to the numbering of amino acid positions corresponding to those set forth in SEQ ID NO:3.

[0095] Other definitions Where applicable, terms used in the singular may also include the plural and vice versa.

[0096] The term "about," when used in conjunction with a numerical value, is meant to encompass a range of numerical values ​​with a lower limit of 5% below the specified numerical value and an upper limit of 5% above the specified numerical value. As used herein, the term "and / or" means any one of any items, or more than one or all of any items.

[0097] As used herein, the term "comprise" or "include," or any portion of that term, means the inclusion of the stated elements, integers, or steps, but not the exclusion of any other elements, integers, or steps. In this context, when the term "comprise" or "include" is used, unless otherwise specified, the term "comprise" or "include" also covers the situation of consisting of the stated elements, integers, or steps. For example, when referring to a protein "comprising" a particular sequence, it is intended to also cover proteins consisting of that particular sequence.

[0098] When reference is made to "first" and "second" in this product, it is only to distinguish between two domains or two chains and does not in any way indicate the position of the two domains.

[0099] As used herein, the term "hybrid protein" is used interchangeably with "chimeric polypeptide" and refers to a larger polypeptide formed by the fusion of at least two heterologous polypeptide sequences, optionally through a linker. Hybrid proteins can be produced by recombinant expression. Reference to a hybrid protein can also include mutants or variants of the hybrid protein.

[0100] The terms "complement C5" or "C5 protein" or "complement C5 protein" or "C5 complement protein" are used interchangeably and refer to various types of complement C5 proteins. Human complement C5 (Uniprot entry P01031) is a secreted multidomain glycoprotein composed of an α chain (999 amino acids) and a β chain (655 amino acids) connected by a disulfide bridge. The peptide bond between Arg751 and Leu752 of the α chain is cleaved by C5 convertase, resulting in the generation of a small 74-amino acid C5a fragment and a large C5b fragment (1580 amino acids). The conversion of C5 to C5b involves a major conformational change, followed by C6 binding. For example, in some embodiments, human C5 has the sequence set forth in SEQ ID NO: 38. The term "antigen" refers to a molecule that elicits an immune response. This immune response can involve antibody production, activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule can be used as an antigen, including essentially any protein or peptide.In addition, antigens can be derived from recombinant DNA or genomic DNA.As used herein, the term "epitope" refers to the part of an antigen that specifically interacts with an antibody molecule.

[0101] As used herein, the term "antigen-binding region" refers to the portion of the fusion protein that binds to a specific antigen. The antigen-binding region may be, for example, an antibody, an immunoglobulin itself, or an antibody fragment. Such an antigen-binding region may or may not have a tertiary structure independent of the rest of the fusion protein, and may or may not bind to that antigen as a separate entity.

[0102] When it is referred to as "the antigen-binding region is derived from an antibody," it means that the binding domain that constitutes the antigen-binding region is or is derived from the binding domain of an antibody that specifically binds to an antigen, e.g., the heavy and / or light chain variable regions of the antigen-binding region are or are derived from the heavy and / or light chain variable regions of said antibody, or one, two, three, four, five or six CDRs of the target-binding region are the CDRs of said antibody.

[0103] The term "derived from" means that the antigen-binding region fragment is substantially the same as the fragment of the antibody from which it is derived, but has mutations, such as substitutions, deletions, or additions, at one or more sites. In a specific embodiment, the mutations are not in the CDRs of the antibody.

[0104] The terms "whole antibody," "full-length antibody," "complete antibody," and "intact antibody" are used interchangeably herein to refer to a naturally occurring glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further divided into hypervariable regions (complementarity-determining regions (CDRs)) and more conserved regions (framework regions (FR)) interposed between them. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions are not directly involved in binding the antibody to an antigen, but exhibit various effector functions. In some embodiments, the heavy chain constant region HC of the antibody of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, preferably the heavy chain constant region of IgG1. The term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0105] The term "antigen-binding fragment" of an antibody refers to a portion or segment of a full-length antibody or a whole antibody that has fewer amino acid residues than the whole antibody or a full-length antibody, but is capable of binding to an antigen or competing with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for binding to an antigen. Antigen-binding fragments can be prepared by recombinant DNA techniques or by enzymatic or chemical cleavage of whole antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabodies, single-domain antibodies (sdAbs), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. In addition, F(ab')2, a dimeric, bivalent antibody fragment of Fab', can be generated by digesting a whole antibody under the disulfide bond in the hinge region with pepsin. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bond in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab fragment with the hinge region. An Fv fragment is composed of the VL and VH domains of a single arm of an antibody. The two domains, VL and VH, of an Fv fragment can be encoded by separate genes, but recombinant methods can also be used to connect the two domains using a synthetic connecting peptide, so that the two domains are produced as a single protein chain, and the VL and VH domains pair in the single protein chain to form a single-chain Fv (scFv).

[0106] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions.

[0107] The "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" of an antibody is a region of an antibody variable domain (VH or VHH) that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an antigen epitope. The CDRs of heavy and light chains are numbered sequentially from the N-terminus and are usually referred to as CDR1, CDR2, and CDR3. The CDRs located in the variable domain of an antibody heavy chain are also referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. For a given amino acid sequence of a light chain variable region or heavy chain variable region, its CDR sequences can be determined using a variety of schemes known in the art. For example: Chothia, which is based on the three-dimensional structure of antibodies and the topology of CDR loops; Kabat, which is based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987)); AbM (University of Bath); Contact (University College London); International ImMunoGeneTics database (IMGT) (International Immunogenetics Information System, World Wide Web imgt.cines.fr / ); and North CDR definition, which is based on affinity propagation clustering using multiple crystal structures (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, pp. 228-256 (2011)).

[0108] Unless otherwise specified, the term "CDR" or "CDR sequence" in the present invention encompasses CDR sequences determined by any of the above methods. CDR can also be determined based on the same Kabat numbering position as the reference CDR sequence. Unless otherwise specified, in the present invention, when referring to the position of the residue in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), the residue position refers to the numbering position according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0109] In one embodiment, the CDRs in an antibody molecule of the invention are determined by the Chothia numbering convention. The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. A naturally occurring immunoglobulin "Fc domain" or "Fc region" comprises two or three constant domains: a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a naturally occurring antibody, the immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of the heavy chain derived from antibodies of the IgG, IgA, and IgD classes, or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from antibodies of the IgM and IgE classes. Unless otherwise specified herein, amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interests, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In this context, the terms "Fc region," "Fc portion," and "Fc fragment" do not include the heavy chain variable region (VH) and light chain variable region (VL), and the heavy chain constant region (CH1) and light chain constant region (CL) of an immunoglobulin, but may optionally include a hinge region at the N-terminus of the heavy chain constant region. Examples of immunoglobulin "effector functions" include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0110] The term "chimeric antibody" refers to an antibody molecule in which (a) the constant region or a portion thereof has been altered, replaced, or exchanged so that the antigen-binding site is linked to a constant region of a different or altered class with a different effector function and / or of a different species, or to an entirely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that confers new properties to the chimeric antibody, or (b) the variable region or a portion thereof has been altered, replaced, or exchanged with a variable region having a different or altered antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. As a result of the replacement with the human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced immunogenicity in humans compared to the original mouse antibody.

[0111] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a murine monoclonal antibody) but is less immunogenic when administered to humans as a therapeutic. This can be done, for example, by retaining the non-human antigen-binding site and replacing the remainder of the antibody with its human counterpart (i.e., replacing the constant region and portions of the variable region not involved in binding with corresponding portions of a human antibody).

[0112] As used herein, the term "fusion protein" refers to a larger polypeptide formed by the fusion of at least two heterologous polypeptide sequences, optionally via a linker. Fusion proteins can be produced by recombinant expression.

[0113] In this regard, antibody constant regions or domains, including the CH1, CL, and Fc domains, as well as the CH2, CH3, and optional CH4 domains that make up the Fc domain, can be selected according to the intended function of the antibody molecule. For example, the constant region can be an IgA, IgD, IgE, IgG, or IgM region, particularly an immunoglobulin constant domain of human IgG, e.g., a human IgG1, IgG2, IgG3, or IgG4 constant domain, preferably a human IgG2, IgG4, or IgG2 / 4 hybrid form constant domain. The immunoglobulin constant region can have a native sequence or a variant sequence.

[0114] The term "hybrid heavy chain constant region" refers to a fragment of a heavy chain constant region, for example, a fragment of a heavy chain constant region in which the constant region domains (e.g., CH1, CH2, CH3, and optionally CH4) constituting the constant region or portions thereof are derived from different IgG, IgA, and IgD class antibodies. For example, an IgG2 / IgG4 hybrid heavy chain constant region refers to a heavy chain constant region in which CH1 and portions of CH2 of the constant region are derived from IgG2, and portions of CH2 and CH3 are derived from IgG4, such as the heavy chain constant regions of the C5 antibody drugs Soliris and Ultomiris.

[0115] As used herein, the term "linker" refers to any molecule that allows for the direct connection of different portions of a bispecific binding molecule. Examples of linkers that establish a covalent connection between different molecular portions include peptide linkers and non-protein polymers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, the linker is a peptide linker (also referred to as a "connecting peptide"), which refers to a short amino acid sequence composed of amino acids such as glycine (G) and / or serine (S) and / or threonine residues (T), or a hinge region from an immunoglobulin, used alone or in combination to connect the amino acid sequence of a first portion of a fusion protein molecule to a second portion of the molecule. For example, a peptide linker can connect the polypeptides of an antibody molecule and a fusion protein molecule. For example, a peptide linker can also connect one portion of an antibody to another portion of an antibody, such as connecting a light chain variable region to a heavy chain variable region. Preferably, the peptide linker has a length sufficient to connect the two entities in a manner that maintains their conformation relative to one another so as not to interfere with the desired activity. Useful linkers also include glycine-alanine polymers, glycine-serine polymers, alanine-serine polymers, and other flexible linkers. In one embodiment, the connecting peptide is 1 to 50 amino acids in length, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in length. In one embodiment, the linker contains one or more glycines, e.g., G n wherein n=an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, or 6. In one embodiment, the linker is, for example, GS, GSG, G n S n , (G n S) n , or (G4S) nIn one embodiment, the connecting peptide is a glycine-serine polymer such as "(GSG)" (where n=an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, or 6). In one embodiment, the connecting peptide is one or more glycines or "(GSG)" ( n " or (G4S) n where n is an integer greater than or equal to 1, e.g., n is an integer of 2, 3, 4, 5, 6, or 7. Suitable flexible connecting peptides can be rationally designed using computer programs that simulate the three-dimensional structures of proteins and peptides or by phage display methods.

[0116] As used herein, the terms "antibody," "binding," or "specific binding" mean that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as radioimmunoassay (RIA) or thin-layer biofilm interferometry or MSD assay or surface plasmon resonance (SPR).

[0117] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for partner Y is usually expressed by the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (kdis and kkon, respectively). Affinity can be measured by conventional methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay described herein.

[0118] In this regard, amino acid mutations can be amino acid substitutions, deletions, insertions, and additions. Any combination of substitutions, deletions, insertions, and additions can be made to obtain an optimized mutant protein with desired properties (e.g., improved solubility). Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxy termini of the polypeptide sequence, as well as deletions and insertions within the polypeptide sequence. Some amino acids may be added at the C-terminus of the hybrid protein. In some embodiments, preferred amino acid mutations are amino acid substitutions, such as single amino acid substitutions or combinations of several amino acid substitutions.

[0119] In this context, reference to an amino acid position to be mutated in optimizing a hybrid protein is determined based on the amino acid sequence set forth in SEQ ID NO: 20. Corresponding amino acid positions on hybrid proteins or polypeptides having other amino acid sequences can be determined by amino acid sequence alignment with SEQ ID NO: 20. For example, reference to "L56" refers to the leucine L at position 56 of SEQ ID NO: 20, or the amino acid residue at the corresponding position in the amino acid sequence of another hybrid protein by alignment.

[0120] In this regard, when referring to hybrid protein mutants or mutations thereof, single amino acid substitutions are described in the following manner: [original amino acid residue / position / amino acid residue to be substituted] or [position / amino acid residue to be substituted]. For example, a substitution of leucine (or other corresponding amino acid) at position 56 with glutamic acid can be represented as L56E or 56E. When various alternative amino acid substitutions (e.g., D, S) exist at a given position (e.g., position L59), the amino acid substitution can be represented as L59 / D / S or 59 / D / S. Thus, individual single amino acid substitutions can be linked by a plus sign "+" or "-" to indicate combined mutations at multiple given positions. For example, a combined mutation of 59S, 82D, and 110T can be represented as 59S+82D+110T or 59S-82D-110T.

[0121] As used herein, "hydrophilic amino acid or residue" refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of less than 0 according to the standardized consensus hydrophobicity scale of Eisenberg et al. (Eisenberg et al., J. Mol. Biol., 179:125-142

[1984] ). Exemplary hydrophilic amino acids are selected from Thr (T), Ser (S), L-His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K), and Arg (R).

[0122] As used herein, "hydrophobic amino acid or residue" refers to an amino acid or residue having a side chain exhibiting a hydrophobicity greater than 0 according to the standardized consensus hydrophobicity scale of Eisenberg et al. (Eisenberg et al., J. Mol. Biol., 179:125-142

[1984] ). Exemplary hydrophobic amino acids are selected from Pro (P), Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), or Tyr (Y).

[0123] "An amino acid that is more hydrophilic than the original amino acid" means that the substituting amino acid is more hydrophilic than the original amino acid, and "an amino acid that is more hydrophilic than the original amino acid" is not necessarily a "hydrophilic amino acid", for example, if Y is more hydrophilic than F, then F can be substituted with Y.

[0124] "Percentage (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a specific amino acid sequence described herein, after aligning the candidate sequence with the specific amino acid sequence described herein and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions that are included as part of the sequence identity. In some embodiments, the present invention contemplates variants of the proteins or polypeptides of the present invention having a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more identity, compared to the polypeptides or proteins specifically disclosed herein. Variants can include conservative changes.

[0125] In the context of polypeptide sequences, "conservative changes" include substitutions, deletions, or additions to a polypeptide sequence that do not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often involve replacing an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Eight groups of amino acids that are conservatively substituted for one another are listed below: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conservative sequence changes" is used to refer to amino acid modifications that do not significantly affect or alter the activity of the parent hybrid protein. For example, conservatively modified variants retain at least 80%, 85%, 90%, 95%, 98%, 99% or more, e.g., 100-110% or more, of the activity of the parent polypeptide or hybrid protein.

[0126] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its derived progeny. Host cells are any type of cell line that can be used to produce the hybrid proteins of the invention, including eukaryotic cells such as mammalian cells, insect cells, yeast cells, and prokaryotic cells such as Escherichia coli (E. coli) cells. Host cells include cultured cells and cells within transgenic animals, transgenic plants, or cultured plant or animal tissue.

[0127] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, viruses, and adeno-associated viruses) into which the recombinant polynucleotide has been incorporated.

[0128] The terms "individual" and "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual is a human.

[0129] The term "treating" includes administering a composition or hybrid polypeptide to prevent or delay the onset of symptoms, complications, or biochemical manifestations of a disease, to alleviate symptoms, or to prevent or inhibit further progression of a disease, condition, or disorder. The term "prevention" includes inhibiting the onset or progression of a disease or disorder, or symptoms of a particular disease or disorder.

[0130] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, with which an active substance is administered.

[0131] The term "pharmaceutical composition" refers to a composition that is in a form effective to enable the biological activity of the active ingredient contained therein and that does not contain additional ingredients that would be unacceptably toxic to the subject to which the composition is administered.

[0132] The term "effective amount" refers to an amount or dosage of a hybrid protein of the present invention or a nucleic acid encoding same, or a composition or combination thereof, which, when administered to a patient in a single dose or multiple doses, produces the desired effect in a patient in need of treatment or prevention.

[0133] A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of the hybrid protein or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, and even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100%, compared to untreated subjects.

[0134] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Generally, since a prophylactic dose is administered to a subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0135] As used herein, the term "complement C5-associated disease or condition" refers to a disease or condition in which unregulated C5 function can result in a disease phenotype due to dysregulated C5 activation, e.g., elevated C5 activation.

[0136] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to an agent (e.g., a polynucleotide probe or antibody) and that facilitates detection of the agent to which it is conjugated or fused. The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical alteration of a substrate compound or composition that is detectable. The term is intended to cover direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of a probe or antibody by reaction with another reagent that is directly labeled. In some embodiments, the label is His or biotin.

[0137] A "subject / patient / individual sample" refers to a collection of cells or bodily fluids obtained from a patient, subject, or individual. The source of the tissue or cell sample can be solid tissue, such as from a fresh, frozen, and / or preserved organ or tissue sample or biopsy or aspirate sample; blood or any blood component; bodily fluid, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; or cells from any stage of a subject's pregnancy or development. In some embodiments, the sample is blood or serum. In some embodiments, the tissue sample may contain compounds that are not naturally associated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0138] These and other aspects and embodiments of the present invention are described in the accompanying drawings (brief description of the drawings immediately following) and the following detailed description of the invention, and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, although it should be understood that these examples are set forth by way of illustration rather than limitation, and that various modifications may occur to those skilled in the art.

[0139] III. Hybrid Proteins or Mutants Thereof The present invention relates to hybrid proteins of CFH and DAF, which contain at least one functional unit derived from CFH and at least one functional unit derived from DAF.

[0140] A.DAF functional unit In certain embodiments, the hybrid protein of the present invention preferably comprises a functional unit derived from DAF. Such a functional unit can dissociate C3 convertase and C5 convertase in the classical pathway and / or the alternative pathway, and / or accelerate the decay-accelerating activity of C3 convertase. In some embodiments, the DAF functional unit comprises CCP3 and CCP4 of DAF.

[0141] The amino acid sequence of such a CCP may be identical to the native or naturally occurring amino acid sequence of DAF. Alternatively, the amino acid sequence of such a CCP may be slightly altered, particularly at the amino or carboxy terminus. This alteration occurs when a restriction enzyme site is incorporated into the polynucleotide encoding the CCP. Such alterations also occur when amino acids are deleted from the N- or C-terminus of the functional unit. For example, in some embodiments, one to two amino acids may be deleted at the N-terminus of CCP3. As another example, in some embodiments, one to two amino acids may be deleted at the C-terminus of CCP4.

[0142] Some amino acid substitutions, preferably conservative substitutions, can also be introduced into the sequence without affecting functional activity. Conservative substitutions can be made, for example, by substituting charged amino acids for each other, or hydrophilic amino acids for each other, hydrophobic amino acids for each other, and amino acids of similar mass for each other.

[0143] In one embodiment, the DAF functional unit comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to CCP3 and / or 4 of the human DAF protein (or CCP3-4 directly linked together by the C-terminus of CCP3 and the N-terminus of CCP4). In some embodiments, the DAF functional unit comprises CCP3 and / or 4 of the human DAF protein (CCP3-4 directly linked together by the C-terminus of CCP3 and the N-terminus of CCP4). In some embodiments, the DAF functional unit comprises CCP3 and 4 of the human DAF protein. In some embodiments, the DAF functional unit is CCP3-4, which are directly linked together by the C-terminus of CCP3 and the N-terminus of CCP4.

[0144] In some embodiments, the human DAF protein is a native human DAF protein. In some embodiments, the native human DAF protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. DAF comprises or consists of the amino acid sequence encoded by the DNA sequence set forth in SEQ ID NO: 2.

[0145] In some embodiments, CCP3 comprises or consists of amino acids 161 to 222 of DAF. In some embodiments, one to two amino acids at the N-terminus of CCP3 may be deleted to allow for the attachment of another functional unit, for example, CCP3 comprises or consists of amino acids 163 to 222 of DAF.

[0146] In some embodiments, CCP3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:7 or 8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:8.

[0147] SEQ ID NO:7:

[0148] [ka]

[0149] SEQ ID NO:8:

[0150] [ka]

[0151] In some embodiments, CCP4 comprises or consists of amino acids 223-285 of DAF. In some embodiments, CCP4 comprises or consists of the amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:9.

[0152] SEQ ID NO:9:

[0153] [ka]

[0154] In some embodiments, CCP3-4 comprises or consists of amino acids 161 to 285 or amino acids 163 to 285 of DAF. In some embodiments, the DAF functional unit CCP3-4 comprises or consists of the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:11, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:11.

[0155] SEQ ID NO: 10: Human DAF CCP3-4

[0156] [ka]

[0157] SEQ ID NO: 11: Human DAF CCP3-4 (2 amino acids missing from the N-terminus of CCP3)

[0158] [ka]

[0159] When referring to an amino acid position in DAF, the position in the amino acid sequence corresponds to the amino acid position in SEQ ID NO:1.

[0160] B.CFH functional unit In certain embodiments, the hybrid proteins of the present invention comprise a functional unit derived from CFH, which functional unit is capable of dissociating the alternative pathway C3 convertase C3bBb and / or binding to C3b. In a preferred embodiment, the CFH functional unit comprises CCP1 of CFH.

[0161] The amino acid sequence of such a CCP of CFH may be identical to the native or naturally occurring amino acid sequence of CFH. Alternatively, the amino acid sequence of such a CCP may be slightly altered, particularly at the amino or carboxy terminus. This alteration occurs when a restriction enzyme site is incorporated into the polynucleotide encoding the CCP. Such alterations also occur when amino acids are deleted from or added to the N- or C-terminus of the functional unit.

[0162] For example, in some embodiments, one to two amino acids may be deleted at the C-terminus of CCP1. In some embodiments, one to two amino acids of CCP2 can be added to the C-terminus of CCP1 to attach a functional unit of DAF; thus, "CCP1" as used herein encompasses CCP1 with amino acids added at the C-terminus (e.g., one to two amino acids of CCP2). For example, if one to two amino acids are deleted from the N-terminus of the functional unit of DAF, one to two amino acids of CCP2 can be added to the C-terminus of CCP1 to link the functional unit of DAF. In some embodiments, KS is deleted from CCP3 of DAF, RP is added to the C-terminus of CCP1 of CFH, and both are ligated to obtain a hybrid protein.

[0163] Some amino acid substitutions in sequence, preferably conservative substitutions, can also be introduced without affecting functional activity.Conservative substitutions can be made, for example, by substituting charged amino acids with each other, or by substituting hydrophilic amino acids with each other, by substituting hydrophobic amino acids with each other, and by substituting amino acids with similar masses with each other.For example, the CCP1 of CFH of the present invention can contain V62I mutation.

[0164] In one embodiment, the CFH functional unit comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to CCP1 of the human CFH protein. In some embodiments, the CFH functional unit comprises CCP1 of the human CFH protein.

[0165] In some embodiments, the human CFH protein is a native human CFH protein. In some embodiments, the native human CFH protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3 or 5. In some embodiments, the CFH comprises or consists of the amino acid sequence encoded by the DNA sequence set forth in SEQ ID NO: 4 or 6.

[0166] In some embodiments, CCP1 comprises or consists of amino acids 19-82 of CFH. In some embodiments, CCP1 comprises or consists of amino acids 19-84 of CFH.

[0167] In some embodiments, the CFH functional unit comprises or consists of an amino acid sequence set forth in SEQ ID NO: 12, 13, 14 or 15, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 12, 13, 14 or 15.

[0168] SEQ ID NO: 12: Human CFH CCP1 (the C-terminus of CCP1 is appended with the two N-terminal amino acids RP of CCP2)

[0169] [ka]

[0170] SEQ ID NO: 13: Human CFH CCP1 (CCP1)

[0171] [ka]

[0172] SEQ ID NO: 14: Human CFH CCP1 (the C-terminus of CCP1 is appended with the two N-terminal amino acids RP of CCP2 and contains V62I)

[0173] [ka]

[0174] SEQ ID NO: 15: Human CFH CCP1 (CCP1 contains V62I)

[0175] [ka]

[0176] When referring to an amino acid position of CFH, the amino acid sequence position corresponds to the amino acid position in SEQ ID NO:3.

[0177] C. Other units Optionally, the hybrid protein or mutant thereof of the present invention can further comprise a tag, preferably about 2 to 10 amino acids, added to the amino or carboxy terminus of the hybrid protein or mutant thereof, e.g., to the carboxy terminus. Typically, such an addition is made to stabilize the protein or to facilitate secretory expression or purification of the hybrid protein or mutant thereof. Such tags are known in the art. Representative examples of such tags include a sequence encoding a stretch of histidine residues (e.g., 2 to 10 histidines, e.g., 2, 3, 4, 5, 6, or 7 histidines), the epitope tag FLAG, herpes simplex glycoprotein D, β-galactosidase, maltose-binding protein, or glutathione S-transferase.

[0178] In some embodiments, the tag is a histidine residue, which can be added at the amino or carboxy terminus, for example, the carboxy terminus, of the hybrid protein or mutant thereof.

[0179] In some embodiments, the tag is a 6xHis tag of GHHHHHH (SEQ ID NO: 16). Optionally, the hybrid proteins of the invention can also include a signal peptide, such as MGWSCIILFLVATATGVHS (SEQ ID NO: 17).

[0180] The present invention also encompasses hybrid proteins or mutants thereof in which one or more amino acids have been modified by post-translational processes or synthetic methods, including, but not limited to, glycosylation, iodination, myristoylation, and pegylation.

[0181] D. Examples of Hybrid Proteins In some embodiments, the hybrid proteins of the invention comprise at least one functional unit derived from CFH and at least one functional unit derived from DAF.

[0182] In some embodiments, a hybrid protein of the invention comprises (i) CCP1 of human complement factor H (CFH) and (ii) CCP3 and / or CCP4 of human decay-accelerating factor (DAF).

[0183] In some embodiments, hybrid proteins of the invention comprise CCP1 of CFH and CCP3 and CCP4 of DAF. In some embodiments, hybrid proteins of the invention consist of CCP1 of CFH and CCP3 and CCP4 of DAF. In some embodiments, hybrid proteins of the invention comprise or consist of CCP1 of CFH and CCP3-4 of DAF.

[0184] In some embodiments, 1-2 amino acids (e.g., 1-2 amino acids at the N-terminus of CCP2) are added to the C-terminus of CCP1 of CFH. In some embodiments, 1-2 amino acids are deleted from the N-terminus of CCP3 or CCP3-4 of DAF.

[0185] In some embodiments, hybrid proteins of the invention comprise CCP1 of CFH and CCP3-4 of DAF, with one to two N-terminal amino acids of CCP2 added to the C-terminus of CCP1 and one to two corresponding amino acids deleted from the N-terminus of CCP3 of DAF. In some specific embodiments, hybrid proteins of the invention comprise CCP1 of CFH and CCP3-4 of DAF, with N-terminal amino acids RP of CCP2 added to the C-terminus of CCP1 and amino acids KS correspondingly deleted from the N-terminus of CCP3 of DAF.

[0186] In some embodiments, the CCP1 of CFH in the hybrid proteins of the invention may contain a V62I mutation. In some embodiments, the hybrid protein of the invention further comprises a signal peptide at the N-terminus, for example the amino acid sequence set forth in SEQ ID NO: 17, and / or a tag at the C-terminus, for example a histidine tag, for example a 6xHis tag, for example GHHHHHH.

[0187] In some embodiments, CCP3 of DAF comprises or consists of amino acids 161-222 of the DAF protein, and / or CCP4 comprises or consists of amino acids 223-285 of the DAF protein, with the amino acid positions numbered corresponding to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, CCP3 of DAF has two amino acids deleted from its N-terminus, e.g., comprises or consists of amino acids 163-222 of the DAF protein, with the amino acid positions numbered corresponding to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, CCP3-4 of DAF comprises or consists of amino acids 161-285 of the DAF protein, with the amino acid positions numbered corresponding to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, CCP3-4 of DAF has two amino acids deleted at its N-terminus, e.g., comprises or consists of amino acids 163-285 of the DAF protein, with the amino acid positions numbered corresponding to the amino acid positions set forth in SEQ ID NO: 1.

[0188] In some embodiments, CCP1 of CFH comprises or consists of amino acids 19-82 of the CFH protein, wherein the amino acid positions are numbered corresponding to the amino acid positions set forth in SEQ ID NO: 3. In some embodiments, CCP1 of CFH comprises or consists of amino acids 19-84 of the CFH protein, wherein the amino acid positions are numbered corresponding to the amino acid positions set forth in SEQ ID NO: 3.

[0189] In some embodiments, CCP3-4 of DAF comprise or consist of amino acids 161-285 of the DAF protein, with the amino acid positions numbered corresponding to those set forth in SEQ ID NO: 1. CCP1 of CFH comprises or consists of amino acids 19-82 of the CFH protein, with the amino acid positions numbered corresponding to those set forth in SEQ ID NO: 3.

[0190] In some embodiments, CCP3-4 of DAF comprises or consists of amino acids 163-285 of the DAF protein, with the amino acid positions numbered corresponding to those set forth in SEQ ID NO: 1. CCP1 of CFH comprises or consists of amino acids 19-84 of the CFH protein, with the amino acid positions numbered corresponding to those set forth in SEQ ID NO: 3.

[0191] In some specific embodiments, the DAF is a human DAF protein, such as a native human DAF protein. (i) the amino acid sequence set forth in SEQ ID NO: 1; (ii) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:2; (iii) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of (i) or (ii); or comprising or consisting of It consists of an amino acid sequence set forth in any one of (i) to (iii).

[0192] In some specific embodiments, the CFH is a human CFH protein, such as a native human CFH protein. (i) the amino acid sequence set forth in SEQ ID NO: 3 or 5; (ii) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 4 or 6; (iii) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of (i) or (ii); Contains, or It consists of an amino acid sequence set forth in any one of (i) to (iii).

[0193] In some embodiments, the hybrid proteins of the present invention comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 18-21, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the above amino acid sequences.

[0194] >Human CFH CCP1 or SCR1-Human DAF / CD55 CCP3-4 or SCR3-4 (in bold):

[0195] [ka]

[0196] > Human CFH CCP1 or SCR1-Human DAF / CD55 CCP3-4 or SCR3-4 (in bold), containing the V62I mutation:

[0197] [ka]

[0198] In some embodiments, the hybrid proteins of the invention comprise or consist of an amino acid sequence according to any one of SEQ ID NOs: 22-25, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the above amino acid sequence.

[0199] > Human CFH CCP1 or SCR1-Human DAF / CD55 CCP3-4 or SCR3-4 (in bold)-6xHis (in italics):

[0200] [ka]

[0201] > Human CFH CCP1 or SCR1-Human DAF / CD55 CCP3-4 or SCR3-4 (in bold)-6xHis (in italics): Contains the V62I mutation:

[0202] [ka]

[0203] In some embodiments, the hybrid proteins of the invention comprise or consist of an amino acid sequence according to any one of SEQ ID NOs: 26-29, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the above amino acid sequence.

[0204] >Signal peptide-human CFH CCP1 or SCR1-human DAF / CD55 CCP3-4 or SCR3-4 (in bold)-6xHis (in italics):

[0205] [ka]

[0206] >Signal peptide-human CFH CCP1 or SCR1-human DAF / CD55 CCP3-4 or SCR3-4 (in bold)-6xHis (in italics): Contains the V62I mutation:

[0207] [ka]

[0208] In some embodiments, the hybrid proteins of the present invention comprise or consist of an amino acid sequence according to any one of SEQ ID NOs: 30-33, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the above amino acid sequence.

[0209] >Signal peptide-human CFH CCP1 or SCR1-human DAF / CD55 CCP3-4 or SCR3-4 (in bold):

[0210] [ka]

[0211] >Signal peptide-human CFH CCP1 or SCR1-human DAF / CD55 CCP3-4 or SCR3-4 (in bold): Contains the V62I mutation:

[0212] [ka]

[0213] E. Hybrid Protein Mutants The present invention also relates to hybrid protein mutants with optimized properties, which are obtained by introducing mutations based on the hybrid proteins defined above. Thus, the hybrid proteins defined above of the present invention can be used as parents to obtain hybrid protein mutants.

[0214] Fusion proteins containing the optimized hybrid proteins can maintain the activity of the original hybrid proteins and have better solubility compared to hybrid proteins that do not have the optimizing mutations introduced.

[0215] In some embodiments, the hybrid proteins and mutants thereof of the present invention have better complement inhibitory activity, including the activity of inhibiting the alternative pathway (AP), the classical pathway (CP), and / or C3b deposition.

[0216] In some embodiments, the hybrid proteins of the invention or mutants thereof are effective in blocking the hemolytic activity of human complement. In some embodiments, the present invention relates to hybrid protein mutants that have improved purity, aggregation characteristics, or expression levels compared to the parent hybrid protein. For example, hybrid protein mutants of the present invention have a reduced proportion of (large) aggregates or improved purity compared to the parent hybrid protein. As another example, hybrid protein mutants of the present invention have a reduced proportion of large aggregates but improved expression levels compared to the parent hybrid protein. As another example, hybrid protein mutants of the present invention have increased expression levels compared to the parent hybrid protein. In some embodiments, the present invention relates to hybrid protein mutants that have a reduced proportion of (large) aggregates, increased expression levels, and substantially equivalent activity (e.g., complement inhibitory activity) compared to the parent hybrid protein.

[0217] In some embodiments, the hybrid protein mutants of the present invention may reduce aggregate formation, improve aggregate formation, or increase protein purity compared to the hybrid protein. In some embodiments, the hybrid protein mutants of the present invention have equivalent or improved complement inhibitory activity compared to the hybrid protein. In some embodiments, the hybrid protein mutants of the present invention have equivalent or even increased protein expression levels compared to the hybrid protein.

[0218] In some embodiments, any combination of substitutions, deletions, insertions, and additions can be made to obtain a final mutant protein construct with desired properties (e.g., a reduced percentage of aggregates of the hybrid protein). Amino acid deletions or insertions include deletions or insertions at the amino and / or carboxy termini of the polypeptide sequence, as well as deletions or insertions within the polypeptide sequence. In some embodiments, preferred amino acid mutations are amino acid substitutions, such as single amino acid substitutions or substitutions of segments of the amino acid sequence.

[0219] In some embodiments, mutant hybrid proteins of the invention are effective in inhibiting human classical complement pathway hemolysis and / or human alternative complement pathway hemolysis, hi some embodiments, mutant hybrid proteins of the invention are effective in inhibiting C3b deposition.

[0220] In some embodiments, the hybrid protein mutants of the invention comprise at least one mutation compared to the parent hybrid protein, optionally wherein the mutation is: Improves the formation of large aggregates, Improve the monomer purity of the mutants, Increase protein expression levels, and / or It does not significantly reduce complement inhibitory activity (eg, reduce AP inhibitory activity).

[0221] In some embodiments, a parent hybrid protein of the invention can be a hybrid protein described herein, for example, a hybrid protein described in Section II-D.

[0222] In some embodiments, optimized hybrid proteins, i.e., hybrid protein mutants, may be obtained by introducing one or more mutations into the hybrid protein for optimization.

[0223] As used herein, "optimizing mutations" refers to mutations that further optimize a hybrid protein with respect to properties such as the solubility of a fusion protein that contains the hybrid protein. When referring to optimizing mutations herein, the amino acid positions indicated are those numbered corresponding to the amino acid positions set forth in SEQ ID NO: 20, whether or not indicated.

[0224] In some embodiments, optimizing mutations refer to mutating amino acids in a hybrid protein to structurally similar, more hydrophilic amino acids, e.g., mutating hydrophobic amino acids (e.g., amino acids predicted by software to mediate aggregate formation) to structurally similar, more hydrophilic amino acids, e.g., mutating hydrophobic amino acids to hydrophilic amino acids. In some embodiments, optimizing mutations include the addition of one or more (e.g., 1-5, e.g., 1, 2, 3, 4, or 5) amino acids at the carboxy terminus, e.g., hydrophilic amino acids, e.g., lysine or serine.

[0225] In one embodiment, the optimizing mutations are selected from, for example, mutations at positions 56, 59, 82, 86, 107, 109 and / or 110, numbered corresponding to the amino acid positions set forth in SEQ ID NO: 20, e.g., substitution or addition of one or more (e.g., 1 to 5, e.g., 1, 2, 3, 4, or 5) amino acids at the C-terminus.

[0226] In some embodiments, the optimized hybrid protein comprises: (1) Addition of one or more (e.g., 1 to 5, e.g., 1, 2, 3, 4, or 5) amino acids (e.g., hydrophilic amino acids) at the C-terminus; (2) Substitution of one or more amino acid positions selected from positions 56, 59, 82, 86, 107, 109, or 110, which are numbered corresponding to the amino acid positions set forth in SEQ ID NO: 20 (e.g., substitution with an amino acid that is more hydrophilic than the original amino acid, e.g., a structurally similar, more hydrophilic amino acid, or substitution with a hydrophilic amino acid, e.g., a hydrophilic amino acid that is structurally similar to the amino acid to be substituted). Includes.

[0227] In some embodiments, the optimized hybrid protein comprises: (1) Addition of one or more (e.g., 1 to 5, e.g., 1, 2, 3, 4, or 5) amino acids at the C-terminus; (2) substitutions at positions 59, 82, and 110, numbered corresponding to the amino acid positions set forth in SEQ ID NO:20, and optionally (3) Substitutions at positions 56, 86, 107, and / or 109, numbered corresponding to the amino acid positions set forth in SEQ ID NO: 20 Includes.

[0228] In some embodiments, the optimized hybrid protein comprises: (1) Addition of one or more (e.g., 1 to 5, e.g., 1, 2, 3, 4, or 5) amino acids at the C-terminus; (2) Substitutions at positions 56, 59, 82, and 110, numbered corresponding to the amino acid positions set forth in SEQ ID NO: 20 Includes.

[0229] In some embodiments, the amino acids added at the C-terminus are KS. In some embodiments, the substitution can be to replace the original amino acid with an amino acid that is more hydrophilic than the original amino acid, for example, replacing F with Y.

[0230] In some embodiments, the substitution may be a substitution of an amino acid for a hydrophilic amino acid, such as D, E, S, or T. In some embodiments, the substitution at position 56 is 56E, the substitution at position 59 is 59D or 59S, the substitution at position 82 is 82D, the substitution at position 86 is 86Y, the substitution at position 107 is 107E, the substitution at position 109 is 109D or 109E, and / or the substitution at position 110 is 110T.

[0231] In some embodiments, the optimized hybrid protein comprises a mutation selected from the group consisting of 56E, 59D / S, 82D, 86Y, 107E, 109D / E, 110T or the addition of C-terminal amino acids KS, or a combination thereof.

[0232] In some embodiments, the optimized hybrid protein comprises: (1) the addition of the amino acid KS at the C-terminus, and (2) 56E, 59D / S, 82D, 86Y, 107E, 109D / E, 110T, 59D / 82D / 110T, 59S / 82D / 110T, 59D / 82D / 110T / 56E, or 59S / 82D / 110T / 56E a substitution or combination of substitutions having any one of: The combination of mutations includes a combination of mutations selected from the group consisting of:

[0233] In some embodiments, the hybrid protein mutant of the present invention comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 53-54, 56-58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88-96, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0234] >F1D34-KS (SEQ ID NO: 60)

[0235] [ka]

[0236] >F1D34-KS-L56E (SEQ ID NO: 62)

[0237] [ka]

[0238] >F1D34-KS-L59D (SEQ ID NO: 64)

[0239] [ka]

[0240] >F1D34-KS-L59S (SEQ ID NO: 66)

[0241] [ka]

[0242] >F1D34-KS-G82D (SEQ ID NO: 68)

[0243] [ka]

[0244] >F1D34-KSF86Y (SEQ ID NO: 70)

[0245] [ka]

[0246] >F1D34-KS-F107E (SEQ ID NO: 72)

[0247] [ka]

[0248] >F1D34-KS-L109D (SEQ ID NO: 74)

[0249] [ka]

[0250] >F1D34-KS-L109E (SEQ ID NO: 76)

[0251] [ka]

[0252] >F1D34-KS-I110T (SEQ ID NO: 78)

[0253] [ka]

[0254] >F1D34-KS-59D / 82D / 110T (SEQ ID NO: 80)

[0255] [ka]

[0256] >F1D34-KS-59S / 82D / 110T (SEQ ID NO: 82)

[0257] [ka]

[0258] >F1D34-KS-59D / 82D / 110T / 56E (SEQ ID NO: 84)

[0259] [ka]

[0260] >F1D34-KS-59S / 82D / 110T / 56E (SEQ ID NO: 86)

[0261] [ka]

[0262] In some embodiments, the hybrid protein mutant of the invention comprises: (1) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 60, and including an additional C-terminal KS; (2) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 62, and including a C-terminal KS addition and a 56E mutation; (3) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 64, and including a C-terminal KS addition and a 59D mutation; (4) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 66, and including a C-terminal KS addition and a 59S mutation; (5) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 68, and including a C-terminal KS addition and an 82D mutation; (6) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 70, and including a C-terminal KS addition and an 86Y mutation; (7) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 72, and including a C-terminal KS addition and a 107E mutation; (8) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 74, and including a C-terminal KS addition and a 109D mutation; (9) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 76, and including a C-terminal KS addition and a 109E mutation; (10) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 78, and including a C-terminal KS addition and a 110T mutation; (11) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 80, and including C-terminal KS and 59D-82D-110T mutations; (12) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 82, and including C-terminal KS and 59S-82D-110T mutations; (13) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 84, and including a C-terminal KS and 59D-82D-110T-56E mutation; (14) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 86, and including the C-terminal KS and 59S-82D-110T-56E mutations.

[0263] In some embodiments, the hybrid protein mutant of the present invention comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 53-54, 56-58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88-96.

[0264] In some embodiments, the hybrid protein mutant of the present invention further comprises a signal peptide at the N-terminus, such as the amino acid sequence set forth in SEQ ID NO: 17, and / or a tag at the C-terminus, such as a histidine tag, such as a 6xHis tag, e.g., GHHHHHH.

[0265] III. Fusion Proteins In some embodiments, the present invention relates to a fusion protein comprising an anti-C5 antibody or antigen-binding fragment thereof and a hybrid protein or mutant thereof of the present invention.

[0266] In some embodiments, the fusion protein comprises an anti-C5 antibody or antigen-binding fragment thereof and a hybrid protein or mutant thereof, wherein one or more hybrid proteins or mutants thereof are connected (at their N-terminus or at their C-terminus) to the N-terminus and / or C-terminus of the heavy chain and / or light chain, respectively, of the anti-C5 antibody or antigen-binding fragment thereof, with or without a linker.

[0267] In some embodiments, the fusion protein comprises an anti-C5 antibody or antigen-binding fragment thereof and a hybrid protein or mutant thereof, wherein one or two hybrid proteins or mutants thereof (e.g., at their N-termini) are each connected to the C-terminus of the heavy chain of the anti-C5 antibody or antigen-binding fragment thereof.

[0268] In some embodiments, the fusion protein comprises a full-length anti-C5 antibody. In some embodiments, in the fusion protein, the anti-C5 antibody or antigen-binding fragment thereof is connected to the hybrid protein or mutant thereof with or without a linker. In some embodiments, the linker is one or more glycines (G). n , G.S., G. n S, G n S n , (G n S) n or (GSG) n or (G4S) n wherein n is an integer greater than or equal to 1, e.g., n is an integer of 2, 3, 4, 5, 6, or 7. In some embodiments, the linker is G, GSG, or G4S.

[0269] In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof or fusion protein comprises an Fc region. In some embodiments, the fusion protein comprises a dimeric Fc formed by dimerization of the Fc regions. In some embodiments, the first Fc region and the second Fc region are the same. In other embodiments, the first Fc region and the second Fc region are different, and the two are paired to heterodimerize. In some embodiments, the Fc region of the fusion protein is connected at its C-terminus to the hybrid protein of the invention or a mutant thereof (e.g., at its N-terminus), e.g., the first Fc region and / or the second Fc region is connected to the hybrid protein of the invention or a mutant thereof via or without a linker.

[0270] The Fc region fragment suitable for the anti-C5 antibody or fusion protein can be any antibody Fc region. The Fc region can include native-sequence Fc regions and variant Fc regions. Native-sequence Fc domains cover various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, October 20, 2014, doi:10.3389 / fimmu.2014.00520). For example, the Fc region of an antibody of the present invention can comprise two or three constant domains: a CH2 domain, a CH3 domain, and an optional CH4 domain. In some embodiments, the antibody Fc region can also have an IgG hinge region or a portion of an IgG hinge region at the N-terminus, such as an IgG1 hinge region or a portion of an IgG1 hinge region. Mutations can be contained in the hinge region.

[0271] Preferably, the Fc region contained in the antibody of the fusion protein of the invention comprises CH2-CH3 from the N-terminus to the C-terminus, or hinge region-CH2-CH3 from the N-terminus to the C-terminus. In some embodiments, the Fc region suitable for the antibody or fusion protein of the invention is human IgG Fc, such as human IgG1 Fc, human IgG2 Fc, human IgG3, or human IgG4 Fc, such as the Fc region of human IgG2 or human IgG4, or an Fc region in the form of a human IgG2 / IgG4 hybrid (e.g., a portion of CH2 is derived from IgG2, and a portion of CH2 and CH3 are derived from IgG4, e.g., the Fc regions of the C5 antibody drugs Soliris and Ultomiris).

[0272] The Fc region of the fusion proteins of the invention can be mutated to obtain desired properties. Mutations to Fc regions are known in the art. In one embodiment, the Fc region comprises a mutation that improves antibody stability, particularly the stability of the IgG4 form of the antibody, such as S228P.

[0273] In one embodiment, the Fc region is modified with respect to the properties of the effector function of the Fc region (e.g., complement activation function of the Fc region). In one embodiment, the effector function is reduced or eliminated compared to a wild-type isotype Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from: using an Fc isotype that naturally has reduced or eliminated effector function, and modifying the Fc region.

[0274] In a preferred embodiment, the Fc region has a reduced effector function mediated by the Fc region, such as a reduced or eliminated ADCC or ADCP or CDC effector function, e.g., comprises mutations that achieve the above functions.

[0275] As will be appreciated by those skilled in the art, depending on the intended use of the fusion protein molecules of the invention, the fusion protein molecules of the invention can also include modifications in the Fc domain that alter the binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor.

[0276] In some embodiments, the Fc region comprises a mutation that reduces binding to an Fcγ receptor. For example, in some embodiments, an Fc region used in the present invention has L234A / L235A mutations (LALA mutations, e.g., in an IgG1 Fc region) that reduce binding to an Fcγ receptor.

[0277] In one embodiment, the Fc region comprises a mutation that extends the half-life (e.g., in vivo half-life) of the antibody, e.g., the Fc region comprises a mutation that increases binding to the FcRn receptor. For example, in some embodiments, the Fc region used in the present invention has mutations to increase binding to the FcRn receptor, e.g., one or more of the following: YTE mutations (M252Y / S254T / T256E), LA mutations (M428L / N434A), or LS mutations (M428L / N434S). In some embodiments, the Fc region used in the present invention has LA mutations, YTE mutations, and LS mutations that reduce binding to Fcγ receptors.

[0278] In one embodiment, the Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity thereto.

[0279] In some embodiments, the Fc region comprising a mutation that increases binding to the FcRn receptor comprises an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99% or more identity, to SEQ ID NO: 59 and comprises M428L and N434A.

[0280] In some embodiments, an Fc region suitable for the present invention comprises an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99% or more identity, to SEQ ID NO: 59 and comprises the mutations S228P, M428L and N434A.

[0281] When the Fc regions are different, mutations that facilitate heterodimerization may be contained in the Fc regions comprised by the fusion proteins of the present invention. In one embodiment, mutations are introduced into the CH3 regions of the two Fc regions. Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 region of a first Fc region and the CH3 region of a second Fc region are engineered in a complementary manner, such that each CH3 region (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the other CH3 region engineered in a complementary manner (as a result, the first and second CH3 regions heterodimerize, and no homodimers are formed at all between the two first CH3 regions or between the two second CH3 regions). Preferably, corresponding knob and hole mutations are introduced into the first and second monomeric Fc regions, respectively, based on the knob-in-hole technique. This technique is described, for example, in Merchant, AM et al. (1998), "An efficient route to human bispecific IgG," Nat Biotechnol 16(7): 677-681.

[0282] In some embodiments of the invention, the invention provides a fusion protein comprising a full-length anti-C5 antibody and a hybrid protein or mutant thereof, wherein the hybrid protein or mutant thereof is connected to the anti-C5 antibody (with or without a linker).

[0283] In some embodiments of the invention, the invention provides a fusion protein comprising a full-length anti-C5 antibody and a hybrid protein or mutant thereof, wherein the hybrid protein is connected at its C-terminus or N-terminus (with or without a linker) to an anti-C5 antibody heavy chain.

[0284] In some embodiments of the invention, the invention provides a fusion protein comprising a full-length anti-C5 antibody and a hybrid protein or mutant thereof, an anti-C5 antibody joined at the C-terminus of its Fc region to the N-terminus of the hybrid protein or mutant thereof to form the heavy chain of the fusion protein (with or without a linker); the light chain of the anti-C5 antibody forms the light chain of the fusion protein; For example, the linker may contain one or more glycines (G) n , G.S., G. n S, G n S n , (G n S) n or (GSG) n or (G4S) n wherein n is an integer greater than or equal to 1, e.g., n is an integer of 2, 3, 4, 5, 6, or 7, e.g., the linker is G, GSG, or G4S.

[0285] In some embodiments, the fusion protein comprises two heavy chains and two light chains, preferably wherein the two heavy chains are identical and / or the two light chains are identical. In some embodiments, the heavy chain of the fusion protein comprises or consists of an amino acid sequence set forth in SEQ ID NO: 55, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 or 87, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0286] In some embodiments, the light chain of the fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0287] In some embodiments, the fusion protein comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 55, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, or 87, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0288] In some embodiments, the fusion protein comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 55, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, or 87, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50.

[0289] In some embodiments, the fusion protein of the invention also comprises a signal peptide (eg, at its N-terminus, eg, the N-terminus of the heavy chain), such as the amino acid sequence set forth in SEQ ID NO:17. IV. Anti-C5 antibody In some embodiments, an anti-C5 antibody or antigen-binding fragment thereof suitable for the fusion protein of the invention can be any anti-C5 antibody or antigen-binding fragment thereof.

[0290] In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that specifically binds to C5, such as the anti-C5 antibody or antigen-binding fragment thereof disclosed in CN113754763A, or eculizumab, ravulizumab, pozelimuab, crovalimab, tesidolumab, or antigen-binding fragments thereof. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises one, two, three, four, five, or six CDRs of an antibody that specifically binds to C5, such as the anti-C5 antibody disclosed in CN113754763A, eculizumab, ravulizumab, pozelimuab, crovalimab, or tesidolumab. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises the CDRs of one, two, or three heavy chain variable regions, i.e., HCDR1, HCDR2, and HCDR3, of an antibody that specifically binds to C5, such as the anti-C5 antibodies disclosed in CN113754763A, eculizumab, ravulizumab, pozelimuab, tesidolumab, or clovalimab. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises the CDRs of one, two, or three light chain variable regions, i.e., LCDR1, LCDR2, and LCDR3, of an antibody that specifically binds to C5, such as the anti-C5 antibodies disclosed in CN113754763A, eculizumab, ravulizumab, pozelimuab, tesidolumab, or clovalimab. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises the three heavy chain variable region CDRs and three light chain variable region CDRs of an antibody that specifically binds to C5, such as the anti-C5 antibodies eculizumab, ravulizumab, pozelimuab, tesidolumab, or clovalimab disclosed in CN113754763A. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises the heavy chain variable region of an antibody that specifically binds to C5, such as the anti-C5 antibodies eculizumab, ravulizumab, pozelimuab, tesidolumab, or clovalimab disclosed in CN113754763A. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises the light chain variable region of a known antibody that specifically binds to C5, such as the anti-C5 antibodies eculizumab, ravulizumab, pozelimuab, tesidolumab, or clovalimab disclosed in CN113754763A.In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises the heavy chain variable region and light chain variable region of an antibody that specifically binds to C5, such as the anti-C5 antibodies disclosed in CN113754763A, eculizumab, ravulizumab, pozelimuab, tesidolumab, or clovalimab. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises the heavy chain of an antibody that specifically binds to C5, such as the anti-C5 antibodies disclosed in CN113754763A, eculizumab, ravulizumab, pozelimuab, tesidolumab, or clovalimab. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises the light chain of an antibody that specifically binds to C5, such as the anti-C5 antibodies disclosed in CN113754763A, eculizumab, ravulizumab, pozelimuab, tesidolumab, or clovalimab. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises the heavy and light chains of an antibody that specifically binds to C5, such as the anti-C5 antibodies disclosed in CN113754763A, eculizumab, ravolizumab, pozelimuab, tesidolumab, or clovalimab.

[0291] In some embodiments, the anti-C5 antibodies applicable to the fusion proteins of the present invention are anti-C5 antibodies as defined below, e.g., have a longer duration of in vivo efficacy or dissociate from C5 more rapidly under acidic conditions than under neutral conditions.

[0292] In some embodiments, anti-C5 antibodies applicable to the fusion proteins of the present invention can effectively inhibit the classical human complement pathway or the alternative human complement pathway (e.g., inhibit hemolysis of the pathway). In some embodiments, C5 antibodies of the present invention can effectively inhibit the classical human complement pathway and the alternative human complement pathway (e.g., inhibit hemolysis of the pathway). In some embodiments, C5 antibodies of the present invention have the activity of persistently inhibiting the classical human complement pathway or the alternative human complement pathway (e.g., inhibiting hemolysis of the pathway). In some embodiments, C5 antibodies of the present invention have the activity of persistently inhibiting the classical and alternative human complement pathways (e.g., inhibiting hemolysis of the pathway).

[0293] In some embodiments, an anti-C5 antibody applicable to the fusion protein of the present invention has pH-dependent antigen-binding specificity. In some embodiments, the anti-C5 antibody has a weakened antigen-binding ability (i.e., a faster dissociation rate) under acidic conditions, e.g., a pH less than about 7, 6.5, or 6, e.g., a pH between about 4 and 7.0, 4.5 and 6.5, or 4.5 and 6.0. In some embodiments, the anti-C5 antibody has a higher dissociation rate under acidic conditions (e.g., a pH less than about 7, 6.5, or 6, e.g., a pH between about 4 and 7.0, 4.5 and 6.5, 4.5 and 6.0, 5.0 and 6.0, or 5.5 and 6.0, e.g., about pH 5.8) than under neutral conditions (e.g., a pH between about 7 or about 7 and 8, e.g., about pH 7.4). In some embodiments, the antibody has an extended drug half-life, e.g., an extended in vivo antibody drug half-life.

[0294] In some embodiments, the antibody or antigen-binding fragment thereof has improved stability. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (HCDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3.

[0295] In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (LCDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region.

[0296] In some aspects, the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH). In some aspects, the anti-C5 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL). In some aspects, the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3.

[0297] In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof further comprises an antibody heavy chain constant region, H C. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof further comprises an antibody light chain constant region, L C. In some embodiments, the anti-C5 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, H C and a light chain constant region, L C.

[0298] In some embodiments, the heavy chain variable region of the anti-C5 antibody comprises: (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 39; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 39; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 39, and preferably, the amino acid changes do not occur in the CDR regions.

[0299] In some embodiments, the light chain variable region of the anti-C5 antibody comprises: (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 45; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 45; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 45, and preferably, the amino acid changes do not occur in the CDR regions.

[0300] In some embodiments, the three complementarity determining regions (HCDRs), HCDR1, HCDR2 and HCDR3, from the heavy chain variable region of the anti-C5 antibody are (i) the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH set forth in SEQ ID NO: 39; or (ii) a sequence containing at least one and no more than five, four, three, two, or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions compared to any one of the sequences in (i). is selected from For example, the CDRs are determined by the Chothia scheme.

[0301] In some embodiments, the three complementarity determining regions (LCDRs), LCDR1, LCDR2 and LCDR3, from the light chain variable region of the anti-C5 antibody are (i) the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in the VL of SEQ ID NO: 45; or (ii) a sequence containing at least one and not more than 5, 4, 3, 2, or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions compared to any one of the sequences in (i). is selected from For example, the CDRs are determined by the Chothia scheme.

[0302] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 40, or HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 40.

[0303] In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 41, or HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 41.

[0304] In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 42, or the HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 42.

[0305] In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 46, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 46.

[0306] In some embodiments, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 47, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 47.

[0307] In some embodiments, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 48, or LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 48.

[0308] In some specific embodiments of the present invention, the anti-C5 antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the VL comprises, consists of, or consists of the amino acid sequence set forth in SEQ ID NO: 45, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0309] In some specific embodiments of the present invention, the anti-C5 antibody or antigen-binding fragment thereof comprises an HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 40, an HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 41, an HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 42, an LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 46, an LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 47, and an LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 48.

[0310] In some specific embodiments of the present invention, the anti-C5 antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39, and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 45.

[0311] In some embodiments of the present invention, the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain constant region. In some embodiments, the heavy chain constant region is derived from IgG1, IgG2, IgG3, or IgG4, preferably from the heavy chain constant region of IgG2 or the heavy chain constant region of IgG4, or from an IgG2 / IgG4 hybrid heavy chain constant region (e.g., the CH1 region, hinge region, and part of the CH2 region are derived from IgG2, and part of the CH2 region and the CH3 region are derived from IgG4, e.g., the heavy chain constant regions of the C5 antibody drugs Soliris and Ultomiris). In some embodiments, the heavy chain constant region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 43; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 43; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) compared to SEQ ID NO: 43.

[0312] In some embodiments of the present invention, the anti-C5 antibody or antigen-binding fragment thereof comprises a light chain constant region. In some embodiments, the light chain constant region of the anti-C5 antibody is a lambda light chain constant region or a kappa light chain constant region, such as a kappa light chain constant region. In some embodiments, the light chain constant region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 49; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 49; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) compared to SEQ ID NO: 49.

[0313] In some embodiments of the invention, the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain. In some embodiments of the invention, the anti-C5 antibody or antigen-binding fragment thereof comprises a light chain. In some embodiments of the invention, the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain.

[0314] In some specific embodiments, the heavy chain of the anti-C5 antibody is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 44; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 44; or (iii) comprises or consists of an amino acid sequence that has one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) compared to SEQ ID NO: 44, preferably the amino acid changes occur outside the CDR regions, and preferably the amino acid changes occur outside the heavy chain variable region.

[0315] In some specific embodiments, the light chain of the anti-C5 antibody is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 50; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 50; or (iii) comprises or consists of an amino acid sequence that has one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) compared to SEQ ID NO: 50, preferably the amino acid changes occur outside the CDR regions, and preferably the amino acid changes occur outside the heavy chain variable region.

[0316] In some specific embodiments, the anti-C5 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:44, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:50, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0317] In some specific embodiments, the anti-C5 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:44 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:50.

[0318] In one embodiment of the present invention, the amino acid changes in the anti-C5 antibodies described herein include amino acid substitutions, insertions, or deletions. In a preferred embodiment, the amino acid changes described in the present invention occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes described in the present invention occur in regions outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid changes described in the present invention are amino acid substitutions, preferably conservative substitutions.

[0319] In some embodiments, an anti-C5 antibody or antigen-binding fragment thereof applicable to the fusion protein of the present invention has one or more of the following properties: (i) exhibit the same or similar binding affinity and / or specificity for C5 as the antibodies of the present invention; (ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the invention to C5; (iii) binds to the same or overlapping epitope as an antibody of the present invention; (iv) competes with an antibody of the invention for binding to C5; (v) Possess one or more biological properties of the antibodies of the present invention.

[0320] In some embodiments, the anti-C5 antibody applicable to the fusion protein of the present invention is an antibody in the form of IgG1, or an antibody in the form of IgG2, or an antibody in the form of IgG3, or an antibody in the form of IgG4, for example, an antibody in the form of IgG1. In some embodiments, the light chain constant region of the anti-C5 antibody applicable to the present invention is a lambda light chain constant region or a kappa light chain constant region, such as a kappa light chain constant region.

[0321] In some embodiments, the anti-C5 antibody is a monoclonal antibody. In some embodiments, the anti-C5 antibody is humanized. In some embodiments, the anti-C5 antibody is a chimeric antibody.

[0322] In one embodiment, the anti-C5 antibody of the present invention also encompasses an antibody fragment thereof (e.g., an antigen-binding fragment), preferably an antibody fragment selected from the following: Fab, Fab', Fab'-SH, Fv, single chain antibody (e.g., scFv), (Fab')2, single domain antibody such as VHH, dAb (domain antibody), or linear antibody.

[0323] In one aspect, the present invention also provides an immunoconjugate comprising a fusion protein molecule of the present invention, wherein the molecule of the present invention is conjugated to another agent (e.g., an antihemolytic agent) or a label to form the immunoconjugate.

[0324] V. Polynucleotides, Vectors, and Host Cells The present invention provides nucleic acids encoding any of the above hybrid protein mutants or fusion proteins of the present invention. Vectors containing the nucleic acids are also provided. In one embodiment, the vector is an expression vector (e.g., a pCDNA vector, e.g., pCDNA3.1). Host cells containing the nucleic acids or vectors are also provided. In one embodiment, the host cell is a eukaryote. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells, e.g., 293FT cells or Expi293 cells). In another embodiment, the host cell is a prokaryote.

[0325] In one aspect, the present invention provides a nucleic acid encoding any of the above-described hybrid proteins or mutants thereof. The polypeptide encoded by the nucleic acid, when expressed from an appropriate expression vector, can have DAF function and / or CFH function, e.g., can dissociate C3 convertase and / or C5 convertase, can accelerate decay-accelerating activity for classical pathway C3 convertase and / or alternative pathway C3 convertase, can dissociate alternative pathway C3bBb or bind to C3b, and can have complement inhibitory activity, including activity that inhibits the alternative pathway (AP), classical pathway (CP), and / or C3b deposition.

[0326] The present invention also provides nucleic acids encoding any of the above fusion proteins. To facilitate production and purification, the hybrid protein mutants or fusion proteins of the present invention may be fused at the N-terminus to a secretory signal peptide and / or to a tag peptide that facilitates purification, such as a hexahistidine tag or a biotin marker.

[0327] As will be apparent to one of skill in the art, due to codon degeneracy, the polypeptide amino acid sequence of each chain of each fusion protein may be encoded by multiple nucleic acid sequences. In some embodiments, the nucleic acids of the present invention include nucleic acids encoding any one of the amino acid sequences set forth in SEQ ID NOs: 39, 44, 45, 50, 53-58, and 60-96, or nucleic acids encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 39, 44, 45, 50, 53-58, and 60-96.

[0328] Nucleic acid sequences encoding the hybrid protein mutants of the invention or the fusion protein molecules of the invention can be produced by methods well known in the art, such as by de novo solid phase DNA synthesis or by PCR amplification.

[0329] In one embodiment, one or more vectors are provided that contain the nucleic acid of the present invention.In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector.Vector includes, but is not limited to, a virus, a plasmid, a cosmid, a phage lambda or a yeast artificial chromosome (YAC).In a preferred embodiment, the expression vector is pCDNA, such as pCDNA3.1.

[0330] In one embodiment, a host cell is provided that contains one or more polynucleotides of the present invention. In some embodiments, a host cell is provided that contains an expression vector of the present invention. As used herein, the term "host cell" refers to any type of cell line that can be engineered to produce an antibody molecule of the present invention. Host cells suitable for supporting the replication and expression of an antibody molecule of the present invention are well known in the art. If necessary, such cells can be transfected or transduced with a particular expression vector, and a large number of cells containing the vector can be cultured to inoculate a large-scale fermenter to obtain sufficient quantities of the molecule of the present invention for clinical use.

[0331] Suitable host cells include prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK293, 293F, 293FT, or Expi293 cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human liver cells (HeP G2), CHO cells, NSO cells, and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0. Suitable mammalian host cell lines for producing antibodies are known in the art. In a preferred embodiment, the host cells are CHO cells or HEK293 cells or 293FT cells or Expi293 cells.

[0332] V. Production and Purification of the Molecules of the Invention In yet another aspect, the present invention provides a method for producing a hybrid protein mutant of the present invention, the method comprising culturing a host cell comprising a nucleic acid molecule encoding the hybrid protein mutant under conditions suitable for expression of said hybrid protein mutant, and optionally further comprising isolating the hybrid protein mutant from the host cell or host cell culture medium and / or purifying the hybrid protein mutant.

[0333] In another aspect, the invention provides a method for producing a fusion protein of the invention, the method comprising culturing a host cell containing polynucleotides encoding the polypeptide chains of said fusion protein molecule under conditions suitable for expressing said polypeptide chains, and optionally further comprising assembling the polypeptide chains to produce said fusion protein molecule under conditions suitable for assembly of said polypeptide chains into said molecule.

[0334] For recombinant production, the polynucleotides encoding the polypeptide chains of the hybrid protein mutants of the present invention or the fusion protein molecules of the present invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Methods well known to those skilled in the art can be used to construct expression vectors. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, phage lambda, or yeast artificial chromosomes (YACs). Once an expression vector containing one or more polynucleotides of the present invention for expression has been prepared, the expression vector can be transfected or introduced into an appropriate host cell. Various techniques can be used to achieve this goal, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, liposome-based transfection, or other conventional techniques.

[0335] Molecules prepared as described herein may be purified by known prior art techniques, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A affinity chromatography), size exclusion chromatography, etc. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc.

[0336] The purity of the molecules of the invention may be determined by any of a variety of well-known analytical methods, including size-exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, etc. The physical / chemical properties and / or biological activities of the antibody molecules provided herein may be identified, screened, or characterized by a variety of assays known in the art.

[0337] VI. Assay Methods The hybrid protein mutants or fusion proteins provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0338] The hemolysis inhibitory effect of the hybrid or fusion protein of the present invention can be measured by methods known in the art, such as in vitro assays and / or in vivo animal experiments. For example, hemolysis assays such as those described in Examples 4.1 and 4.2 and Example 10 can be used to test the hemolysis inhibitory effect of the molecules on the classical and / or alternative and / or lectin pathways of complement immunity.

[0339] The inhibitory activity of the hybrid protein of the present invention against complement C3b deposition can be measured by methods known in the art, such as in vitro assays and / or in vivo animal experiments. For example, a hemolytic assay, such as the method described in Example 4.3, can be used to test the inhibitory effect of the above molecules against C3b deposition on the surface of red blood cells.

[0340] The inhibitory activity of the fusion protein of the present invention against complement C3b deposition can be measured by methods known in the art, such as in vitro assays and / or in vivo animal experiments. For example, a hemolytic assay such as that described in Example 9 or Wieslab can be used to test the inhibitory effect of the molecule on C3b deposition on the surface of erythrocytes.

[0341] The binding and dissociation properties of the fusion protein molecules of the present invention with human complement C5 protein can be determined by methods known in the art, such as ELISA, Western blot, ForteBio, and the like.

[0342] VII. PHARMACEUTICAL COMPOSITIONS, PHARMACEUTICAL COMBINATIONS, AND KITS In one aspect, the invention provides a composition, e.g., a pharmaceutical composition, medicament, or formulation, comprising a molecule of the invention (e.g., a hybrid protein mutant or fusion protein, or an immunoconjugate, etc.).

[0343] In one embodiment, the composition further comprises a pharmaceutical excipient, such as a pharmaceutical carrier, including a buffer, as known in the art. As used herein, "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonicity agents, and absorption retardants. For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th Edition, RC Rowe, PJ Seskey and SCOwen, Pharmaceutical Press, London, Chicago.

[0344] The composition or drug or formulation of the present invention can be in various forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injection solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0345] The compositions or medicaments or formulations of the present invention can also contain, in addition to one or more molecules of the present invention, other therapeutic agents necessary for the particular indication being treated and preferably do not adversely affect each other's activity. Thus, in one embodiment, the composition or formulation or medicament, e.g., pharmaceutical composition, contains a combination of one or more molecules of the present invention and one or more other therapeutic agents.

[0346] The composition or drug or formulation of the present invention can be in various forms.These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injection solutions), powders or suspensions, liposomes and suppositories.The composition or drug or formulation of the present invention is suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).The preferred form depends on the intended mode of administration and therapeutic use.

[0347] The present invention also provides pharmaceutical combinations or pharmaceutical combination products comprising the molecules of the present invention (fusion proteins or immunoconjugates thereof). Optionally, the pharmaceutical combinations or pharmaceutical combination products also include one or more other therapeutic agents (e.g., antihemolytic agents).

[0348] The term "pharmaceutical combination" or "combination product" refers to a non-fixed or fixed combination product, including, but not limited to, a kit. The term "non-fixed combination" means that the active ingredients are administered to a patient simultaneously as separate entities, either without specific time restrictions or sequentially at the same or different time intervals, such administration providing prophylactically or therapeutically effective levels of the two or more active agents in the patient. In some embodiments, the hybrid protein of the present invention and other therapeutic agents used in the pharmaceutical combination are administered at levels equal to or lower than the levels at which they would be administered alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosages and / or time intervals of the two or more active agents are preferably selected so that the combined use of each part provides a greater effect in treating a disease or condition than would be obtained by using either component alone. Each component may be in a separate formulation, which may be the same or different.

[0349] The present invention also provides kits containing the pharmaceutical combination, e.g., the kits may include, in the same package: - a first container containing a pharmaceutical composition comprising a molecule of the invention (a fusion protein or an immunoconjugate thereof), - a second container containing a pharmaceutical composition optionally containing one or more other therapeutic agents (e.g., an antihemolytic agent) (in some embodiments, two or more other therapeutic agents are in the same container or in different containers); Contains:

[0350] VIII. Uses and Methods In one aspect, the present invention provides a method for preventing or treating a complement system-related disease or condition in a subject, the method comprising the step of administering to the subject an effective amount of a molecule of the present invention (e.g., a hybrid protein mutant or fusion protein or an immunoconjugate thereof, etc.), or a composition or medicament or formulation comprising same.

[0351] In some embodiments, the complement system-associated disease is caused by abnormal activation of the complement system or dysregulation of the complement system. In some embodiments, the abnormal activation of the complement system or dysregulation of the complement system is due to, for example, microbial infection or increased autoimmune antibodies, or due to a decrease, loss, impairment, or functional interference or disruption of complement regulatory proteins. In some embodiments, treatment of the disease would benefit from inhibiting the activity of the complement system.

[0352] In some embodiments, the complement system-related disease or condition is a complement C5-related disease or condition. In some embodiments, the subject has an elevated level of complement C5 protein (e.g., at the nucleic acid or protein level) (e.g., compared to a sample from a healthy subject). In some embodiments, the subject has an elevated level of complement C5 protein (e.g., at the nucleic acid or protein level) in their blood or blood cells (e.g., compared to the blood or blood cells of a healthy subject). In some embodiments, the treatment of the disease will benefit from inhibiting complement C5 protein at the nucleic acid or protein level.

[0353] In some embodiments, the complement C5-associated disease or disorder can be a disease requiring inhibition of hemolysis, such as a disease requiring inhibition of hemolysis of the classical pathway of complement immunity and / or the alternative pathway of complement immunity.

[0354] In some embodiments, the complement system-associated disease or disorder is a disease in which inhibition of C3b deposition activity is desirable, such as dense deposition disease (DDD). In some embodiments, the hybrid protein mutant or fusion protein molecule or immunoconjugate of the invention or a composition or medicament or formulation comprising same will delay the onset of the condition and / or symptoms associated with the condition.

[0355] In some embodiments, the hybrid protein mutants or fusion protein molecules or immunoconjugates, or nucleic acids encoding them, or compositions or medicaments or formulations comprising the proteins or nucleic acids of the invention may be administered in combination with one or more other therapies, e.g., treatment modalities and / or other therapeutic agents, for the uses described herein, e.g., for the prevention and / or treatment of the associated diseases or disorders mentioned herein.

[0356] The route of administration of the hybrid protein mutants or fusion protein molecules or immunoconjugates, or the nucleic acids encoding them, or compositions or medicaments or formulations comprising the proteins or nucleic acids of the invention, is dependent on known methods, e.g., injection or infusion.

[0357] In another aspect, the invention provides the use of a hybrid protein mutant or fusion protein molecule or immunoconjugate, or nucleic acids encoding them, or a composition or medicament or formulation comprising a protein or nucleic acid of the invention, in the production or preparation of a medicament for a use described herein, e.g., for preventing or treating a complement C5-related disease or condition referred to herein.

[0358] In other aspects, the present invention also relates to hybrid protein mutants or fusion protein molecules or immunoconjugates, or nucleic acids encoding them, or compositions or medicaments or formulations comprising the proteins or nucleic acids of the invention, for use in therapy, e.g., to prevent or treat complement C5-related diseases or conditions mentioned herein.

[0359] IV. Diagnosis and Detection In certain embodiments, the fusion proteins or immunoconjugates thereof provided herein can be used to detect the presence of complement C5 in a biological sample.

[0360] As used herein, the term "detection" includes quantitative or qualitative detection, and exemplary detection methods may include immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR techniques (e.g., RT-PCR). In certain embodiments, the biological sample is a body fluid.

[0361] In certain embodiments, the method comprises contacting a biological sample with a fusion protein molecule described herein under conditions that allow the fusion protein molecule described herein to bind to complement C5, and detecting whether a complex is formed between the molecule and complement C5, where the formation of the complex indicates the presence of complement C5. The method may be an in vitro method or an in vivo method. In one embodiment, the fusion protein molecule of the present invention is used to select subjects suitable for treatment with an inhibitor of complement C5 (e.g., an antibody against complement C5, such as an anti-C5 antibody or antigen-binding fragment thereof, or a fusion protein thereof, or an immunoconjugate thereof, of the present invention), e.g., complement C5 is a biomarker for selecting subjects.

[0362] In certain embodiments, a labeled fusion protein or immunoconjugate thereof of the invention is provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels) and moieties, such as enzymes or ligands, that are indirectly detected, for example, by enzymatic reaction or molecular interaction.

[0363] In some embodiments, the label is a label such as biotin or a His tag. In some embodiments provided herein, the sample is obtained prior to treatment with a molecule of the invention or a composition, drug, or formulation comprising the same. In some embodiments, the sample is obtained prior to treatment with another therapy. In some embodiments, the sample is obtained during or after treatment with another therapy.

[0364] In some embodiments, complement C5 is detected pre-treatment, eg, before the start of treatment or pre-treatment after a treatment interval. In some embodiments, a method of treating a disease of the present invention is provided, the method comprising the steps of testing a subject (e.g., a sample) (e.g., a subject sample) for the presence of complement C5, thereby determining a complement C5 level, comparing the complement C5 level to a control value (e.g., a value in a normal individual), and if the complement C5 level is greater than the control value, administering a therapeutically effective amount of a molecule of the present invention or a composition, drug, or formulation comprising same to the subject, optionally in combination with one or more other therapies, thereby treating the disease. [Example]

[0365] Example 1. Molecular construction of hybrid proteins To construct protein expression vectors for the hybrid molecules of the present invention and the prior art complement regulatory molecules, DNA encoding each protein was subcloned into the pcDNA3.1 expression vector (purchased from Biofeng) using synthetic gene DNA (GENEWIZ Inc., Suzhou / Sangon Biotech (Shanghai) Co., Ltd.) as a template using conventional molecular cloning techniques. The plasmids were verified by sequencing and then used for transient protein expression. The signal peptide MGWSCIILFLVATATGVHS was added to the N-terminus of each encoded protein, and a GHHHHHH6xHis tag was added to the C-terminus to facilitate subsequent secretory expression in mammalian cells and nickel column purification.

[0366] The amino acid sequences of each protein are as follows: >CR13m (human CR1 CCP1-3 or SCR1-3, containing N29K, S37Y, G79D and D109N mutations, sequence from patent US9988611_B2): SEQ ID NO: 34

[0367] [ka]

[0368] >D24 (human DAF / CD55 CCP2-4 or SCR2-4, the sequence can be found in Hui-fen Zhang et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 276, No. 29, published July 20, pp. 27290-27295, 2001): SEQ ID NO: 35

[0369] [ka]

[0370] >FH15 (human CFH CCP1-5 or SCR1-5, including the V62I mutation, for the sequence see Masha Fridkis-Hareli et al., Blood. 2011 Oct. 27; 118(17): 4705-4713 and Agustin Tortajada et al., Hum Mol Genet. 2009 Sep. 15; 18(18): 3452-3461): SEQ ID NO: 36

[0371] [ka]

[0372] >F1D34-1 (human CFH CCP1 or SCR1-human DAF / CD55 CCP3-4 or SCR3-4): SEQ ID NO: 22 >F1D34-2i (human CFH CCP1 or SCR1-human DAF / CD55 CCP3-4 or SCR3-4, containing the V62I mutation): SEQ ID NO: 24 >CR15D34 (human CR1 CCP15 or SCR15-human DAF / CD55 CCP3-4 or SCR3-4): SEQ ID NO: 37

[0373] [ka]

[0374] >FH13 (human CFH CCP1-3 or SCR1-3, containing a V62I mutation, with two more amino acids at the C-terminus for attaching a His purification tag): SEQ ID NO: 51

[0375] [ka]

[0376] >D14 (human DAF / CD55 CCP1-4 or SCR1-4, no leader sequence, with two more amino acids at the C-terminus to attach a His purification tag): SEQ ID NO: 52

[0377] [ka]

[0378] Example 2. Expression and purification of hybrid proteins After amplifying the plasmid in strain DH5 alpha (Yeasen Biotechnology), the plasmid was prepared using NucleoBond Xtra Midi Plus (MACHEREY-NAAGEL; see the product manual for details). The prepared plasmid was transfected into Expi293 cells (purchased from Thermo Fisher) using PEI (Polyscience) for transient protein expression (see Jager, V. et al., BMC Biotechnol 13, 52, 2013 for transfection methods). The collected supernatant was purified by affinity chromatography on a nickel ion pre-packed column (GE Life Sciences, HisTrap HP; see the product manual for details). The protein sample was first washed with 20 mM imidazole buffer to remove impurities, and then eluted with 250 mM or 500 mM imidazole buffer. The protein sample was then buffer-exchanged into PBS buffer using an ultrafiltration concentrator (Amicon® Ultra, Merck Millipore). Protein samples after liquid exchange were filter sterilized through a 0.2 μm filter, and protein quantification was performed according to the theoretical extinction coefficient using the NanoDrop (Thermofisher) A280 method.

[0379] Example 3. Protein Purity Detection SDS-PAGE 2.5 μg of protein was added proportionally to loading buffer (Sangon Biotech) with and without reducing agent, respectively. Samples in loading buffer containing reducing agent were boiled at 95°C for 5 minutes to completely denature the protein. Samples using loading buffer without reducing agent were not heat-treated. Electrophoresis was performed using 10% Precast-GLgel Tris-Glycine precast gels (Sangon Biotech) on a Mini-PROTEAN® Tetra electrophoresis system (BioRad). After electrophoresis was completed, the gels were stained with 0.1% Coomassie Brilliant Blue staining solution and destained with ethanol-glacial acetic acid solution.

[0380] The results of SDS-PAGE electrophoresis showed that, except for the slight diffusion of the D14 protein band, the other protein bands were relatively clear, indicating that the purity of the protein was good under both reducing and non-reducing conditions (as shown in Figure 2). In addition, there was a certain discrepancy between the electrophoretic molecular weight and the theoretical molecular weight due to differences in the reduction, denaturation, and modification states of the protein and the electrophoresis conditions.

[0381] SEC-HPLC Protein purity analysis was performed using size exclusion chromatography (SEC). Purified protein samples in PBS were applied to a TSKgel Super SW3000 column (300 × 4.6 mm, 5 μm; TOSOH). SEC was performed using a U3000 HPLC instrument (DIONEX). Elution was performed isocratically at a flow rate of 0.25 mL / min. All proteins were detected using UV detection at wavelengths of 280 nm and 214 nm. Component analysis was performed using the instrument's built-in software.

[0382] The results of the analysis showed that the monomer purities of the hybrid proteins F1D34-1 and F1D34-2i in solution reached 92% and 95%, respectively (FIG. 3A / B), indicating good monomer purity.

[0383] Example 4. Detection of complement inhibitory activity of hybrid proteins 4.1 Detection of inhibitory activity against the classical complement pathway (CP) In this experiment, the degree of inhibition of hemolysin-mediated hemolysis of sheep red blood cells caused by the complement activity of normal human serum was measured by testing the test proteins at various concentrations, and the inhibitory activity of each protein against complement CP was analyzed and compared.

[0384] The experimental process was briefly described as follows: (1) Gradient dilution of protein: Protein was prepared into a 12000 nM solution using GVB++ buffer (Gelatin Flora Buffer containing Ca2+ and Mg2+, Cat. No.: 25-02080, TIANDZ), followed by 4-fold gradient dilution, for a total of 7 gradients.

[0385] (2) Serum dilution: Normal human serum NHS (Shanghai Schbio Co., Ltd.) was removed from a −70° C. refrigerator and then naturally thawed at 4° C., and the serum was then diluted to 4% with GVB++ buffer.

[0386] (3) Activation of sheep red blood cells: Sheep red blood cells (Nanjing SenBeiJia Biological Technology Co., Ltd.) were washed with GVB++ buffer until the supernatant was clear, then resuspended in GVB++ buffer. Hemolysin (BM351Y, Beijing Bersee Technology Co., Ltd.) was added to the red blood cell suspension at a ratio of 1:4000, followed by incubation at 4°C for 15 minutes to activate the sheep red blood cells. The activated sheep red blood cells were washed twice with GVB++ and resuspended in GVB++ buffer.

[0387] (4) Incubation: 25 μL each of diluted protein and serum was mixed in a 96-well plate, and then 50 μL of the erythrocyte suspension obtained in step (3) was added, resulting in an initial final protein concentration of 3000 nM and a final serum concentration of 1%. The mixture was thoroughly blended and placed in a constant temperature incubator at 37°C for 1 hour, along with a negative control (containing only serum) and a positive control (containing only serum and erythrocytes).

[0388] (5) Termination: After incubation, 100 μL of 20 mM EDTA-GVB buffer (GVBE) was added to each reaction well to terminate the reaction. (6) Reading: The 96-well plate was centrifuged at 3000 rpm for 5 minutes. 100 μL of the supernatant was added to a new 96-well flat-bottom plate. The absorbance value at OD405 nm was measured using a multi-function plate reader, and the data was saved.

[0389] (7) Data processing: The obtained OD405nm reading was substituted into the following formula to calculate the erythrocyte hemolysis inhibition rate. Hemolysis inhibition rate (%) = (positive control reading - experimental reading) / (positive control reading - negative control reading) * 100% The IC50 value of the protein was then calculated by plotting a four-parameter fitting curve using the final protein concentration as the horizontal axis and the hemolysis inhibition rate as the vertical axis model. The results are shown in Figure 4.

[0390] Experimental results for CP hemolysis inhibitory activity showed that both the CFH and DAF hybrid proteins F1D34-1 and F1D34-2i had potent CP inhibitory activity, comparable to that of the CR1 activity-enhancing fragment CR13m and the DAF fragment D24, significantly better than that of the CFH fragment FH15 and the DAF fragment D14, and far superior to that of the CR1 and DAF hybrid protein CR15D34. Only weak CP inhibitory activity was detected for CR15D34, whereas the CFH fragment FH13 had almost no activity (as shown in Figure 4), indicating that the hybrid proteins obtained through domain hybridization had excellent CP inhibitory activity.

[0391] 4.2 Detection of inhibitory activity against the alternative complement pathway (AP) In this experiment, the degree of inhibition of rabbit erythrocyte hemolysis caused by the complement activity of normal human serum was measured by testing the test proteins at various concentrations, and the inhibitory activity of each protein against complement AP was analyzed and compared.

[0392] The experimental process was briefly described as follows: (1) Gradient dilution of protein: Protein was prepared into a 28,000 nM solution using GVBMG buffer (Gelatin Flora Buffer containing Mg2+ and EGTA, Cat. No.: 25-02090, TIANDZ), followed by 4-fold gradient dilution, for a total of 7 gradients.

[0393] (2) Serum dilution: Normal human serum NHS (Shanghai Schbio Co., Ltd.) was removed from a −70°C refrigerator and then naturally thawed at 4°C, and the serum was diluted to 60% with GVBMG.

[0394] (3) Preparation of rabbit erythrocytes: Rabbit erythrocytes (Nanjing SenBeiJia Biological Technology Co., Ltd.) were washed with GVBMG until the supernatant was clear, and then resuspended in GVBMG.

[0395] (4) Incubation: 25 μL each of diluted protein and serum was mixed in a 96-well plate, and then 50 μL of the erythrocyte suspension obtained in step (3) was added, resulting in an initial final protein concentration of 7000 nM and a final serum concentration of 15%. The mixture was thoroughly blended and placed in a constant temperature incubator at 37°C for 1 hour, along with a negative control (containing only serum) and a positive control (containing only serum and erythrocytes).

[0396] (5) Termination: After incubation, 100 μL of 10 mM EDTA-GVB buffer (GVBE) was added to each reaction well to terminate the reaction. (6) Reading: The 96-well plate was centrifuged at 3000 rpm for 5 minutes. 100 μL of the supernatant was added to a new 96-well flat-bottom plate. The absorbance value at OD405 nm was measured using a multi-function plate reader, and the data was saved.

[0397] (7) Data processing: The obtained OD405nm reading was substituted into the following formula to calculate the erythrocyte hemolysis inhibition rate. Hemolysis inhibition rate (%) = (positive control reading - experimental reading) / (positive control reading - negative control reading) * 100% The IC50 value of the antibody was then calculated by plotting a four-parameter fitting curve using the final protein concentration on the horizontal axis and the hemolysis inhibition rate on the vertical axis. The results are shown in Figure 5.

[0398] The experimental test results for AP hemolysis inhibitory activity showed that both the CFH and DAF hybrid proteins F1D34-1 and F1D34-2i had strong AP inhibitory activity, which was better than that of the CFH fragments FH15 and FH13 and the DAF fragments D24 and D14, and also better than that of the CR1 and DAF hybrid protein CR15D34 and the CR1 activity-enhancing fragment CR13m (as shown in Figure 5), indicating that the hybrid proteins obtained through domain hybridization had better AP inhibitory activity.

[0399] 4.3. Detection of inhibitory activity on complement C3b deposition In this experiment, we detected the inhibition of C3b deposition on the surface of rabbit erythrocytes by various complement regulatory proteins after human complement activation, and analyzed and compared the inhibitory activity of each protein on C3b deposition.

[0400] The experimental process was briefly described as follows: (1) Gradient dilution of protein: Protein was prepared to a 1400 nM solution using GVBMG buffer (Gelatin Flora Buffer containing Mg2+ and EGTA, Cat. No.: 25-02090, TIANDZ), followed by 3-fold gradient dilution, for a total of 7 gradients.

[0401] (2) Serum dilution: To prevent complement activation from causing hemolysis and destruction of rabbit red blood cells, which would prevent accurate detection of C3b deposition on the cell surface, C5-depleted normal human serum was used for the experiment. C5-Dpl NHS (Complement Technology) was removed from a -70°C refrigerator and then thawed at 4°C. The serum was diluted to 40% with GVBMG.

[0402] (3) Preparation of rabbit red blood cells: 4% rabbit red blood cells (Nanjing SenBeiJia Biological Technology Co., Ltd.) were washed with GVBMG until the supernatant was clear, and then resuspended in GVBMG to a cell density of 1 × 10 7 The cell density was adjusted to 100 cells / mL.

[0403] (4) Incubation: 25 μL each of the diluted protein and serum was mixed in a 96-well plate, and then 50 μL of the red blood cell suspension obtained in step (3) was added, resulting in an initial final protein concentration of 350 nM and a final serum concentration of 10%. The mixture was thoroughly blended and placed in a constant temperature incubator at 37°C for 1 hour, along with a negative control (containing only complement-inactivated serum and red blood cells) and a positive control (containing only normal serum and red blood cells).

[0404] (5) Termination: After incubation, 100 μL of 10 mM EDTA-GVB buffer (GVBE) was added to each reaction well to terminate the reaction. (6) Detection: The incubated cells were washed three times with PBS, and fluorescein-labeled anti-human C3b / iC3b antibody (APC anti-complement C3b / iC3b antibody, Biolegend) was added for staining, followed by incubation at 4°C for 30 minutes and washing three times again. The APC fluorescence MFI (mean fluorescence intensity) of each sample was measured using a flow cytometer (Cytoflex, Beckman), and the data were saved.

[0405] (7) Data processing: The C3b deposition rate on the surface of red blood cells was calculated using the following formula. Deposition rate (%) = MFI value of experimental group / MFI value of positive control * 100% The IC50 values ​​of the antibodies were then calculated by plotting a four-parameter fit curve using the final protein concentration on the horizontal axis and the deposition rate on the vertical axis. The results are shown in Figure 6.

[0406] The experimental test results on C3b deposition showed that both the CFH and DAF hybrid proteins F1D34-1 and F1D34-2i had potent C3b deposition inhibitory activity, which was better than that of the CFH fragments FH15 and FH13 and the DAF fragments D24 and D14, and also better than that of the CR1 and DAF hybrid protein CR15D34 and the CR1 activity-enhancing fragment CR13m (as shown in Figure 6), indicating that the hybrid proteins obtained through domain hybridization had better C3b deposition inhibitory activity.

[0407] Example 5: Preparation and activity of anti-C5 antibodies 5.1 Preparation of anti-C5 antibody Anti-human C5 antibody was obtained through mouse immunization, hybridoma screening and humanization transformation. For its sequence and preparation method, please refer to CN113754763A (16H46L39am).

[0408] 5.2 Manipulation of the pH Dependence of Anti-C5 Antibody Previous reports have shown that molecules with pH-dependent dissociation properties can rapidly dissociate from target molecules during endocytosis, thereby reducing target-mediated clearance (TMDD) in vivo and extending half-life, and that such molecules with pH-dependent properties can be rapidly obtained through "histidine substitution" (Casim A. Sarkar et al., Nat Biotechnol. 2002 Sep; 20(9):908-13. doi:10.1038 / nbt725; Tomoyuki Igawa et al., Nat Biotechnol. 2010 Nov; 28(11):1203-7. doi:10.1038 / nbt.1691).

[0409] After screening for "histidine substitution" in the CDR region of the antibody, a C5 antibody phAb with pH-dependent dissociation properties was finally obtained, the sequence of which is shown in Table 1.

[0410] ForteBio Octet K2 was used to detect the pH-dependent dissociation properties of the antibody. The detection process is briefly described as follows. Using an ANTI-HUMAN IgG FC sensor (FORTEBIO, 18-5060), antibodies were loaded in a pH-neutral buffer at an antibody concentration of 10 μg / mL. After baseline measurement, the antibodies were bound to C5 antigen (Complement Technology, A120) at an antigen concentration of 10 μg / mL, followed by dissociation in pH 7.4 and pH 5.4 buffers, respectively. The pH 5.4 buffer can be obtained by adjusting the pH using phosphate or citrate buffers. The parameters were: loading 180 seconds, baseline 60 seconds, binding 180 seconds, and dissociation 500 seconds. For detailed methodology, please refer to the ForteBio Octet instruction manual.

[0411] The detection results are shown in FIG. 7, which show that the modified C5 antibody phAb dissociates from C5 more rapidly in pH 5.4 buffer than in pH 7.4 buffer. 5.3 In vivo activity detection of pH-dependent C5 antibody To verify the complement inhibitory activity of the pH-dependent C5 antibody in animals, human C5 transgenic mice were used for the study. Specifically, serum was collected separately from the human C5 transgenic mice (Southern Model Animal Center), and the human C5 content in the serum was detected using ELISA. According to the human C5 content in the serum, 1 mg of C5 antibody was administered per 105 μg / mL of C5 injected via the tail vein. The phAb is a C5 antibody with pH-dependent properties of the present application, and the Ab is 16H46L39am disclosed in CN113754763A. Both use the same human IgG4 constant region sequence (including S228P and LA mutations (M428L / N434A), SEQ ID NO: 59). Serum was collected before and after injection (1 hour, 3 days, 6 days, 9 days, 12 days, 15 days, 20 days, and 23 days), and serum complement activity was tested according to the following process.

[0412] (1) Chicken red blood cells (SBJ-RBC-C003, Nanjing SenBeiJia Biological Technology Co., Ltd.) were taken in 1 mL and washed three times with GVB++ (gelatin Flora buffer containing Ca2+ and Mg2+, Cat. No.: 25-02080, Tianenze). After resuspension, anti-chicken red blood cell antibody (1 μg / mL, anti-cRBC, Cat. No.: 203-4139, Rockland) was added, and the mixture was incubated at 4°C for 15 minutes to activate the chicken red blood cells. They were then centrifuged, washed three times with GVB++, and the red blood cells were resuspended in 1 mL of GVB++.

[0413] (2) C5-depleted human serum (C5-depleted NHS, CompTech) was mixed with a transgenic mouse serum sample at a final concentration of 6.25%. Both diluents were GVB++. Control 1 contained only serum (ODctrl1) and GVB++. Control 2 contained a serum sample (ODctrl2) before antibody injection, and its hemolysis level was used as a control for 100% hemolysis.

[0414] (3) 30 μl of activated chicken red blood cells was pipetted into the serum and incubated at 37° C. for 1 hour. After centrifugation, 80 μl of the supernatant was taken and the OD405 was read using a multi-function plate reader.

[0415] Hemolysis rate (%) = (OD sample - ODctrl1) / (ODctrl2 - ODctrl1) x 100 The test results are shown in Figure 8. The test results show that the pH-dependent C5 antibody phAb has a longer-lasting complement inhibitory activity.

[0416] [Table 3]

[0417] Example 6 Construction of Hybrid Protein Mutants and Fusion Protein Molecules Comprising Hybrid Protein Mutants 6.1 Molecular Construction and Mutant Design To obtain a candidate molecule with better complement inhibitory activity, we fused the resulting hybrid protein molecule with the C5 antibody (the structure of the fusion protein is shown in Figure 9 ), resulting in a hybrid protein-antibody fusion protein (Ab-F1D34: heavy chain: SEQ ID NO: 60, light chain: antibody light chain: SEQ ID NO: 50). Transient expression results showed that the Ab-F1D34 protein was fully expressed and had very high monomer purity in solution. However, the protein solution became cloudy when stored at 4°C, suggesting that there may be some defect in the protein's solubility that is not conducive to the development of high-concentration pharmaceutical formulations. Ab-F15 was also prepared as a control fusion protein (Ab-F15: heavy chain: SEQ ID NO: 97, light chain: antibody light chain: SEQ ID NO: 50).

[0418] Because the antibody itself has good solubility, we first optimized the solubility of the hybrid protein. First, a structural model of the hybrid protein F1D34 was constructed based on the previously reported crystal structures of CFH (PDB: 5o35) and DAF (PDB: 7do4) (as shown in Figure 1). Next, we used the computer-aided molecular design software Discovery Studio 2020 (BIOVIA) to predict aggregation sites (as shown in Figure 10, the red regions are hydrophobic amino acid sites that may mediate aggregation). Selected amino acid sites that may mediate protein aggregation were then mutated. Finally, the mutated protein model was re-predicted to obtain beneficial mutations that reduce protein aggregation, i.e., the amino acids were mutated to structurally similar, more hydrophilic amino acids. The mutation list is as follows (Tables 4 and 5).

[0419] [Table 4]

[0420] Here, C'-KS indicates that KS amino acids have been added at the C' terminus of the fusion protein F1D34, ie, the fusion protein contains an extended DAF sequence, with the remaining mutant amino acid positions corresponding to the F1D34 sequence.

[0421] [Table 5]

[0422] The amino acid sequences of each mutant are as follows: >F1D34-KS (SEQ ID NO: 60) >F1D34-KS-6×His (italics): SEQ ID NO: 56

[0423] [ka]

[0424] >F1D34-KS-L56E (SEQ ID NO: 62) >F1D34-KS-L56E-6×His (italics): SEQ ID NO: 57

[0425] [ka]

[0426] >F1D34-KS-L59D (SEQ ID NO: 64) >F1D34-KS-L59D-6×His (italics): SEQ ID NO: 58

[0427] [ka]

[0428] >F1D34-KS-L59S (SEQ ID NO: 66) >F1D34-KS-L59S-6×His (italics): SEQ ID NO: 88

[0429] [ka]

[0430] >F1D34-KS-G82D (SEQ ID NO: 68) >F1D34-KS-G82D-6×His (italics): SEQ ID NO: 89

[0431] [ka]

[0432] >F1D34-KSF86Y (SEQ ID NO: 70) >F1D34-KSF86Y-6×His (italics): SEQ ID NO: 91

[0433] [ka]

[0434] >F1D34-KS-F107E (SEQ ID NO: 72) >F1D34-KS-F107E×His (italics): SEQ ID NO: 92

[0435] [ka]

[0436] >F1D34-KS-L109D: SEQ ID NO: 74 >F1D34-KS-L109D×His (italics): SEQ ID NO: 93

[0437] [ka]

[0438] >F1D34-KS-L109E (SEQ ID NO: 76) >F1D34-KS-L109E-6×His (italics): SEQ ID NO: 94

[0439] [ka]

[0440] >F1D34-KS-I110T (SEQ ID NO: 78) >F1D34-KS-I110T-6×His (italics): SEQ ID NO: 90

[0441] [ka]

[0442] The 59D, 59S, 82D, 110T and 56E mutations were selected for combination to construct a hybrid protein mutant with combined mutations, the sequence of which is as follows:

[0443] >F1D34-KS-59D / 82D / 110T (SEQ ID NO: 80) >F1D KS-59D / 82D / 110T-659D / 8 (italics): SEQ ID NO: 95

[0444] [ka]

[0445] >F1D34-KS-59S / 82D / 110T: SEQ ID NO: 82 >F1D34-KS-59S / 82D / 110T-6×His (italics): SEQ ID NO: 96

[0446] [ka]

[0447] >F1D34-KS-59D / 82D / 110T / 56E (SEQ ID NO: 84) >F1D34-KS-59D / 82D / 110T / 56E-6×His (italics): SEQ ID NO: 54

[0448] [ka]

[0449] >F1D34-KS-59S / 82D / 110T / 56E (SEQ ID NO: 86) >F1D34-KS-59S / 82D / 110T / 56E-6×His (italics): SEQ ID NO: 53

[0450] [ka]

[0451] The hybrid protein mutant molecules were fused with the C5 antibody (the structure of the fusion protein is shown in Figure 9) to obtain hybrid protein-antibody fusion proteins, where the heavy chain amino acid sequence of each fusion protein is as follows:

[0452] >Ab-F1D34-HC

[0453] [ka]

[0454] (SEQ ID NO: 55, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 60) >Ab-F1D34 C'-KS-HC

[0455] [ka]

[0456] (SEQ ID NO: 61, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 60) Each fusion protein mutant was then constructed based on Ab-F1D34 C'-KS-HC, with the sequences as follows:

[0457] >Ab-F1D34 L56E-HC

[0458] [ka]

[0459] (SEQ ID NO: 63, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 62) >Ab-F1D34 L59D-HC

[0460] [ka]

[0461] (SEQ ID NO: 65, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 64) >Ab-F1D34 L59S-HC

[0462] [ka]

[0463] (SEQ ID NO: 67, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 66) >Ab-F1D34 G82D-HC

[0464] [ka]

[0465] (SEQ ID NO: 69, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 68) >Ab-F1D34 F86Y-HC

[0466] [ka]

[0467] (SEQ ID NO: 71, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 70) >Ab-F1D34 F107E-HC

[0468] [ka]

[0469] (SEQ ID NO: 73, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 72) >Ab-F1D34 L109D-HC

[0470] [ka]

[0471] (SEQ ID NO: 75, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 74) >Ab-F1D34 L109E-HC

[0472] [ka]

[0473] (SEQ ID NO: 77, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 76) >Ab-F1D34 I110T-HC

[0474] [ka]

[0475] (SEQ ID NO: 79, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 78) A combinatorial mutant fusion protein was constructed, the heavy chain amino acid sequence of which was as follows:

[0476] >Ab-F1D34 59D / 82D / 110T-HC

[0477] [ka]

[0478] (SEQ ID NO: 81, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 80) >Ab-F1D34 59S / 82D / 110T-HC

[0479] [ka]

[0480] (SEQ ID NO: 83, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 82) >Ab-F1D34 59D / 82D / 110T / 56E-HC

[0481] [ka]

[0482] (SEQ ID NO: 85, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 84) >Ab-F1D34 59S / 82D / 110T / 56E-HC

[0483] [ka]

[0484] (SEQ ID NO: 87, the underlined part is the optimized hybrid protein, the amino acid sequence of which is SEQ ID NO: 86) The hybrid protein mutant was constructed and the protein was expressed and purified in the manner of Examples 1 and 2, where the construct is constructed as signal peptide (SEQ ID NO: 17) + hybrid protein mutant + 6xHis tag (SEQ ID NO: 16).

[0485] For fusion proteins, genes encoding the fusion proteins were synthesized by GENEWIZ and Nanjing Tsingke Biotechnology Co., Ltd. For secretory expression, a signal peptide was added to the N-terminus of the protein. The signal peptide sequence was MGWSCIILFLVATATGVHS (SEQ ID NO: 17). Using standard molecular biology techniques, the genes were cloned into a eukaryotic expression vector (pCDNA3.1, purchased from Miaoling Biology). Mutants were constructed using overlap PCR, and after sequence verification, the constructed plasmids for protein expression were prepared using a plasmid midi kit (NucleoBond Xtra Midi Plus, MACHEREY-NAGEL).

[0486] Example 7. Fusion Protein Analysis 7.1 Expression, purification, and rough assessment of solubility of fusion proteins For fusion protein expression, transient transfection and expression Expi293 cells (purchased from Thermofisher) were used. The prepared heavy and light chain plasmids were co-transfected into Expi293 cells at a ratio of 1:1.5 using PEI-MW40000 (PolySciences) to transiently express the desired proteins. For PEI preparation and use, please refer to the PolySciences PEI MAX instruction manual. For Expi293 cell culture and transfection procedures, please refer to the Thermofisher Expi293 Expression System instruction manual. Five to seven days after transient transfection, the cell supernatant was collected by centrifugation. The supernatant was filtered through a 0.45 μm filter for protein purification.

[0487] Transiently expressed proteins were purified using Protein A pre-packed columns (GE Lifesciences), see manufacturer's instructions for procedure.

[0488] The collected eluted protein samples were neutralized with Tris buffer and then placed in a refrigerator at 4°C. After 24 hours, the samples were visually observed for the amount of precipitate and the turbidity of the solution, and the solubility was roughly evaluated on a scale of 1 to 10, with 10 representing the most precipitate or most turbidity and 1 representing the least precipitate or most clear. Based on the rough evaluation of solubility, a score for each fusion protein is given in the table below (Table 6). Based on the observed solubility, each mutation improved the solubility of the fusion protein to various degrees, using the C'-KS mutant fusion protein as a benchmark.

[0489] [Table 6]

[0490] After centrifugation or filtration to remove precipitates, the solution was dialyzed or ultrafiltered, exchanged into a PBS buffer system, and finally sterilized by filtration through a 0.22 μm filter to obtain the protein sample to be tested. The protein sample was quantified using a NanoDrop™ spectrophotometer (Thermo Scientific) in combination with the theoretical extinction coefficient of the protein.

[0491] 7.2 Analysis of fusion protein purity The purity of the fusion protein was analyzed using size-exclusion chromatography (SEC) by applying the purified sample in PBS to a TSKgel SuperSW3000 300 × 4.6 mm, 5 μm column (TOSOH). SEC was performed using a Model U3000 HPLC instrument (DIONEX). All proteins were detected using UV detection at wavelengths of 280 nm and 214 nm. Elution was isocratic at a flow rate of 0.25 mL / min. The analytical results showed that the fusion protein purified in one step using Protein A had good monomer purity in solution (Table 7), indicating the physical homogeneity and stability of the fusion protein in solution as a monomeric protein.

[0492] [Table 7]

[0493] 7.3 Solubility evaluation The clarified fusion protein solution samples exchanged into PBS were concentrated by centrifugation at 4000 g at 10° C. using ultrafiltration tubes (Amicon® Ultra 30 Kda). The samples were mixed after each 15-minute centrifugation and then centrifuged again until a precipitate appeared or the volume of the sample solution no longer changed significantly. The concentration of the protein samples was measured using a NanoDrop™ spectrophotometer (Thermo Scientific) in combination with the theoretical extinction coefficient of the protein. The concentrations of each concentrated combination mutant fusion protein are shown in Table 8 below. The measurement results showed that the concentration of the concentrated fusion protein containing the 59D mutation was higher than that of the concentrated fusion protein containing the 59S mutation, the concentration of the concentrated fusion protein containing four mutations was higher than that of the concentrated fusion protein containing three mutations, and the concentrated fusion protein containing the 59D / 82D / 110T / 56E mutation had the highest concentration at approximately 140 mg / mL, indicating better solubility.

[0494] [Table 8]

[0495] Example 8 Hemolysis inhibitory activity of fusion proteins 8.1 Alternative pathway (AP) hemolysis inhibitory activity The method for detecting the complement AP inhibitory activity of the fusion protein was the same as in 4.2.

[0496] AP activity of fusion proteins with single point mutations To test the AP inhibitory activity of fusion proteins with single point mutations, a 4-fold gradient dilution was performed starting from a final protein concentration of 1000 nM, resulting in a total of seven concentration points, with a final serum concentration of 15%. The test results showed that the activity of each point mutant fusion protein in inhibiting AP hemolysis was essentially the same and was not significantly different from the unmutated fusion protein Ab-F1D34, indicating that each mutation did not affect the AP inhibitory activity. The results are shown in Figure 11 (the hybrid protein mutations were used to list the fusion proteins with the antibodies fused to them).

[0497] AP activity of fusion proteins with combined mutations To test the AP inhibitory activity of the fusion proteins with the combined mutations, a three-fold gradient dilution was performed starting from a final protein concentration of 400 nM, or a three-fold gradient dilution was performed starting from a final protein concentration of 300 nM or 900 nM, resulting in a total of seven concentration points, with a final serum concentration of 10%. The test results showed that the activity of each of the combined mutant fusion proteins in inhibiting AP hemolysis was essentially the same and was not significantly different from the fusion protein Ab-F1D34 before mutation, indicating that each of the combined mutations did not affect the AP inhibitory activity. The results are shown in Figure 13A (the hybrid protein mutations were used to list the fusion proteins with the antibodies fused to them in the figure).

[0498] Compared with the prior art eculizumab monoclonal antibody, the fusion protein with the 59D / 82D / 110T / 56E combination mutations had better AP inhibitory activity, solving the problem that eculizumab monoclonal antibody cannot completely inhibit AP hemolysis (as shown in Figure 14A). In addition, compared with the C5 antibody-CFH CCP1-5 fusion protein Ab-F15, the AP inhibitory activity of the fusion protein with the 59D / 82D / 110T / 56E combination mutations was also slightly better (as shown in Figure 14B) (59D / 82D / 110T / 56E in Figure 14 represents the fusion protein of the hybrid protein mutant with 59D / 82D / 110T / 56E and the antibody).

[0499] 8.2 Classical Pathway (CP) Hemolysis Inhibitory Activity The method for detecting the complement AP inhibitory activity of the fusion protein was the same as in 4.1. CP activity of fusion proteins with single point mutations To test the CP inhibitory activity of fusion proteins with single point mutations, a 4-fold gradient dilution was performed starting from a final protein concentration of 12.5 nM or 200 nM, resulting in a total of seven concentration points, with a final serum concentration of 1%. The test results showed that the activity of each point mutant fusion protein to inhibit CP hemolysis was different. The fusion proteins with C'-KS, L56E, L59D, L59S, G82D, and I110T mutations were not significantly different from the original Ab-F1D34 fusion protein, while the fusion proteins with F86Y, L109D, L109E, and F107E mutations were slightly reduced, indicating that different mutations had different effects on CP inhibitory activity. The results are shown in Figure 12 (the hybrid protein mutations were used to list the fusion proteins with the antibodies fused to them).

[0500] CP activity of fusion proteins with combined mutations To test the CP inhibitory activity of the fusion proteins with the combined mutations, a three-fold gradient dilution was performed from a final protein concentration of 25 nM or 150 nM, resulting in a total of seven or eight concentration points, with a final serum concentration of 1% or 5%. The test results showed that the activity of each combined mutant fusion protein in inhibiting CP hemolysis was essentially the same and was not significantly different from the fusion protein Ab-F1D34 before mutation, indicating that each combined mutation did not affect the CP inhibitory activity. The results are shown in Figure 13B (the hybrid protein mutations were used to list the fusion proteins with the antibodies fused to them in the figure).

[0501] Compared with the prior art eculizumab monoclonal antibody, the fusion protein with the 59D / 82D / 110T / 56E combined mutations had better CP hemolysis inhibitory activity against sheep red blood cells (sRBCs), with IC50 values ​​of the monoclonal antibody and fusion protein of 5.874 nM and 0.4767 nM, respectively (Figure 15A). In addition, compared with the C5 antibody-CFH CCP1-5 fusion protein Ab-F15, the fusion protein with the 59D / 82D / 110T / 56E combined mutations also had significantly stronger CP hemolysis inhibitory activity against sheep red blood cells (sRBCs), with IC50 values ​​of the two fusion proteins of 8.158 nM and 0.8537 nM, respectively (Figure 15B).

[0502] Example 9 C3b deposition inhibitory activity 9.1 Rabbit red blood cell method The experimental procedure was the same as in 4.3, except that a 3-fold gradient dilution was performed starting from 900 nM protein, resulting in a total of 7 gradients. To prevent C3b-deposited red blood cells from being disrupted due to lysis by the membrane attack complex (MAC), which would prevent accurate detection, C6- or C7-depleted human serum (C6- or C7-depleted NHS, CompTech) was used for incubation. The incubated red blood cells were stained with anti-human C3b APC (BioLegend) and detected using a flow cytometer (Beckman Cytoflex). The final data were calculated using the MFI (median fluorescence intensity) value of the red blood cell group without complement inhibitory proteins as 100%, and the inhibitory activity of each complement inhibitory protein in inhibiting C3b deposition on the red blood cell surface was compared.

[0503] The detection results show that eculizumab monoclonal antibody has no C3b deposition inhibitory activity, while the fusion protein with the 59D / 82D / 110T / 56E combined mutations has good C3b deposition inhibitory activity (Figure 16A), which is also obviously better than that of the C5 antibody-CFH CCP1-5 fusion protein Ab-F15, with the IC50 of the two fusion proteins being 24.58 nM and 152 nM, respectively (Figure 16B).

[0504] 9.2 Wieslab method Using the WIESLAB® Complement System Alternative Pathway kit (Svar, COMPLAP330RUO), the final C5b-9 (MAC) detection antibody was replaced with a C3b antibody (prepared in a similar manner as the C5 antibody, see SEQ ID NOs: 1 and 5 in US2012 / 0128674A1 for sequences) to detect C3b deposition generated after activation of the complement system at the bottom of the microplate, and the effect of adding antibodies or fusion proteins on C3b generation or C3b deposition at the bottom of the plate.

[0505] The detection process is briefly described as follows: Normal human serum (NHS, Shanghai Schbio Co., Ltd.) and the test protein samples were diluted using the buffer provided with the kit. 50 mL of the diluted NHS and test protein samples (59D / 82D / 110T / 56E fusion protein, eculizumab, and Ab-F15) were taken, mixed well, and added to the microplate provided with the kit. The final concentration of serum was 5%, and the final concentrations of the test protein samples were 900, 300, 100, 33.33, 11.11, and 3.7 nM, while the blank, PC, and NC were set as indicated. After incubation at 37°C for 1 hour, the mixture was washed three times with the kit's washing buffer. Mouse C3b antibody was added at a final concentration of 1 μg / mL, and the mixture was incubated at 37°C for 1 hour. The mixture was then washed three times with washing buffer. Next, goat anti-mouse IgG-Fc secondary antibody (HRP) (1:2000 dilution, SinoBiological, SSA006) was added, and the mixture was washed three times with washing buffer. Substrate was added to develop and stop the reaction. The absorbance at OD405nm was measured using a microplate reader, and the inhibition rate was calculated.

[0506] % of complement inhibition: [1-(sample-NC) / (PC-NC)] x 100 The experimental results show that the fusion protein 59D / 82D / 110T / 56E containing the hybrid protein mutants has stronger activity, which is obviously better than the fusion protein Ab-F15 containing CFH CCP1-5, while eculizumab has no C3b deposition inhibitory activity (as shown in Figure 17), which is substantially consistent with the results of the assay using rabbit erythrocyte method, further verifying the advantage of the fusion protein containing the hybrid protein in the activity of inhibiting C3b deposition.

[0507] Example 10 Biotin pathway (AP) hemolysis inhibitory activity Wieslab method The WIESLAB® Complement System Alternative Pathway kit (Svar, COMPLAP330RUO) was used for detection, and detailed methods are provided in the product manual.

[0508] The detection process is briefly described as follows: Normal human serum (PC, included in the kit) and the test protein samples were diluted using the buffer provided with the kit. 50 mL of diluted NHS and test protein samples (59D / 82D / 110T / 56E, eculizumab, and Ab-F15) were mixed well and added to the microplate provided with the kit. The final serum concentration was 1%, and the final concentrations of the test protein samples were 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 nM, while the blank, PC, and NC were set as indicated. After incubation at 37°C for 1 hour, the mixture was washed three times with the kit's washing solution, and the secondary antibody included in the kit was added. The mixture was incubated at 37°C for 1 hour, washed three times with the washing solution, and then substrate was added to develop and stop the color. The OD405 nm absorbance was detected using a microplate reader, and the inhibition rate was calculated.

[0509] % of complement inhibition: [1-(sample-NC) / (PC-NC)] x 100 The experimental results show that the fusion protein 59D / 82D / 110T / 56E containing the hybrid protein mutants has stronger LP inhibitory activity, which is obviously better than the fusion protein Ab-F15 containing eculizumab and CFH CCP1-5 (as shown in Figure 18).

[0510] Example 11. Purity analysis of hybrid protein mutants Protein purity analysis was performed using size exclusion chromatography (SEC). Purified protein samples in PBS from the same batch, expressed and purified using identical conditions, were applied to a TSKgel Super SW3000 column (300 × 4.6 mm, 5 μm, TOSOH). SEC was performed using a U3000 HPLC instrument (DIONEX). Elution was performed isocratically at a flow rate of 0.25 mL / min. All proteins were detected using UV detection at wavelengths of 280 nm and 214 nm. Component analysis was performed using the instrument's built-in software.

[0511] The analysis results showed that the monomer purity of the hybrid proteins F1D34-1 and F1D34-2i in solution reached 92% and 95%, respectively (Figure 19A / B), indicating good monomer purity. However, both proteins exhibited distinct aggregates at around 13.4 RT / min, which is the peak appearance time, indicating their large aggregate form. The percentage of F1D34-1 aggregates exceeded 5% (Figure 19A), while the percentage of F1D34-2i aggregates exceeded 3% (Figure 19B).

[0512] To compare the aggregation improvement of each hybrid protein mutant, we statistically summarized the percentage of aggregates near the peak appearance time of the large aggregate peak (12–15 RT / min) (Figure 19A–H). The results showed that adding a KS to the C'-terminus of the hybrid protein F1D34-2i significantly reduced the formation of large aggregates (F1D34-KS, a 3 percentage point reduction compared to F1D34-1, 2.5% vs. 5.6%, and a roughly 1 percentage point reduction compared to F1D34-2i, 2.5% vs. 3.5%). Meanwhile, other KS-based mutations also improved the formation of large aggregates to varying degrees. Among them, the L59D and L59S mutations showed the most significant improvement, resulting in a reduction of F1D34-KS-L59D and F1D34-KS-59S aggregates to approximately 1.3%. Furthermore, the aggregates of F1D34-KS-L56E and F1D34-KS-I110T obtained by the L56E and I110T mutations were reduced by 0.7% and 0.5%, respectively, compared to the aggregates of F1D34-KS with the KS mutation. The aggregates of F1D34-KS-G82D with the G82D mutation were substantially identical to those of F1D34-KS with the KS mutation (2.55% vs. 2.54%), indicating that the formation of large aggregates cannot be further reduced by KS (Figure 20A). Therefore, the monomer purity of each mutant was also improved to various degrees (Figure 20B), which is also consistent with the improved aggregates.

[0513] Example 12. Comparison of expression levels of mutant proteins To evaluate the effect of mutations on protein expression levels, the expression levels of each mutant expressed and purified in the same batch under the same conditions were compared with the expression level of F1D34-1 (100%) by transient transfection. The expression and quantification methods were as described in Example 2. The results are shown in Figure 21. The expression level of F1D34-2i was approximately 50% higher than that of F1D34-1, and F1D34-KS, obtained by adding the amino acid KS to the C' terminus of the hybrid protein F1D34-2i, improved the expression level by approximately 1-fold. His-tagged F1D34-KS-L56E, F1D34-KS-L59D, or F1D34-KS-L59S, obtained by the L56E, L59D, or L59S mutations based on the addition of KS, respectively, further improved the expression level, while F1D34-KS-G82D or F1D34-KS-I110T, obtained by the G82D or I110T mutations, could not further improve the expression level based on F1D34-KS obtained by the KS mutation.

[0514] Example 13. Comparison of complement inhibitory activity of mutant proteins 13.1 Detection of inhibitory activity against the classical pathway of complement (CP) To evaluate the effect of mutations on protein CP inhibitory activity, each mutant was tested for its complement CP inhibitory activity using the same experimental procedure as in 4.1, except that a final serum concentration of 5% was used and 4-fold dilutions were performed from a final hybrid protein concentration of 1000 nM. The results of activity detection are shown in Figure 22A. Compared with the hybrid proteins F1D34-1 and F1D34-2i, each mutant did not significantly reduce CP inhibitory activity.

[0515] 13.2 Detection of inhibitory activity against the alternative complement pathway (AP) To evaluate the effect of mutations on protein AP inhibitory activity, each mutant was tested for its complement AP inhibitory activity using the same experimental procedure as in 4.2, except that a final serum concentration of 15% was used and a 4-fold dilution was performed from a final hybrid protein concentration of 7000 nM (from a final F1D34-KS-I110T concentration of 11534 nM). The results of activity detection are shown in Figure 22A. Compared with the hybrid proteins F1D34-1 and F1D34-2i, each mutant did not significantly reduce CP inhibitory activity.

[0516] [Table 9-1]

[0517] [Table 9-2]

[0518] [Table 9-3]

[0519] [Table 9-4]

[0520] [Table 9-5]

[0521] [Table 9-6]

[0522] [Table 9-7]

[0523] [Table 9-8]

[0524] Table 9-9

Claims

1. A hybrid protein mutant, wherein the parent hybrid protein is (a) CCP1 of human complement factor H (CFH); (b) CCP3 and CCP4 of human decay-accelerating factor (DAF), and optionally comprising or consisting of a signal peptide and / or tag, Compared to the parent hybrid protein, the mutant: Mutations, e.g., substitutions, at positions 56, 59, 82, 86, 107, 109, and / or 110, e.g., substitutions with amino acids that are more hydrophilic than the original amino acids, and / or mutations at the C-terminus, e.g., addition of one or more amino acids at the C-terminus. and one or more mutations selected from the group consisting of: The above hybrid protein mutant, wherein the positions of the amino acid mutations are numbered corresponding to the amino acid positions set forth in SEQ ID NO:

20.

2. The hybrid protein mutant of claim 1, wherein the optimized hybrid protein comprises substitutions at positions 59, 82 and 110, numbered corresponding to the amino acid positions set forth in SEQ ID NO: 20, and / or addition of one or more amino acids at the C-terminus.

3. 3. The hybrid protein mutant of claim 2, comprising substitutions at positions 56, 86, 107 and / or 109, numbered corresponding to the amino acid positions set forth in SEQ ID NO:

20.

4. 3. The hybrid protein mutant of claim 2, comprising substitutions at positions 56, 59, 82 and / or 110, numbered corresponding to the amino acid positions set forth in SEQ ID NO: 20, for example, substitutions at 59, 82 and 110, or substitutions at 56, 59, 82 and 110.

5. The mutation, Substitution of the amino acid at positions 56, 59, 82, 86, 107, 109 and / or 110, numbered corresponding to the amino acid positions set forth in SEQ ID NO: 20, with an amino acid that is more hydrophilic than the original amino acid, for example, with D, E, S, Y or T; or Addition of 1 to 5 amino acids, e.g., 2 amino acids, at the C-terminus The hybrid protein mutant according to any one of claims 1 to 4, selected from the group consisting of:

6. The mutation, 56D / E, 59D / S, 82D, 86Y, 107E, 109D / E, 110T, or the addition of C-terminal amino acids KS, or combinations thereof 6. The hybrid protein mutant of any one of claims 1 to 5, selected from the group consisting of:

7. 56E, 59D / S, 82D, 86Y, 107E, 109D / E, 110T, 59D-82D-110T, 59S-82D-110T, 59D-82D-110T-56E, 59S-82D-110T-56E, or Addition of the amino acid KS at the C-terminus 7. The hybrid protein mutant of claim 6, comprising:

8. The hybrid protein mutant of any one of claims 1 to 7, wherein CCP3 and CCP4 of DAF are directly linked together to form CCP3-4.

9. 9. The hybrid protein mutant of any one of claims 1 to 8, wherein CCP1 of human CFH comprises or consists of the amino acid sequence of positions 19 to 82 of the human CFH protein, optionally wherein CCP1 comprises a V62I mutation, and the amino acid positions are numbered corresponding to the amino acid positions set forth in SEQ ID NO:

3.

10. 10. The hybrid protein mutant of any one of claims 1 to 9, wherein CCP1 of human CFH comprises or consists of the amino acid sequence of positions 19 to 84 of the human CFH protein, optionally wherein CCP1 comprises a V62I mutation, and the amino acid positions are numbered corresponding to the amino acid positions set forth in SEQ ID NO:

3.

11. A hybrid protein mutant according to any one of claims 1 to 10, wherein CCP3 of human DAF comprises or consists of the amino acid sequence of positions 161 to 222 of the human DAF protein, and / or CCP4 of human DAF comprises or consists of the amino acid sequence of positions 223 to 285 of the human DAF protein, the amino acid positions being numbered corresponding to the amino acid positions set forth in SEQ ID NO:

1.

12. A hybrid protein mutant according to any one of claims 1 to 11, wherein CCP3 of human DAF comprises or consists of the amino acid sequence of positions 163 to 222 of the human DAF protein, and / or CCP4 of human DAF comprises or consists of the amino acid sequence of positions 223 to 285 of the human DAF protein, the amino acid positions being numbered corresponding to the amino acid positions set forth in SEQ ID NO:

1.

13. 13. The hybrid protein mutant of any one of claims 1 to 12, wherein CCP3-4 of human DAF comprises or consists of the amino acid sequence of positions 161 to 285 of the human DAF protein, the amino acid positions being numbered corresponding to the amino acid positions set forth in SEQ ID NO:

1.

14. 14. The hybrid protein mutant of any one of claims 1 to 13, wherein CCP3-4 of human DAF comprises or consists of the amino acid sequence of positions 163 to 285 of the human DAF protein, the amino acid positions being numbered corresponding to the amino acid positions set forth in SEQ ID NO:

1.

15. (1) CCP1 of human CFH comprises or consists of the amino acid sequence of positions 19 to 82 of the human CFH protein, CCP3 of human DAF comprises or consists of the amino acid sequence of positions 161 to 222 of the human DAF protein, and CCP4 of human DAF comprises or consists of the amino acid sequence of positions 223 to 285 of the human DAF protein; (2) CCP1 of human CFH comprises or consists of the amino acid sequence of positions 19 to 84 of the human CFH protein, CCP3 of human DAF comprises or consists of the amino acid sequence of positions 163 to 222 of the human DAF protein, and CCP4 of human DAF comprises or consists of the amino acid sequence of positions 223 to 285 of the human DAF protein; (3) CCP1 of human CFH comprises or consists of the amino acid sequence of positions 19 to 82 of the human CFH protein, and CCP3-4 of human DAF comprises or consists of the amino acid sequence of positions 161 to 285 of the human DAF protein; or (4) CCP1 of human CFH comprises or consists of the amino acid sequence of positions 19 to 84 of the human CFH protein, and CCP3-4 of human DAF comprises or consists of the amino acid sequence of positions 163 to 285 of the human DAF protein; A hybrid protein mutant according to any one of claims 1 to 14, wherein the amino acid positions of the human CFH protein are numbered corresponding to the amino acid positions set forth in SEQ ID NO: 3, and the amino acid positions of the human DAF protein are numbered corresponding to the amino acid positions set forth in SEQ ID NO:

1.

16. The hybrid protein mutant of any one of claims 1 to 15, wherein CCP1 of human CFH has a V62I mutation.

17. the human CFH protein is a human native CFH protein, or (i) an amino acid sequence set forth in SEQ ID NO: 3 or 5; (ii) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 4 or 6; (iii) an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of (i) or (ii); Contains, or A hybrid protein mutant according to any one of claims 1 to 16, consisting of an amino acid sequence according to any one of (i) to (iii).

18. the human DAF protein is a native human DAF protein, or (i) the amino acid sequence set forth in SEQ ID NO: 1; (ii) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:2; (iii) an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of (i) or (ii); Contains, or A hybrid protein mutant according to any one of claims 1 to 17, consisting of an amino acid sequence according to any one of (i) to (iii).

19. CCP1 of human CFH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, 13, 14 or 15, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 12, 13, 14 or 15; CCP3 of human DAF comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7 or 8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8; CCP4 of human DAF comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 9; and / or 19. A hybrid protein mutant according to any one of claims 1 to 18, wherein CCP3-4 of human DAF comprises or consists of an amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO:

11.

20. 20. The hybrid protein mutant of any one of claims 1 to 19, wherein the tag is a purification tag, such as a hexahistidine tag or a biotin marker, for example comprising the amino acid sequence set forth in SEQ ID NO:

16.

21. The hybrid protein mutant according to any one of claims 1 to 20, wherein the signal peptide is a secretory signal peptide comprising, for example, the amino acid sequence set forth in SEQ ID NO:

17.

22. 22. The hybrid protein mutant of any one of claims 1 to 21, wherein the parent hybrid protein comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 18 to 33, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence.

23. 23. The hybrid protein mutant of claim 22, wherein the parent hybrid protein comprises or consists of the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence.

24. 24. The hybrid protein mutant of any one of claims 1 to 23, comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 53 to 54, 56 to 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 to 96, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

25. (1) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 60, and including an additional C-terminal KS; (2) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 62, and including a C-terminal KS addition and a 56E mutation; (3) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 64, and including a C-terminal KS addition and a 59D mutation; (4) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 66, and including a C-terminal KS addition and a 59S mutation; (5) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 68, and including a C-terminal KS addition and an 82D mutation; (6) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 70, and including a C-terminal KS addition and an 86Y mutation; (7) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 72, and including a C-terminal KS addition and a 107E mutation; (8) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 74, and including a C-terminal KS addition and a 109D mutation; (9) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 76, and including a C-terminal KS addition and a 109E mutation; (10) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 78, and including a C-terminal KS addition and a 110T mutation; (11) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 80, and including C-terminal KS and 59D-82D-110T mutations; (12) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 82, and including C-terminal KS and 59S-82D-110T mutations; (13) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 84, and including C-terminal KS and 59D-82D-110T-56E mutations; (14) An amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence set forth in SEQ ID NO: 86, and C-terminal KS and 59S-82D-110T-56E mutations.

24. The hybrid protein mutant of any one of claims 1 to 23, comprising:

26. A fusion protein comprising an anti-C5 antibody or antigen-binding fragment thereof and a hybrid protein mutant according to any one of claims 1 to 25, wherein the antibody or antigen-binding fragment thereof is linked to the hybrid protein mutant with or without a linker.

27. 27. The fusion protein of claim 26, wherein one or more hybrid protein mutants are connected (at their N-terminus or at their C-terminus) to the N-terminus and / or C-terminus of the heavy chain and / or light chain of an anti-C5 antibody, respectively, with or without a linker.

28. The fusion protein of any one of claims 1 to 27, wherein the anti-C5 antibody is a humanized or chimeric antibody.

29. The fusion protein of any one of claims 1 to 28, wherein the anti-C5 antibody is a monoclonal antibody.

30. The antigen-binding fragment of the anti-C5 antibody may be Fab, Fab', Fab'-SH, Fv, single chain antibody (e.g., scFv), (Fab') ２ 30. The fusion protein of any one of claims 1 to 29, selected from a single domain antibody, such as a VHH, a dAb (domain antibody), or a linear antibody.

31. The fusion protein of any one of claims 1 to 30, wherein the anti-C5 antibody or antigen-binding fragment thereof comprises an Fc region, and preferably the Fc region is connected at its C-terminus to the N-terminus of the hybrid protein, with or without a linker.

32. The linker may contain one or more glycines (G) ｎ , G.S., G. ｎ S, G ｎ S ｎ , (G ｎ S) ｎ or (GSG) ｎ or (G ４ S) ｎ wherein n is an integer of 1 or greater, e.g., n is an integer of 2, 3, 4, 5, 6, or 7, e.g., the linker is selected from G, GSG, or G ４ The fusion protein of any one of claims 1 to 31, wherein the fusion protein is S.

33. including full-length anti-C5 antibodies and hybrid proteins, an anti-C5 antibody is joined at the C-terminus of its Fc region to the N-terminus of the hybrid protein to form the heavy chain of the fusion protein (with or without a linker); The fusion protein of any one of claims 1 to 32, wherein the light chain of the anti-C5 antibody forms the light chain of the fusion protein.

34. The anti-C5 antibody or antigen-binding fragment thereof i) an anti-C5 antibody or an antigen-binding fragment thereof disclosed in CN113754763A; or ii) eculizumab, ravulizumab, pozelimab, crovalimab, tesidolumab, or antigen-binding fragments thereof The fusion protein according to any one of claims 1 to 33, selected from:

35. The anti-C5 antibody or antigen-binding fragment thereof The fusion protein according to any one of claims 1 to 33, comprising three complementarity determining regions (CDRs) of a heavy chain variable region (VH), HCDR1, HCDR2 and HCDR3, and three CDRs of a light chain variable region (VL), LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2 and HCDR3 are the three CDRs contained in the VH set forth in SEQ ID NO: 39, and LCDR1, LCDR2 and LCDR3 are the three CDRs contained in the VL set forth in SEQ ID NO:

45.

36. The anti-C5 antibody or antigen-binding fragment thereof - HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 40, - an HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 41, - an HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 42, - an LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 46, - an LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 47, and - LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 48 The fusion protein of any one of claims 1 to 33, comprising:

37. The anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH, wherein the heavy chain variable region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 39; or (ii) A fusion protein according to claim 35 or 36, comprising or consisting of the amino acid sequence of SEQ ID NO:

39.

38. The anti-C5 antibody or antigen-binding fragment thereof comprises a light chain variable region VL, wherein the light chain variable region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 45; or (ii) A fusion protein according to any one of claims 35 to 37, comprising or consisting of the amino acid sequence of SEQ ID NO:

45.

39. the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL; the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; - the fusion protein of claim 35 or 36, wherein the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

40. The fusion protein of claim 35 or 36, wherein the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH comprises or consists of the amino acid sequence of SEQ ID NO: 39 and VL comprises or consists of the amino acid sequence of SEQ ID NO:

45.

41. The fusion protein of any one of claims 35 to 40, wherein the anti-C5 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region HC, for example, the heavy chain constant region (HC) is an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, preferably an IgG2 or IgG4 heavy chain constant region, or an IgG2 / IgG4 hybrid form heavy chain constant region.

42. The heavy chain constant region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 43; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 43; or (iii) The fusion protein of claim 41, comprising or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:

43.

43. 43. The fusion protein of claim 41 or 42, wherein the heavy chain constant region comprises one or more mutations that enhance FcRn binding, such as YTE mutations (M252Y / S254T / T256E), LA mutations (M428L / N434A), or LS mutations (M428L / N434S), preferably LA mutations, and / or comprises a mutation that enhances stability, such as S228P.

44. The fusion protein of any one of claims 35 to 43, wherein the anti-C5 antibody or antigen-binding fragment thereof comprises a light chain constant region, for example, the light chain constant region is a lambda or kappa constant region.

45. The light chain constant region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 49; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 49; or (iii) The antibody or antigen-binding fragment thereof described in claim 44, comprising or consisting of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:

49.

46. the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain; The heavy chain (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 44; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 44; or (iii) The fusion protein of any one of claims 35 to 45, comprising or consisting of an amino acid sequence which has one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 44, preferably the amino acid changes occur outside the CDR regions, preferably the amino acid changes occur outside the heavy chain variable region.

47. the anti-C5 antibody or antigen-binding fragment thereof comprises a light chain; The light chain (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 50; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 50; or (iii) The fusion protein of any one of claims 35 to 46, comprising or consisting of an amino acid sequence which has one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 50, preferably the amino acid changes occur outside the CDR regions, preferably the amino acid changes occur outside the heavy chain variable region.

48. the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; - the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, 48. The fusion protein according to claim 46 or 47, wherein the light chain comprises or consists of an amino acid sequence of SEQ ID NO: 50, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

49. The fusion protein of claim 48, wherein the anti-C5 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:

50.

50. 34. The fusion protein of claim 33, comprising two heavy chains and two light chains.

51. 51. The fusion protein of claim 33 or 50, wherein the heavy chain of the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 or 87, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and / or the light chain of the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

52. A nucleic acid molecule encoding a hybrid protein mutant according to any one of claims 1 to 25 or a fusion protein according to any one of claims 26 to 51.

53. 53. An expression vector comprising the nucleic acid molecule of claim 52, preferably pcDNA3.

1.

54. 54. A host cell comprising a nucleic acid molecule according to claim 52 or an expression vector according to claim 53, which is preferably a prokaryote or a eukaryote such as a CHO cell, a 293 cell, e.g. an Expi293 cell.

55. 10. A method for preparing a hybrid protein mutant according to any one of claims 1 to 25 or a fusion protein according to any one of claims 26 to 51, comprising culturing a host cell according to claim 54 under conditions suitable for expression of the hybrid protein mutant or fusion protein, and optionally further comprising the step of separating the hybrid protein mutant or fusion protein from the host cell or host cell culture medium and / or purifying the hybrid protein mutant or fusion protein.

56. An immunoconjugate comprising the fusion protein of any one of claims 26 to 51 and another agent, such as an antihemolytic agent or a label.

57. A pharmaceutical composition or medicament or formulation comprising a hybrid protein mutant according to any one of claims 1 to 25 or a fusion protein according to any one of claims 26 to 51, and optionally a pharmaceutical excipient.

58. A hybrid protein mutant according to any one of claims 1 to 25 or a fusion protein according to any one of claims 26 to 51, and Further therapeutic agents 1. A pharmaceutical combination product comprising:

59. 57. A method for preventing or treating a disease or condition associated with the complement system in a subject, comprising administering to the subject an effective amount of a hybrid protein mutant according to any one of claims 1 to 25, or a fusion protein according to any one of claims 26 to 51, or an immunoconjugate according to claim 56, or a pharmaceutical composition or formulation according to claim 57, or a pharmaceutical combination product according to claim 58.

60. 60. The method of claim 59, wherein the disease or condition is caused by abnormal activation or dysregulation of the complement system, or is a complement C5-associated disease or condition.

61. 61. The method of claim 60, wherein the abnormal activation of the complement system or dysregulation of the complement system is due to, for example, microbial infection or increased autoimmune antibodies, or due to a decrease, loss, impairment or functional interference or disruption of a complement regulatory protein.

62. 61. The method of claim 60, wherein the complement C5-associated disease or condition comprises a disease phenotype caused by unregulated C5 function, such as due to dysregulated C5 activation, e.g., elevated C5 activation.

63. The method of claim 60, wherein the complement C5-associated disease or condition is a disease or condition in which a subject has an elevated level of complement C5 protein (e.g., at the nucleic acid or protein level) (e.g., compared to a healthy subject), or in which a subject has an elevated level of complement C5 protein (e.g., at the nucleic acid or protein level) in their blood or blood cells (e.g., compared to the blood or blood cells of a healthy subject).

64. The method of claim 63, wherein the complement system-related disease or disorder is selected from diseases requiring inhibition of hemolysis, such as diseases requiring inhibition of hemolysis of the classical pathway of complement immunity and / or the alternative pathway of complement immunity, or diseases requiring inhibition of C3b deposition activity, such as dense deposition disease (DDD).

65. 52. A method for detecting the presence of complement C5 in a biological sample, comprising contacting the biological sample with the fusion protein of any one of claims 26 to 51 under conditions that allow binding to complement C5, and detecting whether a complex is formed between the antibody or antigen-binding fragment thereof or the fusion protein and complement C5, wherein the formation of a complex indicates the presence of complement C5.

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