Anti-human complement c5 antibodies and fusion proteins thereof

Abstract

The present application relates to an antibody specifically binding to complement C5, and fusion proteins thereof. The present application also relates to polynucleotides encoding the antibody or fusion protein, expression vectors and host cells, as well as pharmaceutical compositions thereof, and methods and uses for treating diseases associated with C5 protein.

Description

[0001] This invention relates to an antibody that specifically binds to complement C5, and its fusion protein. The invention also relates to polynucleotides encoding said antibody or fusion protein, expression vectors and host cells, pharmaceutical compositions thereof, and methods and uses for treating C5 protein-related diseases. Background Technology

[0002] The complement system, composed of over 30 soluble protein molecules, is part of the innate immune system. Its components include intrinsic complement components, various regulatory factors, and complement receptors. The complement system can be activated through three relatively independent yet interconnected pathways, thereby exerting a variety of biological effects, such as opsonizing phagocytosis, lysing cells, mediating inflammation, modulating the immune system, and clearing immune complexes. These effects include enhancing phagocytosis, increasing the chemotaxis of phagocytes, increasing vascular permeability, neutralizing viruses, lysing cells, and regulating the immune response. While complement activation provides valuable first-line defense against potential pathogens, complement activation that promotes protective inflammatory responses can also pose a potential threat to the host. Complement activation and its deposition on target structures can also indirectly cause cell or tissue damage. Complement activation products mediating 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 comprise proteins designated C1 through C9, and these proteins are sequentially activated via three distinct pathways (classical, lectin, and alternative / bypass pathways) to elicit an immune response. Complement C5, the fifth component of complement, plays a crucial role in inflammation and cytotoxicity. This protein consists of α- and β-peptide chains linked by disulfide bonds. The activated peptide C5a is an anaphylatoxin that induces inflammatory responses in various cells via C5aR (CD88) and C5L2 (GPR77), exhibiting potent spasmodic and chemotactic activity, and is derived from the α-peptide through cleavage with C5 convertase. The macromolecular cleavage product of C5b can form a complex with the C6 complement component, which forms the basis of the membrane attack complex (MAC), which includes additional complement components. If the complement system is not properly controlled or is overactivated, it can exert potent cytotoxicity on host cells.

[0004] Numerous studies have shown that complement activation is associated with a variety of diseases, such as those related to human complement hemolytic activity.

[0005] The anti-C5 monoclonal antibody ekulizumab (Soliris, a registered trademark) exhibits high affinity for complement C5 and inhibits complement activation by suppressing the cleavage of C5 into C5a / C5b and the associated formation of the membrane attack complex. Thus, ekulizumab exhibits inhibitory activity against hemolysis and is therefore used as a treatment for paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Furthermore, ekulizumab is known as a treatment for generalized myasthenia gravis (gMG).

[0006] Although eculizumab showed good hemolysis inhibition effect via the classical pathway in in vitro hemolysis inhibition activity assays, its insufficient activity in blocking the alternative complement pathway (AP) meant that its efficacy could not fully meet the needs of patients. For example, some PNH patients who had undergone eculizumab (Soliris) treatment were still unable to get rid of transfusion dependence due to extravascular hemolysis.

[0007] Furthermore, since lack of regulation or improper activation of complement activation can lead to host tissue damage, the complement system is also tightly 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 accelerator factor (DAF; CD55), membrane cofactor protein (MCP; CD46), and complement receptor 1 (CR1; CD35), as well as liquid phase proteins such as factor H (FH or CFH) and C4b-binding protein (C4BP). Structurally, RCA proteins consist of complement control protein repeat (CCP) modules, where 2-4 consecutive modules contribute to regulatory functions known as decay accelerator activity (DAA) and cofactor activity (CFA). RCA proteins function by targeting the central enzymes of the complement pathway, C3 / C5 convertases. To inactivate these enzymes, RCA proteins bind to these convertases or their non-catalytic subunits and inactivate them. In DAA, the RCA protein binds to the convertase and irreversibly dissociates it into its subunits, while in CFA, the RCA protein binds to the non-catalytic subunits of the convertase (C3b / C4b) and recruits serine protease factor I (FI) to cleave and inactivate them, thereby stopping their ability to form C3 convertase. Factor H inhibits C3b amplification. It is a cofactor for factor I-catalyzed cleavage of C3b into iC3b and is an opsonin and ligand for complement receptors 2 and 3. It accelerates the irreversible dissociation of C3bBb in the alternative pathway C3 convertase and may also compete with factor B for binding to C3b during convertase formation. Factor H is soluble and important for protecting surfaces, including the extracellular matrix (ECM). Factor H binds to adrenomedullin (a peptide hormone) and may prevent its degradation. The role of FH in managing cellular senescence, stress, or damage is through interactions with C-reactive protein, pentamin, DNA, histones, annexin II, malondialdehyde adducts of proteins, and oxidized lipids. FHL1 also possesses factor I cofactor activity and C3bBb accelerated decay activity (The Complement Facts Book. Edited by: Scott Barnum and Theresa Schein, Copyright). © 2018 Elsevier Ltd. All rights reserved. https: / / doi.org / 10.1016 / C2015-0-06595-9 (Chapter 30).

[0008] DAF essentially protects host cells from autocomplement attack by inhibiting C3 and C5 cleavage through blocking the formation of classical and alternative C3 and C5 convertases and accelerating their decay. When purified DAF is added to cells, it integrates into the cell membrane and exhibits functional activity. DAF has also been found to modulate T cell tolerance, thereby negatively regulating animal models of various autoimmune diseases (The Complement Facts Book. Edited by: Scott Barnum and Theresa Schein, Copyright). © 2018 Elsevier Ltd. All rights reserved. https: / / doi.org / 10.1016 / C2015-0-06595-9 (Chapter 25).

[0009] Therefore, there is a need to develop new anti-C5 antibodies, as well as fusion proteins based on anti-C5 antibodies and complement regulatory proteins, to improve the activity and efficacy of C5 antibodies. Summary of the Invention:

[0010] This invention relates to an antibody capable of efficiently binding to human complement C5 protein. In some embodiments, the antibody can effectively block human complement hemolytic activity.

[0011] In some implementations, the antibodies of the present invention can effectively inhibit hemolysis via the classical complement pathway and the alternative complement pathway.

[0012] In some embodiments, the present invention relates to a fusion protein comprising an anti-C5 antibody and a complement regulatory protein (e.g., a hybrid protein of complement regulatory proteins). In some embodiments, compared to the anti-C5 antibody, the fusion protein (1) further enhances the blocking activity of the classical complement pathway (CP), thereby increasing efficacy; and (2) significantly improves the blocking activity of the alternative complement pathway (AP). In a further embodiment, the fusion protein has a stronger inhibitory activity against C3b deposition on the cell surface compared to known C5 antibody fusion proteins in the art, wherein C3b deposition is considered a major cause of extravascular hemolysis in PNH patients.

[0013] In some embodiments, the present invention relates to the following specific implementations:

[0014] 1. An anti-C5 antibody or its antigen-binding fragment, comprising three 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 complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:39; and LCDR1, LCDR2 and LCDR3 are the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:45.

[0015] 2. An anti-C5 antibody or its antigen-binding fragment, comprising HCDR1 containing or consisting of the amino acid sequence shown in SEQ ID NO:40, HCDR2 containing or consisting of the amino acid sequence shown in SEQ ID NO:41, HCDR3 containing or consisting of the amino acid sequence shown in SEQ ID NO:42; LCDR1 containing or consisting of the amino acid sequence shown in SEQ ID NO:46, LCDR2 containing or consisting of the amino acid sequence shown in SEQ ID NO:47, and LCDR3 containing or consisting of the amino acid sequence shown in SEQ ID NO:48.

[0016] 3. An antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH, wherein the heavy chain variable region (i) comprises or is composed of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:39; or

[0017] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:39.

[0018] 4. An antibody or antigen-binding fragment thereof from any of embodiments 1-3, comprising a light chain variable region VL, wherein the light chain variable region...

[0019] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:45; or

[0020] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:45.

[0021] 5. The antibody or its antigen-binding fragment according to implementation method 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein

[0022] The heavy chain variable region comprises the amino acid sequence shown 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 with it, or is composed of said amino acid sequence; and the light chain variable region comprises the amino acid sequence shown 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 with it, or is composed of said amino acid sequence.

[0023] 6. The antibody or antigen-binding fragment thereof of embodiment 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:39, and VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:45.

[0024] 7. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-6 further comprises a heavy chain constant region HC, for example, the antibody heavy chain constant region HC is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of IgG2 or IgG4, or a heavy chain constant region of IgG2 / IgG4 hybrid.

[0025] 8. The antibody or its antigen-binding fragment of embodiment 7, wherein the heavy chain constant region

[0026] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:43; or

[0027] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:43; or

[0028] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:43.

[0029] 9. The antibody or antigen-binding fragment thereof of embodiment 7 or 8, wherein the heavy chain constant region contains a mutation that increases binding to the FcRn receptor, such as one or more of the following: YTE mutation (M252Y / S254T / T256E), LA mutation (M428L / N434A), or LS mutation (M428L / N434S), preferably including an LA mutation; and / or a mutation that increases antibody stability, such as S228P.

[0030] 10. An antibody or antigen-binding fragment thereof from any of embodiments 1-9, comprising a light chain constant region, for example, the light chain constant region being a lambda or kappa light chain constant region.

[0031] 11. The antibody or antigen-binding fragment thereof of embodiment 10, wherein the light chain constant region

[0032] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:49; or

[0033] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:49; or

[0034] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:49.

[0035] 12. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-11, comprising a heavy chain, wherein the heavy chain (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:44; or

[0036] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:44; or

[0037] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:44, preferably, the amino acid alterations do not occur in the CDR region, and preferably, the amino acid alterations do not occur in the heavy chain variable region.

[0038] 13. An antibody or antigen-binding fragment thereof according to any one of embodiments 1-12, comprising a light chain, wherein the light chain (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:50; or

[0039] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:50; or

[0040] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:50, preferably, the amino acid alterations do not occur in the CDR region, preferably, the amino acid alterations do not occur in the light chain variable region.

[0041] 14. The antibody or antigen-binding fragment thereof according to embodiment 12 or 13, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:44, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence, or is composed of the amino acid sequence; and the light chain comprises the amino acid sequence of SEQ ID NO:50, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence, or is composed of the amino acid sequence.

[0042] 15. The antibody or antigen-binding fragment thereof described in Embodiment 14, comprising a heavy chain and a light chain, wherein the heavy chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 44, and the light chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 50.

[0043] 16. The antibody or antigen-binding fragment thereof as described in any one of embodiments 1-15, wherein the antibody is a humanized antibody or a chimeric antibody.

[0044] 17. The antibody or antigen-binding fragment thereof as described in any one of embodiments 1-16, wherein the antibody is a monoclonal antibody.

[0045] 18. The antibody or antigen-binding fragment thereof as described in any one of embodiments 1-17, wherein the antigen-binding fragment is 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.

[0046] 19. A fusion protein comprising an anti-C5 antibody or an antigen-binding fragment thereof and a hybrid protein, wherein the hybrid protein comprises or is composed of the following:

[0047] (i) CCP1 of human complement factor H (CFH),

[0048] (ii) Human decay accelerators (DAFs) CCP3 and CCP4,

[0049] The antibody or its antigen-binding fragment is linked to the hybrid protein via or without a linker;

[0050] Preferably, the anti-C5 antibody or its antigen-binding fragment is selected from...

[0051] i) The antibody or its antigen-binding fragment as described in any one of embodiments 1-18;

[0052] ii) The anti-C5 antibody or its antigen-binding fragment disclosed in CN113754763A; or

[0053] iii) Eculizumab, Ravulizumab, Pozelimab, Crovalimab, Tesidolumab or their antigen-binding fragments.

[0054] 20. The fusion protein described in Implementation Scheme 19, wherein CCP3 and CCP4 of DAF are directly linked together to form CCP3-4.

[0055] 21. The fusion protein of embodiment 19 or 20, wherein the CCP1 of human CFH comprises, or is composed of, the amino acid sequence of human CFH protein from position 19 to 82, and optionally, the CCP1 comprises the V62I mutation, wherein the amino acid position corresponds to the amino acid position number shown in SEQ ID NO:3.

[0056] 22. The fusion protein of embodiment 19 or 20, wherein the CCP1 of human CFH comprises, or is composed of, the amino acid sequence of human CFH protein from position 19 to 84, optionally, the CCP1 comprises the V62I mutation, wherein the amino acid position corresponds to the amino acid position number shown in SEQ ID NO:3.

[0057] 23. A fusion protein of any one of embodiments 19-22, wherein CCP3 of human DAF comprises or is composed of amino acid sequence 161-222 of human DAF protein, and / or CCP4 of human DAF comprises or is composed of amino acid sequence 223-285 of human DAF protein, wherein the amino acid positions correspond to the amino acid position numbers shown in SEQ ID NO:1.

[0058] 24. A fusion protein of any one of embodiments 19-22, wherein CCP3 of human DAF comprises or is composed of amino acid sequence 163-222 of human DAF protein, and / or CCP4 of human DAF comprises or is composed of amino acid sequence 223-285 of human DAF protein, wherein the amino acid positions correspond to the amino acid position numbers shown in SEQ ID NO:1.

[0059] 25. The fusion protein of embodiment 20, wherein the CCP3-4 of human DAF comprises, or is composed of, the amino acid sequence of positions 161-285 of the human DAF protein, wherein the amino acid positions correspond to the amino acid position numbers shown in SEQ ID NO:1.

[0060] 26. A fusion protein of any one of embodiments 20-22, wherein the CCP3-4 of human DAF comprises, or is composed of, the amino acid sequence of positions 163-285 of the human DAF protein, wherein the amino acid positions correspond to the amino acid position number shown in SEQ ID NO:1.

[0061] 27. The fusion protein of implementation scheme 20, wherein...

[0062] (1) CCP1 of human CFH contains or is composed of the amino acid sequence of human CFH protein from position 19 to 82, and CCP3 of human DAF contains or is composed of the amino acid sequence of human DAF protein from position 161 to 222, and CCP4 of human DAF contains or is composed of the amino acid sequence of human DAF protein from position 223 to 285.

[0063] (2) CCP1 of human CFH contains or is composed of the amino acid sequence of human CFH protein from position 19 to 84, and CCP3 of human DAF contains or is composed of the amino acid sequence of human DAF protein from position 163 to 222, and CCP4 of human DAF contains or is composed of the amino acid sequence of human DAF protein from position 223 to 285.

[0064] (3) CCP1 of human CFH contains or is composed of the amino acid sequence of human CFH protein from position 19 to 82, and CCP3-4 of human DAF contains or is composed of the amino acid sequence of human DAF protein from position 161 to 285; or (4) CCP1 of human CFH contains or is composed of the amino acid sequence of human CFH protein from position 19 to 84, and CCP3-4 of human DAF contains or is composed of the amino acid sequence of human DAF protein from position 163 to 285; wherein the amino acid positions of human CFH protein correspond to the amino acid position numbers shown in SEQ ID NO:3, and the amino acid positions of human DAF protein correspond to the amino acid position numbers shown in SEQ ID NO:1.

[0065] 28. The fusion protein of implementation scheme 27, wherein the human CFH CCP1 has a V62I mutation.

[0066] 29. A fusion protein of any one of embodiments 19-28, wherein the human CFH protein is a natural human CFH protein or contains the amino acid sequence shown in (i) SEQ ID NO:3 or 5.

[0067] (ii) The amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO:4 or 6;

[0068] (iii) has an amino acid sequence of at least 95%, 96%, 97%, 98%, or 99% as shown in (i) or (ii);

[0069] Or it may consist of the amino acid sequence shown in any of (i) to (iii).

[0070] 30. A fusion protein of any one of embodiments 19-29, wherein the human DAF protein is a natural human DAF protein or contains the amino acid sequence shown in (i) SEQ ID NO:1.

[0071] (ii) The amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO:2;

[0072] (iii) has an amino acid sequence of at least 95%, 96%, 97%, 98%, or 99% as shown in (i) or (ii);

[0073] Or it may consist of the amino acid sequence shown in any of (i) to (iii).

[0074] 31. The fusion protein of any one of the implementation schemes 19-30, wherein...

[0075] The human CFH CCP1 comprises the amino acid sequence of SEQ ID NO:12, 13, 14 or 15, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% uniformity with the amino acid sequence of SEQ ID NO:12, 13, 14 or 15, or is composed of said sequence;

[0076] The CCP3 of the human DAF contains the amino acid sequence of SEQ ID NO:7 or 8, or contains an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% uniformity with the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8, or is composed of said sequence;

[0077] The human DAF CCP4 comprises the amino acid sequence of SEQ ID NO:9, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence of SEQ ID NO:9, or is composed of said sequence; and / or the human DAF CCP3-4 comprises the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:11, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:11, or is composed of said sequence.

[0078] 32. A fusion protein of any one of embodiments 19-31, wherein the fusion protein comprises a signal peptide, for example, at its N-terminus.

[0079] 33. The fusion protein of embodiment 32, wherein the signal peptide is a secretory signal peptide, for example, comprising the amino acid sequence shown in SEQ ID NO: 17.

[0080] 34. A fusion protein of any one of embodiments 19-33, wherein the hybrid protein comprises an amino acid sequence of any one of SEQ ID NO: 18-33, or comprises or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence.

[0081] 35. A fusion protein of any one of embodiments 19-34, wherein one or more hybrid proteins (at their N-terminus or at their C-terminus) are respectively linked to the N-terminus and / or C-terminus of the heavy chain and / or light chain of the anti-C5 antibody, with or without a linker.

[0082] 36. A fusion protein of any one of embodiments 19-35, wherein the anti-C5 antibody or its antigen-binding fragment comprises an Fc region, wherein the Fc region is attached at its C-terminus to the N-terminus of the hybrid protein, with or without a linker.

[0083] 37. A fusion protein according to any one of embodiments 19-36, wherein the linker is selected from one or more glycine (G)n, GS, G n S, G n S n 、(G n S) n Or (GSG)n or (G4S)n, where n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7, for example, the connector is G, GSG or G4S.

[0084] 38. A fusion protein comprising any one of embodiments 19-37, wherein the fusion protein comprises a full-length anti-C5 antibody and a hybrid protein, wherein...

[0085] The anti-C5 antibody is linked 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 (via or without a linker).

[0086] The light chain of the anti-C5 antibody constitutes the light chain of the fusion protein.

[0087] 39. The fusion protein of embodiment 38, wherein the fusion protein comprises two heavy chains and two light chains.

[0088] 40. The fusion protein of embodiment 38 or 39, wherein the heavy chain of the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 54, 55, 56, 57 or 58, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence, or is composed of the sequence; and / or the light chain of the fusion protein comprises the amino acid sequence shown 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 with the amino acid sequence, or is composed of the sequence.

[0089] 41. A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described in any one of embodiments 1-18, or a fusion protein as described in any one of embodiments 19-40.

[0090] 42. An expression vector comprising the nucleic acid molecule of embodiment 41, preferably, the expression vector being pCDNA3.1.

[0091] 43. A host cell comprising the nucleic acid molecule described in embodiment 41 or the expression vector described in embodiment 42, preferably, the host cell is prokaryotic or eukaryotic, such as CHO cells, 293 cells, such as Expi293 cells.

[0092] 44. A method for preparing an antibody or antigen-binding fragment thereof as described in any one of embodiments 1-18, or a fusion protein as described in any one of embodiments 19-40, the method comprising culturing a host cell of embodiment 43 under conditions suitable for expression of the antibody or fusion protein, and optionally further comprising isolating the protein from the host cell or the host cell culture medium, and / or purifying the protein.

[0093] 45. An immunoconjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of embodiments 1-18, or a fusion protein as described in any one of embodiments 19-40, and other active agents, such as an antihemolytic agent or a label.

[0094] 46. ​​A pharmaceutical composition or drug or formulation comprising an antibody or antigen-binding fragment thereof as described in any one of embodiments 1-18, or a fusion protein as described in any one of embodiments 19-40, and optionally a pharmaceutical excipient.

[0095] 47. Drug combination products, which include

[0096] The antibody or its antigen-binding fragment as described in any one of embodiments 1-18, or the fusion protein as described in any one of embodiments 19-40; and

[0097] Other treatments.

[0098] 48. A method for preventing or treating complement system-related diseases or conditions in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof as described in any one of embodiments 1-18, or a fusion protein as described in any one of embodiments 19-40; or an immunoconjugate of embodiment 45 or a pharmaceutical composition or formulation of embodiment 46; or a pharmaceutical combination product of embodiment 47.

[0099] 49. The method of implementation scheme 48, wherein the disease or condition is caused by abnormal activation or dysregulation of the complement system or is a complement C5-related disease or condition.

[0100] 50. The method of implementation scheme 49, wherein the abnormal activation or dysregulation of the complement system is due to, for example, microbial infection or an increase in autoimmune antibodies, or due to a decrease, absence, dysfunction, or functional interference or blockage of complement regulatory proteins.

[0101] 51. The method of implementation scheme 49, wherein the complement C5-related disease or condition includes a disease phenotype resulting from unregulated C5 function, such as due to dysregulated C5 activation, such as increased C5 activation.

[0102] 52. The method of implementation scheme 49, wherein the complement C5-related disease or condition refers to a disease or condition in which the subject has (e.g., elevated levels, such as nucleic acid or protein levels) complement C5 protein (e.g., compared to a healthy subject) or the subject's blood or blood cells have (e.g., elevated levels, such as nucleic acid or protein levels) complement C5 protein (e.g., compared to the blood or blood cells of a healthy subject).

[0103] 53. The method of implementation scheme 52, wherein the complement system-related disease or condition is selected from diseases requiring hemolysis inhibition, such as diseases requiring inhibition of hemolysis via the classical complement immune pathway and / or the alternative complement immune pathway; or diseases requiring inhibition of C3b deposition activity.

[0104] 54. A method for detecting the presence of complement C5 in a biological sample, comprising contacting the biological sample with an antibody or antigen-binding fragment thereof, as described in any one of embodiments 1-18, or a fusion protein, as described in any one of embodiments 19-40, under conditions that allow it to bind 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 the complex indicates the presence of complement C5. Attached Figure Description

[0105] Figure 1 The crystal structures of CFH CCP1-4 and DAF CCP1-4 and the structural model of the hybrid protein are shown.

[0106] Figure 2 The image shows the SDS-PAGE electrophoresis results of each protein to be tested.

[0107] Figure 3 The results of SEC-HPLC purity analysis of the heterozygous protein are shown.

[0108] Figure 4 The CP inhibitory activity of each test protein was shown.

[0109] Figure 5 The AP inhibitory activity of each test protein is shown.

[0110] Figure 6 The study demonstrated the inhibitory activity of each tested protein in inhibiting C3b deposition on the surface of erythrocytes.

[0111] Figure 7 The dissociation properties of anti-C5 antibody and complement C5 as detected by ForteBio at different pH values ​​are shown.

[0112] Figure 8 The anti-C5 antibody was shown to inhibit the hemolytic activity of serum CP in human C5 transgenic mice.

[0113] Figure 9 A schematic diagram showing the linkage between the anti-C5 antibody heavy chain and the hybrid protein is shown.

[0114] Figure 10 The CP inhibition activity of the tested fusion protein was demonstrated.

[0115] Figure 11 The AP inhibitory activity of the tested fusion protein was demonstrated.

[0116] Figure 12 The assay showed that the fusion protein inhibited the deposition of C3b on the surface of erythrocytes.

[0117] Figure 13 The study demonstrated the CP and AP inhibitory activities of the fusion protein to be tested with the complement inhibitor C5 monoclonal antibody Eculizumab and the bifunctional C5 antibody FH1-5.

[0118] Figure 14 The experiment demonstrated the C3b deposition inhibition activity of the fusion protein to be tested with the complement inhibitory protein C5 monoclonal antibody Eculizumab and the bifunctional C5 antibody FH1-5 fusion protein (Wieslab method). Detailed description of the invention:

[0119] I. Definition:

[0120] It should be understood that the present invention is not limited to the specific methodologies, schemes, and reagents described herein, as these can vary. It should also be 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 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 pertains.

[0121] Decay Accelerator Factor (DAF)

[0122] Decay Accelerator Factor (DAF, CD55) is a membrane-associated regulatory protein that protects the cell from activation by its own complement on its surface. DAF functions by rapidly dissociating C3 and C5 convertases (the central enzymes of the cascade). DAF possesses the most potent decay-accelerating activity against complement-regulated proteins and acts on both classical pathway (C4b2a and C4b2a3b) and alternative pathway (C3bBb and C3BbC3b) enzymes. However, DAF lacks cofactor function.

[0123] Structural analysis of DAF revealed that, starting from its N-terminus, it consists of a unit of 4 to 60 amino acids in length, followed by a highly O-glycosylated serine (S) and threonine (T) fragment (STP), which is then linked to a post-translational glycosinositol phospholipid (GPT) anchor.

[0124] In some implementations, the DAF is a person DAF with the following login numbers: Genbank login numbers M31516, M15799, M64653, S72858, or M643567.

[0125] In some embodiments, the DAF is human DAF. In some embodiments, the human DAF is human natural DAF. In some embodiments, the amino acid sequence of human natural DAF is shown in SEQ ID NO:1, and its individual modules are shown in Table 1 below. Four 60-amino acid-long repeat units are called complement control protein repeats (CCPs) or short common repeats (SCRs). CCP1 includes amino acids 35-96. CCP2 includes amino acids 96-160; CCP3 includes amino acids 161-222, and CCP4 includes amino acids 223-285. They provide all the regulatory activities of the DAF. The highly O-glycosylated region acts as a buffer, positioning the CCP at an appropriate distance above the surface membrane. The GPI anchor allows the DAF to move freely within the plasma membrane plane, enabling it to inactivate the invertase complex anywhere it is assembled. In this document, when referring to the amino acid position of a module of DAF, all references are to the amino acid position number shown in SEQ ID NO:1.

[0126] Table 1: Protein modules of DAF (UniProt partitioning method)

[0127]

[0128] In some implementations, the nucleotide sequence of the cDNA encoding DAF is shown in SEQ.ID NO:2.

[0129] Factor H (CFH or FH)

[0130] "Complement factor H," "factor H," "FH," "CFH protein," or "CFH" are used interchangeably to refer to a protein of approximately 150 kDa 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 is used to regulate the alternative pathways of the complement system, ensuring that the complement system targets pathogens or other dangerous substances without harming host tissues.

[0131] Factor H is primarily monomeric, but exhibits weak self-association (KD = 28 μM) and may oligomerize in the presence of glycosaminoglycans or high concentrations of metal ions. CFH consists of 20 homologous units called complement control protein repeats (CCPs) (SCRs or sushi domains), some of which are involved in cell attachment, 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. Each module forms two disulfide bonds. SCRs 19 and 20 are involved in C3b binding.

[0132] The splice variant FHL-1 consists of the first 7 CCPs, followed by the C-terminal sequence Ser-Pro-Leu-Thr. Each CCP contains approximately 60 residues, including four unchanged cysteine ​​residues, forming Cys. I -Cys III Cys II -Cys IV Disulfides. Adjacent modules are linked by sequences of three to eight residues.

[0133] In negatively stained electron micrographs, FH molecules exhibit multiple conformations, but are primarily folded; analysis of ultracentrifugation, small-angle X-ray scattering (modules deposited in PDB, such as 3GAV), and chemical crosslinking also indicates that the module chains themselves are bent. High-resolution structures of the following FH fragments were determined, either alone or in combination with other molecules (PDB identifiers are provided in parentheses): CCP 1–2 (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 an elongated sphere with a major axis of approximately 4 nm and a minor axis of approximately 2 nm, and contains a β chain in an antiparallel sheet approximately aligned with the major axis. Its N and C ends are located at either end of its long axis, facilitating end-to-end arrangements of tandem CCPs with variable inter-module contact, tilting, and twisting.

[0134] In some embodiments, CFH is human CFH, such as natural human CFH. In some embodiments, the amino acid sequence of CFH has the following accession numbers: HGNC:HGNC:4883, Ensembl:ENSG00000000971, HPRD:00601, MIM:134370, or Vega:OTTHUMG00000035607.

[0135] In some embodiments, the amino acid sequence of CFH is shown in SEQ ID NO:3. In some embodiments, the amino acid positions corresponding to the various modules of CFH are shown in Table 2 below.

[0136] Table 2: Protein modules of CFH (Uniprot partitioning method)

[0137]

[0138] In some implementations, the nucleotide sequence of the cDNA encoding CFH is shown in SEQ.ID NO:4.

[0139] In some embodiments, the protein sequence encoding the splice variant FHL-1 of CFH is shown in SEQ ID NO:5. In some embodiments, the nucleotide sequence of the cDNA encoding the splice variant FHL-1 of CFH is shown in SEQ ID NO:6.

[0140] In this article, when referring to the amino acid position of the CFH module, the amino acid position number corresponds to the one shown in SEQ ID NO:3.

[0141] Other definitions

[0142] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0143] As used herein, the term “and / or” means any one of the options or two or more or all of the options.

[0144] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps. For example, when referring to a protein that “comprising” a specific sequence, it is also intended to cover proteins consisting of that specific sequence.

[0145] When “first” and “second” are mentioned in this article, it is only to distinguish between two structural domains or two chains, and does not indicate the location of the two structural domains in any way.

[0146] As used herein, the terms "hybrid protein" and "chimeric polypeptide" are used interchangeably, referring to a larger polypeptide formed by the optional fusion of at least two heterologous polypeptide sequences through a linker. Hybrid proteins can be produced through recombinant expression.

[0147] The terms “complement C5” or “C5 protein” or “complement C5 protein” or “C5 complement protein” are used interchangeably and refer to complement C5 proteins in different species. 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) linked by disulfide bridges. The peptide bond between Arg751 and Leu752 of the α chain is cleaved by C5 convertase, producing a small 74-amino acid-long C5a fragment and a large C5b fragment (1580 amino acids). The conversion of C5 to C5b involves a large conformational change and leads to subsequent C6 binding. For example, in some embodiments, human C5 has the sequence shown in SEQ ID NO:38. The term “antigen” refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen that specifically interacts with antibody molecules.

[0148] As used herein, the term "antigen-binding region" refers to the portion of a fusion protein that binds to a specific antigen. An antigen-binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. This antigen-binding region may or may not have a tertiary structure independent of the remainder of the fusion protein and may or may not bind its antigen as a standalone entity.

[0149] When it is mentioned that "the antigen-binding region is derived from the antibody", it means that the binding domains constituting the antigen-binding region are or are derived from the binding domains of the antibody that specifically bind to the antigen. For example, the heavy chain variable region and / or light chain variable region of the antigen-binding region are or are derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the target binding region are the CDRs of the antibody.

[0150] The term "derived from" means that the fragment in the antigen-binding region is substantially identical to the fragment from the antibody from which it originated, but has mutations at one or more sites, such as substitution, deletion, or addition. In one specific embodiment, the mutation is not in the antibody's CDR.

[0151] The terms "full-length antibody," "complete antibody," and "intact antibody" are used interchangeably herein to refer to naturally occurring glycoproteins comprising at least two heavy chains (H) and two light chains (L) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions (complementarity-determining regions (CDRs) interspersed with more conserved regions (framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Constant regions do not directly participate in antibody-antigen binding but exhibit various effector functions. In some embodiments, the antibody heavy chain constant region HC 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.

[0152] The term "antigen-binding fragment" in antibody refers to a portion or segment of a full-length antibody with fewer amino acid residues than a full-length antibody, but capable of binding antigens or competing with full-length antibodies (i.e., full-length antibodies from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody antibodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting full-length antibodies with papain. Furthermore, digestion of complete antibodies with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into Fab' monomers. Fab' monomers are essentially Fab fragments with hinge regions. Fv fragments consist of the VL and VH domains of the antibody's single arm. The two domains VL and VH of the Fv fragment can be encoded by independent genes, but they can also be produced as a single protein chain by using a recombinant approach, connecting the two domains with a synthetic linker peptide, and pairing the VL and VH regions in the single protein chain to form a single-chain Fv (scFv).

[0153] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that participates in the binding of the antibody to the antigen. The variable regions of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three complementarity-determining regions.

[0154] The complementarity-determining region (CDR), or hypervariable region, is a highly variable region within the antibody's variable domain (VH or VHH) that forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are sequentially numbered starting from the N-terminus, typically referred to as CDR1, CDR2, and CDR3. CDRs located within the variable domain of the antibody heavy chain are also called HCDR1, HCDR2, and HCDR3, while those located within the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, its CDR sequence can be determined using a variety of schemes known in the art, such as: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop; Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (International Immunogenetic Information System, World Wide Web imgt.cines.fr / ); and North's CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)).

[0155] The following are the area ranges of CDRs defined using the kabat, AbM, Chothia, Contact, and IMGT schemes.

[0156]

[0157] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing manner. In some embodiments, the CDR of this invention is determined based on the Kabat scheme.

[0158] CDRs can also be determined based on having the same Kabat numbering position as the reference CDR sequence. Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5). th The position of the number in Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)

[0159] In one embodiment, the CDR in the antibody molecule of the present invention is determined by the Chothia numbering rule.

[0160] 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 its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" or "Fc region" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, an immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) derived from the heavy chain of IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) derived from two heavy chains of IgM and IgE antibodies. Unless otherwise stated herein, amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In this document, the terms “Fc region,” “Fc portion,” and “Fc fragment” do not include the heavy chain variable region VH and light chain variable region VL, or the heavy chain constant region CH1 and light chain constant region CL of immunoglobulins, but may include, in some cases, the hinge region at the N-terminus of the heavy chain constant region. Examples of “effector functions” of immunoglobulins include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen-presenting cell uptake of antigens, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0161] The term "chimeric antibody" refers to an antibody molecule in which (a) a constant region or a portion thereof is altered, replaced, or exchanged, thereby linking the antigen-binding site to a different or altered class, effector function, and / or species of constant region, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug), which confers new properties to the chimeric antibody; or (b) a variable region or a portion thereof is altered, replaced, or exchanged with a variable region having different or altered antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region derived from human immunoglobulins. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing antigens while exhibiting reduced immunogenicity in humans, as compared to the original mouse antibody.

[0162] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (such as a mouse monoclonal antibody) while exhibiting low immunogenicity when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portion of the antibody with its corresponding human portion (i.e., replacing the non-binding portions of the constant region and variable region with the corresponding portions of the human antibody).

[0163] 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 through recombinant expression.

[0164] In this document, antibody constant regions or antibody constant domains, including CH1, CL, and Fc domains, as well as CH2, CH3, and optionally CH4 domains constituting the Fc domain, can be selected according to the intended function of the antibody molecule. For example, constant regions can be IgA, IgD, IgE, IgG, or IgM regions, especially immunoglobulin constant domains of human IgG, such as constant domains of human IgG1, IgG2, IgG3, or IgG4, preferably constant domains of human IgG2, IgG4, or IgG2 / 4 hybrids. Immunoglobulin constant regions can have native or variant sequences.

[0165] The term "hybrid heavy chain constant region" refers to fragments of a heavy chain constant region, such as constant region domains (e.g., CH1, CH2, CH3, and optionally CH4) or portions thereof that constitute the constant region, derived from different IgG, IgA, and IgD antibodies. For example, an IgG2 / IgG4 hybrid heavy chain constant region refers to a constant region where CH1 and a portion of CH2 are derived from IgG2, and a portion of CH2 and CH3 are derived from IgG4, such as the heavy chain constant regions of the C5 antibody drugs Soliris and Ultomiris.

[0166] As used herein, the term "connector" refers to any molecule that enables the direct connection of different parts of a bispecific binding molecule. Examples of connectors that establish covalent links between different molecular parts include peptide connectors and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyethylene, or copolymers of PEG and polypropylene glycol. In some embodiments, the connector is a peptide connector (also known as a "connecting peptide"), which refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or hinge regions from immunoglobulins, for linking the amino acid sequence of a first part of a fusion protein molecule to a second part of the binding molecule. For example, a peptide connector can link an antibody molecule and a polypeptide of a fusion protein molecule. For example, a peptide connector can also link one part of an antibody to another part of an antibody, such as linking a light chain variable region to a heavy chain variable region. Preferably, the peptide connector has a length sufficient to connect two entities in such a way that they maintain their conformation relative to each other without hindering the desired activity. Useful linkers also include glycine-alanine polymers, glycine-serine polymers, alanine-serine polymers, and other flexible linkers. In one embodiment, the linker peptide has a length of 1-50 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In one embodiment, the linker is one or more glycines, such as (G). n (SEQ ID NO:62), where n = an integer from 1 to 10, such as 1, 2, 3, 4, 5, or 6. In one embodiment, the connector is, for example, a glycine-serine polymer, such as GS (SEQ ID NO:63), G... n S(SEQ ID NO:64),GSG(SEQ ID NO:65),G n S n (SEQ ID NO:66), (G) n S) n (SEQ ID NO:67) or (G4S)n (SEQ ID NO:68), where n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, or 6. In one embodiment, the linker peptide is selected from one or more glycines, or "(GSG) n (SEQ ID NO:69)” or (G4S)n, where n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7, such as GGGGS(G4S, SEQ ID NO:70). Suitable flexible linker peptides can be rationally designed by simulating the three-dimensional structure of proteins and peptides using computer programs or by using phage display methods.

[0167] As used herein, the terms “anti-,” “binding,” or “specific binding” mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific 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), thin-layer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0168] “Affinity” or “binding affinity” refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can usually be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding determination described in this paper.

[0169] The "percentage of identity (%)" for an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the specific amino acid sequence shown in this specification, after comparing the candidate sequence with the specific amino acid sequence shown herein and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. In some embodiments, the invention contemplates variants of the protein or polypeptide of the invention that have a considerable degree of identity with respect to the polypeptide or protein specifically disclosed herein, for example, an identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher. Such variants may contain conserved changes.

[0170] For polypeptide sequences, "conservative alteration" includes substitutions, deletions, or additions to the polypeptide sequence that do not substantially change the desired functional activity of the polypeptide sequence. For example, a conserved substitution often results in an amino acid being replaced by a chemically similar amino acid. Tables of conserved substitutions of functionally similar amino acids are well known in the art. The following lists eight groups of amino acids containing mutually conserved substitutions: 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 "conserved sequence alteration" is used to refer to amino acid modifications that do not significantly affect or alter the activity of the parental heterozygous protein. For example, conserved modified variants retain at least 80%, 85%, 90%, 95%, 98%, 99% or higher, such as 100-110% or higher, of activity relative to the parent polypeptide or heterozygous protein.

[0171] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be any type of cell system that can be used to produce the hybrid protein of the present invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0172] The term "expression vector" refers to a vector containing recombinant polynucleotides and an expression control sequence that effectively links the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including clomids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0173] The terms “individual” or “subject” are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated 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.

[0174] The term "treatment" includes the administration of a composition or hybrid polypeptide to prevent or delay the onset of symptoms, complications, or biochemical indicators of a disease, to alleviate symptoms, or to stop or inhibit the further development of a disease, symptom, or condition. The term "prevention" includes the suppression of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition.

[0175] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0176] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0177] The term "effective amount" refers to such an amount or dose of the hybrid protein or nucleic acid encoding it or a combination thereof, which, when administered to a patient in a single or multiple doses, produces the desired effect in a patient requiring treatment or prevention.

[0178] "Therapeutic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. Therapeutic effective amount is also a amount in which any toxic or harmful effects of the hybrid protein or composition or combination are less than the beneficial therapeutic effect. Relative to an untreated subject, "therapeutic effective amount" preferably inhibits or improves a measurable parameter by at least about 40%, and more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0179] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered to individuals before or at an early stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0180] As used in this article, the term “complement C5-related disease or condition” refers to a disease or condition in which unregulated C5 function may result in a disease phenotype, for example, due to dysregulated C5 activation, such as increased C5 activation.

[0181] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the conjugated or fused reagent. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of probes or antibodies by conjugation (i.e., physical linking) to a detectable substance and indirect labeling of probes or antibodies by reaction with another directly labeled reagent. In some embodiments, the label is His or biotin.

[0182] "Subject / Patient / Individual Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as tears, vitreous fluid, cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of pregnancy or development in the subject. In some embodiments, the sample is blood or serum.

[0183] These and other aspects and embodiments of the invention are described in the accompanying drawings (briefly described below) and the following detailed description of the invention, and are exemplified in the following embodiments. Any or all features discussed above and throughout this application may be combined in various embodiments of the invention. The following embodiments further illustrate the invention; however, it should be understood that the embodiments are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art.

[0184] II. Heterozygous proteins

[0185] The present invention relates to a CFH and DAF hybrid protein comprising at least one functional unit from CFH and at least one functional unit from DAF.

[0186] A.DAF Functional Unit

[0187] In some embodiments, the hybrid protein of the present invention preferably comprises a functional unit derived from DAF. This functional unit is capable of dissociating C3 and C5 convertases and / or accelerating decay-accelerating activity against classical pathway C3 convertases and / or bypass pathway C3 convertases. In some embodiments, the DAF functional unit comprises CCP3 and 4 of DAF.

[0188] The amino acid sequence of such CCPs can be identical to the natural or naturally occurring amino acid sequence of DAFs. Alternatively, the amino acid sequence of such CCPs can be slightly modified, particularly at the amino or carboxyl terminus. This modification occurs when a polynucleotide encoding the CCP is incorporated into the restriction enzyme site. It also occurs when an amino acid is deleted from the N-terminus or C-terminus of the functional unit. For example, in some embodiments, one or two amino acids may be deleted from the N-terminus of CCP3.

[0189] Furthermore, some amino acid substitutions can be introduced into the sequence without affecting functional activity, preferably conservative substitutions. Conservative substitutions can be, for example, substitution of charged amino acids with each other, substitution of hydrophilic amino acids with each other, substitution of hydrophobic amino acids with each other, and substitution of amino acids of similar mass with each other.

[0190] In one embodiment, the DAF functional unit comprises CCP3 and / or 4 of the human DAF protein (or CCP3-4 formed by direct linkage of the C-terminus of CCP3 to the N-terminus of CCP4) having at least 95%, 96%, 97%, 98%, and 99% amino acid sequences. In some embodiments, the DAF functional unit comprises CCP3 and / or 4 of the human DAF protein (CCP3-4 formed by direct linkage of the C-terminus of CCP3 to 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 formed by direct linkage of the C-terminus of CCP3 to the N-terminus of CCP4.

[0191] In some embodiments, the human DAF protein is a naturally occurring human DAF protein. In some embodiments, the naturally occurring human DAF protein comprises or consists of the amino acid sequence shown in SEQ ID NO:1. The DAF comprises or consists of the amino acid sequence encoded by the DNA sequence shown in SEQ ID NO:2.

[0192] In some embodiments, CCP3 comprises or is composed of amino acids 161 to 222 of DAF. In some embodiments, to link other functional units, one or two amino acids at the N-terminus of CCP3 may be omitted, for example, CCP3 comprises or is composed of amino acids 163 to 222 of DAF.

[0193] In some embodiments, CCP3 comprises the amino acid sequence of SEQ ID NO:7 or 8, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8, or is composed of said sequence.

[0194] SEQ ID NO:7:

[0195] KSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECRE

[0196] SEQ ID NO:8:

[0197] CPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECRE.

[0198] In some implementations, CCP4 comprises or consists of amino acids 223 to 285 of DAF.

[0199] In some embodiments, CCP4 comprises the amino acid sequence of SEQ ID NO:9, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% uniformity with the amino acid sequence of SEQ ID NO:9, or is composed of said sequence.

[0200] SEQ ID NO:9:

[0201] IYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRG.

[0202] 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 the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:11, or comprises or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% uniformity with the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:11.

[0203] SEQ ID NO:10: Human DAF CCP3-4

[0204] KSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECRE IYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRG

[0205] SEQ ID NO:11: Human DAF CCP3-4 (CCP3 with two amino acids deleted from its N-terminus)

[0206] CPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECRE IYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRG

[0207] When referring to the amino acid position of DAF, the amino acid sequence position corresponds to the amino acid position of SEQ ID NO:1.

[0208] B.CFH Functional Unit

[0209] In some embodiments, the hybrid protein of the present invention comprises a functional unit derived from CFH. This functional unit is capable of dissociating the bypass pathway C3 convertase C3bBb and / or binding C3b. In a preferred embodiment, the CFH functional unit comprises CCP1 of CFH.

[0210] The amino acid sequence of the CCP of this type of CFH can be identical to the natural or naturally occurring amino acid sequence of CFH. Alternatively, the amino acid sequence of this type of CCP can be slightly modified, particularly at the amino or carboxyl terminus. This modification occurs when a polynucleotide encoding the CCP is incorporated into the restriction enzyme site. It also occurs when an amino acid is deleted or added from the N-terminus or C-terminus of the functional unit.

[0211] For example, in some implementations, one or two amino acids may be deleted from the C-terminus of CCP1.

[0212] In some embodiments, one or two amino acids from CCP2 can be added to the C-terminus of CCP1 to link the functional unit of DAF. Therefore, "CCP1" as used herein encompasses CCP1 with an amino acid (e.g., one or two amino acids from CCP2) added to its C-terminus. For example, if one or two amino acids are missing from the N-terminus of the functional unit of DAF, one or two amino acids from CCP2 can be added to the C-terminus of CCP1 to link the functional unit of DAF. In some embodiments, KS is missing from CCP3 of DAF, while RP is added to the C-terminus of CCP1 of CFH, resulting in a hybrid protein.

[0213] Furthermore, some amino acid substitutions can be introduced into the sequence without affecting functional activity, preferably conservative substitutions. Conservative substitutions can be, for example, substitution of charged amino acids with each other, or substitution of hydrophilic amino acids with each other, substitution of hydrophobic amino acids with each other, and substitution of amino acids of similar mass with each other. For example, the CCP1 of the CFH of the present invention can contain the V62I mutation.

[0214] In one embodiment, the CFH functional unit comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with CCP1 of the human CFH protein. In some embodiments, the CFH functional unit comprises CCP1 of the human CFH protein.

[0215] In some embodiments, the human CFH protein is a naturally occurring human CFH protein. In some embodiments, the naturally occurring human CFH protein comprises or is composed of the amino acid sequence shown in SEQ ID NO:3 or 5. In some embodiments, CFH comprises or is composed of the amino acid sequence encoded by the DNA sequence shown in SEQ ID NO:4 or 6.

[0216] In some embodiments, CCP1 comprises or consists of amino acids 19 to 82 of CFH. In some embodiments, CCP1 comprises or consists of amino acids 19 to 84 of CFH.

[0217] In some embodiments, the CFH functional unit comprises the amino acid sequence of SEQ ID NO:12, 13, 14 or 15, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:12, 13, 14 or 15, or is composed of said sequence.

[0218] SEQ ID NO:12: Human CFH CCP1 (CCP1 with two N-terminal amino acids RP from CCP2 added to its C-terminus)

[0219] EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNP LRKCQKRP

[0220] SEQ ID NO: 13: Human CFH CCP1 (CCP1)

[0221] EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNP LRKCQK

[0222] SEQ ID NO:14: Human CFH CCP1 (CCP1 with two N-terminal amino acids RP added to the C-terminus of CCP2 and containing V62I)

[0223] EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPL RKCQKRP

[0224] SEQ ID NO:15: Human CFH CCP1 (CCP1 includes V62I)

[0225] EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPL RKCQK

[0226] When referring to the amino acid position of CFH, the amino acid sequence position corresponds to the amino acid position of SEQ ID NO:3.

[0227] C. Other Units

[0228] Optionally, the hybrid protein of the present invention may further include a tag, preferably about 2 to 10 amino acids, added to the amino or carboxyl terminus of the hybrid protein, such as the carboxyl terminus. Typically, such addition is made to stabilize the protein or to facilitate secretory expression or purification of the hybrid protein. Such tags are known in the art. Representative examples of such tags include sequences encoding a series of histidine residues (e.g., 2-10 histidines, such as 2, 3, 4, 5, 6, or 7 histidines), epitope tags, herpes simplex glycoprotein D, β-galactosidase, maltose-binding protein, or glutathione S-transferase.

[0229] In some embodiments, the tag is a histidine residue that may be added to the amino terminus or carboxyl terminus of the hybrid protein, such as the carboxyl terminus.

[0230] In some implementations, the label is a 6xHis label of GHHHHHH (SEQ ID NO:16).

[0231] Optionally, the hybrid protein of the present invention may also include a signal peptide, such as MGWSCIILFLVATATGVHS (SEQ ID NO:17).

[0232] This invention also covers hybrid proteins in which one or more amino acids are altered through post-translational processes or synthetic methods. Examples of such modifications include, but are not limited to, glycosylation, iodination, myristylation, and polyethylene glycol modification.

[0233] D. Examples of heterozygous proteins

[0234] In some embodiments, the hybrid protein of the present invention comprises at least one functional unit from CFH and at least one functional unit from DAF.

[0235] In some embodiments, the hybrid protein of the present invention comprises CCP1 of CFH and CCP3 and CCP4 of DAF. In some embodiments, the hybrid protein of the present invention is composed of CCP1 of CFH and CCP3 and CCP4 of DAF. In some embodiments, the hybrid protein of the present invention comprises CCP1 of CFH and CCP3-4 of DAF, or is composed of thereof.

[0236] In some embodiments, CCP1 of CFH has 1-2 amino acids added to its C-terminus (e.g., 1-2 amino acids added to the N-terminus of CCP2). In some embodiments, CCP3 or CCP3-4 of DAF has 1-2 amino acids missing from its N-terminus.

[0237] In some embodiments, the hybrid protein of the present invention comprises CCP1 of CFH and CCP3-4 of DAF, wherein CCP1 has 1-2 amino acids from the N-terminus of CCP2 added to its C-terminus, and CCP3 of DAF has 1-2 amino acids correspondingly deleted from its N-terminus. In some specific embodiments, the hybrid protein of the present invention comprises CCP1 of CFH and CCP3-4 of DAF, wherein CCP1 has RP amino acids from the N-terminus of CCP2 added to its C-terminus, and CCP3 of DAF has RP amino acids correspondingly deleted from its N-terminus.

[0238] In some embodiments, the CCP1 of the CFH of the present invention may contain the V62I mutation.

[0239] In some embodiments, the hybrid protein of the present invention also includes a signal peptide at its N-terminus, such as the amino acid sequence shown in SEQ ID NO:17, and / or a tag at its C-terminus, such as a histidine tag, such as a 6×His tag, such as GHHHHHH.

[0240] In some embodiments, CCP3 of DAF comprises or is composed of amino acids 161-222 of the DAF protein, and / or CCP4 comprises or is composed of amino acids 223-285 of the DAF protein, wherein the amino acid positions correspond to the amino acid position numbers shown in SEQ ID NO:1. In some embodiments, CCP3 of DAF is missing two amino acids at its N-terminus, such as amino acids 163-222 of the DAF protein, or is composed of them, wherein the amino acid positions correspond to the amino acid position numbers shown in SEQ ID NO:1. In some embodiments, CCP3-4 of DAF comprises or is composed of amino acids 161-285 of the DAF protein, wherein the amino acid positions correspond to the amino acid position numbers shown in SEQ ID NO:1. In some embodiments, CCP3-4 of DAF is missing two amino acids at its N-terminus, such as amino acids 163-285 of the DAF protein, or is composed of them, wherein the amino acid positions correspond to the amino acid position numbers shown in SEQ ID NO:1.

[0241] In some embodiments, CCP1 of CFH comprises or is composed of amino acids 19-82 of the CFH protein, wherein the amino acid positions correspond to the amino acid position numbers shown in SEQ ID NO:3. In some embodiments, CCP1 of CFH comprises or is composed of amino acids 19-84 of the CFH protein, wherein the amino acid positions correspond to the amino acid position numbers shown in SEQ ID NO:3.

[0242] In some embodiments, CCP3-4 of DAF comprises or consists of amino acids 161-285 of the DAF protein, wherein the amino acid positions correspond to the amino acid position numbers shown in SEQ ID NO:1; and CCP1 of CFH comprises or consists of amino acids 19-82 of the CFH protein, wherein the amino acid positions correspond to the amino acid position numbers shown in SEQ ID NO:3.

[0243] In some embodiments, CCP3-4 of DAF comprises, or is composed of, amino acids 163-285 of the DAF protein, wherein the amino acid positions correspond to the amino acid position numbers shown in SEQ ID NO:1; and CCP1 of CFH comprises, or is composed of, amino acids 19-84 of the CFH protein, wherein the amino acid positions correspond to the amino acid position numbers shown in SEQ ID NO:3.

[0244] In some specific embodiments, DAF is a human DAF protein, such as the human native DAF protein. In some specific embodiments, the DAF protein comprises...

[0245] (i) The amino acid sequence shown in SEQ ID NO:1

[0246] (ii) The amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO:2;

[0247] (iii) has an amino acid sequence of at least 95%, 96%, 97%, 98%, or 99% as shown in (i) or (ii);

[0248] It may consist of the amino acid sequence shown in any one of (i) to (iii).

[0249] In some specific embodiments, CFH is a human CFH protein, such as the human native CFH protein. In some specific embodiments, the CFH protein comprises...

[0250] (i) The amino acid sequence shown in SEQ ID NO:3 or 5

[0251] (ii) The amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO:4 or 6;

[0252] (iii) has an amino acid sequence of at least 95%, 96%, 97%, 98%, or 99% as shown in (i) or (ii);

[0253] It may consist of the amino acid sequence shown in any one of (i) to (iii).

[0254] In some embodiments, the hybrid protein of the present invention comprises an amino acid sequence of any one of SEQ ID NO:18-21, or comprises or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence.

[0255] > Human CFH CCP1 or SCR1 -Human DAF / CD55 CCP3-4 or SCR3-4 (bold):

[0256]

[0257] Human CFH CCP1 or SCR1 - Human DAF / CD55 CCP3-4 or SCR3-4 (bold body), containing V62I mutation:

[0258]

[0259] In some embodiments, the hybrid protein of the present invention comprises an amino acid sequence of any one of SEQ ID NO:22-25, or comprises or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence.

[0260] > Human CFH CCP1 or SCR1 -Man DAF / CD55 CCP3-4 or SCR3-4 (Bold) -6×His (Italic):

[0261]

[0262] >People CFH CCP1 or SCR1 - Human DAF / CD55 CCP3-4 or SCR3-4 (bold)-6×His (italic), containing V62I mutation:

[0263]

[0264] In some embodiments, the hybrid protein of the present invention comprises an amino acid sequence of any one of SEQ ID NO:26-29, or comprises or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence.

[0265] >Signal peptide- Human CFH CCP1 or SCR1 -Man DAF / CD55 CCP3-4 or SCR3-4 (Bold) -6×His (Italic):

[0266]

[0267] >Signal peptide-human CFH CCP1 or SCR1 -Man DAF / CD55 CCP3-4 or SCR3-4 (Bold) -6×His (Italic):

[0268] Includes V62I mutation:

[0269]

[0270] In some embodiments, the hybrid protein of the present invention comprises the amino acid sequence of any one of SEQ ID NO:30-33, or comprises or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence.

[0271] >Signal peptide- Human CFH CCP1 or SCR1 -Human DAF / CD55 CCP3-4 or SCR3-4 (bold):

[0272]

[0273] >Signal peptide-human CFH CCP1 or SCR1 -Human DAF / CD55 CCP3-4 or SCR3-4 (bold):

[0274] Includes V62I mutation:

[0275]

[0276] III. Anti-C5 antibody

[0277] One aspect of this invention is to provide a C5 antibody that has a longer duration of in vivo efficacy.

[0278] In some embodiments, the anti-C5 antibody or its antigen-binding fragment of the present invention dissociates from C5 more rapidly under acidic conditions than under neutral conditions. In some embodiments, the anti-C5 antibody or its antigen-binding fragment of the present invention has pH-dependent antigen-binding properties, for example, it has a weaker ability to bind to the antigen under slightly acidic conditions (~pH 5.4-6.0), or it dissociates more rapidly. In some embodiments, the anti-C5 antibody or its antigen-binding fragment of the present invention has an extended in vivo half-life.

[0279] In some embodiments, the C5 antibody of the present invention is capable of effectively inhibiting the classical human complement pathway or the alternative human complement pathway (e.g., inhibiting hemolysis of said pathways). In some embodiments, the C5 antibody of the present invention is capable of effectively inhibiting both the classical and alternative human complement pathways (e.g., inhibiting hemolysis of said pathways). In some embodiments, the C5 antibody of the present invention has sustained inhibitory activity against both the classical and alternative human complement pathways (e.g., inhibiting hemolysis of said pathways). In some embodiments, the C5 antibody of the present invention has sustained inhibitory activity against both the classical and alternative human complement pathways (e.g., inhibiting hemolysis of said pathways).

[0280] In some embodiments, the C5 antibody of the present invention has pH-dependent antigen-binding specificity. In some embodiments, the anti-C5 antibody has a reduced ability to bind antigens (i.e., a faster dissociation rate) under acidic conditions, for example, at a pH less than about 7, 6.5, or 6, such as at a pH between about 4-7.0, 4.5-6.5, or 4.5-6.0. In some embodiments, the antibody has an extended drug half-life, for example, an extended in vivo antibody drug half-life.

[0281] In some implementations, the antibody has increased stability.

[0282] In some embodiments, the anti-C5 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, namely HCDR1, HCDR2 and HCDR3.

[0283] In some embodiments, the anti-C5 antibody or its antigen-binding fragment of the present invention comprises three complementary determinant regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3.

[0284] In some embodiments, the anti-C5 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region and three complementarity-determining regions (LCDRs) from the light chain variable region.

[0285] In some aspects, the anti-C5 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH). In some aspects, the anti-C5 antibody or its antigen-binding fragment of the present invention comprises a light chain variable region (VH). In some aspects, the anti-C5 antibody or its antigen-binding fragment of the present invention 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.

[0286] In some embodiments, the anti-C5 antibody or its antigen-binding fragment of the present invention further comprises an antibody heavy chain constant region (HC). In some embodiments, the anti-C5 antibody or its antigen-binding fragment of the present invention further comprises an antibody light chain constant region (LC). In some embodiments, the anti-C5 antibody or its antigen-binding fragment of the present invention further comprises both a heavy chain constant region (HC) and a light chain constant region (LC).

[0287] In some embodiments, the heavy chain variable region of the anti-C5 antibody of the present invention

[0288] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:39; or

[0289] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:39; or

[0290] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:39, preferably, the amino acid alterations do not occur in the CDR region.

[0291] In some embodiments, the light chain variable region of the anti-C5 antibody of the present invention

[0292] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:45; or

[0293] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:45; or

[0294] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:45, preferably, the amino acid changes do not occur in the CDR region.

[0295] In some embodiments, the anti-C5 antibody of the present invention has three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3 selected from...

[0296] (i) the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:39, or

[0297] (ii) A sequence that, relative to any one of (i), contains at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions.

[0298] For example, the CDR is determined using the Chothia scheme.

[0299] In some embodiments, the anti-C5 antibody of the present invention comprises three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3 selected from...

[0300] (i) the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:45, or

[0301] (ii) A sequence that, relative to any one of (i), contains at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions.

[0302] For example, the CDR is determined using the Chothia scheme.

[0303] In some embodiments, HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:40, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:40.

[0304] In some embodiments, HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:41, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:41.

[0305] In some embodiments, HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:42, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:42.

[0306] In some embodiments, LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:46, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:46.

[0307] In some embodiments, LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:47, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:47.

[0308] In some embodiments, LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:48, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:48.

[0309] In some specific embodiments of the present invention, the anti-C5 antibody or its antigen-binding fragment comprises VH and VL, wherein the VH contains the amino acid sequence shown 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 with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown 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 with it, or is composed of said amino acid sequence.

[0310] In some specific embodiments of the present invention, the anti-C5 antibody or its antigen-binding fragment of the present invention comprises HCDR1 containing or consisting of the amino acid sequence shown in SEQ ID NO:40, HCDR2 containing or consisting of the amino acid sequence shown in SEQ ID NO:41, HCDR3 containing or consisting of the amino acid sequence shown in SEQ ID NO:42; LCDR1 containing or consisting of the amino acid sequence shown in SEQ ID NO:46, LCDR2 containing or consisting of the amino acid sequence shown in SEQ ID NO:47, and LCDR3 containing or consisting of the amino acid sequence shown in SEQ ID NO:48.

[0311] In some specific embodiments of the present invention, the anti-C5 antibody or its antigen-binding fragment comprises VH and VL, wherein VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:39, and VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:45.

[0312] In some embodiments of the present invention, the anti-C5 antibody or its antigen-binding fragment comprises a heavy chain constant region. In some embodiments, the heavy chain constant region is derived from IgG1, IgG2, IgG3, or IgG4, preferably a heavy chain constant region of IgG2 or IgG4, or a hybrid heavy chain constant region of IgG2 / IgG4 (e.g., CH1, the hinge region, and a portion of CH2 are derived from IgG2, and a portion of CH2 and CH3 are derived from IgG4, such as the heavy chain constant regions of the C5 antibody drugs Soliris and Ultomiris). In some embodiments, the heavy chain constant region...

[0313] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:43; or

[0314] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:43; or

[0315] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:43.

[0316] In some embodiments of the present invention, the anti-C5 antibody or its antigen-binding fragment comprises a light chain constant region. In some embodiments, the light chain constant region of the anti-C5 antibody of the present invention is a lambda or kappa light chain constant region, such as a Kappa light chain constant region. In some embodiments, the light chain constant region...

[0317] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:49; or

[0318] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:49; or

[0319] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:49.

[0320] In some embodiments of the present invention, the anti-C5 antibody or its antigen-binding fragment of the present invention comprises a heavy chain. In some embodiments of the present invention, the anti-C5 antibody or its antigen-binding fragment of the present invention comprises a light chain. In some embodiments of the present invention, the anti-C5 antibody or its antigen-binding fragment of the present invention comprises both a heavy chain and a light chain.

[0321] In some specific implementation schemes, the heavy chain of the anti-C5 antibody

[0322] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:44; or

[0323] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:44; or

[0324] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:44, preferably, the amino acid alterations do not occur in the CDR region, and preferably, the amino acid alterations do not occur in the heavy chain variable region.

[0325] In some specific implementation schemes, the light chain of the anti-C5 antibody

[0326] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:50; or

[0327] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:50; or

[0328] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:50, preferably, the amino acid alterations do not occur in the CDR region, preferably, the amino acid alterations do not occur in the light chain variable region.

[0329] In some specific embodiments, the anti-C5 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown 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 with the amino acid sequence, or is composed of the amino acid sequence, and the light chain comprises the amino acid sequence shown 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 with the amino acid sequence, or is composed of the amino acid sequence.

[0330] In some specific embodiments, the anti-C5 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 44, and the light chain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 50.

[0331] In one embodiment of the invention, the amino acid alteration of the anti-C5 antibody described herein includes amino acid substitution, insertion, or deletion. In a preferred embodiment, the amino acid alteration described herein occurs in a region outside the CDR (e.g., in the FR). More preferably, the amino acid alteration described herein occurs in a region outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid alteration described herein is an amino acid substitution, preferably a conservative substitution.

[0332] In some embodiments, the anti-C5 antibody or its antigen-binding fragment of the present invention has one or more of the following characteristics:

[0333] (i) exhibits the same or similar binding affinity and / or specificity to C5 as the antibody of the present invention;

[0334] (ii) Inhibit (e.g., competitively inhibit) the binding of the antibody of the present invention to C5;

[0335] (iii) Epitopes that bind to the same or overlapping with the antibodies of the present invention;

[0336] (iv) Competing with the antibody of the present invention to bind to C5;

[0337] (v) Having one or more biological characteristics of the antibody of the present invention.

[0338] In some embodiments, the anti-C5 antibody of the present invention is an antibody in the form of IgG1, IgG2, IgG3, or IgG4, for example, an antibody in the form of IgG1. In some embodiments, the light chain constant region of the anti-C5 antibody of the present invention is a lambda or kappa light chain constant region, for example, a Kappa light chain constant region.

[0339] In some implementations, the anti-C5 antibody is a monoclonal antibody.

[0340] In some implementations, the anti-C5 antibody is humanized.

[0341] In some implementations, the anti-C5 antibody is a chimeric antibody.

[0342] In one embodiment, the anti-C5 antibody of the present invention also encompasses its antibody fragments (e.g., antigen-binding fragments), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), or linear antibodies.

[0343] IV. Fusion protein

[0344] In some embodiments, the present invention also relates to a fusion protein comprising an anti-C5 antibody or an antigen-binding fragment thereof, as well as the hybrid protein of the present invention.

[0345] In some embodiments, the fusion protein comprises an anti-C5 antibody or its antigen-binding fragment and a hybrid protein, wherein one or more of the hybrid proteins (at their N-terminus or their C-terminus) are respectively linked to the N-terminus and / or C-terminus of the heavy chain and / or light chain of the anti-C5 antibody or its antigen-binding fragment, with or without a linker.

[0346] In some embodiments, the fusion protein comprises an anti-C5 antibody or an antigen-binding fragment thereof and a hybrid protein, wherein one or both hybrid proteins (e.g., at their N-terminus) are respectively linked to the C-terminus of the heavy chain of the anti-C5 antibody or the antigen-binding fragment thereof.

[0347] In some implementations, the fusion protein comprises a full-length anti-C5 antibody.

[0348] In some embodiments, in the fusion protein, an anti-C5 antibody or its antigen-binding fragment is linked to the hybrid protein via a linker or without a linker. In some embodiments, the linker is selected from one or more glycine (G)n, GS, G... n S, G n S n 、(G n S) n Or (GSG) n Or (G4S) n Where n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6, or 7. In some embodiments, the connector is G, GSG, or G4S.

[0349] In some embodiments, the anti-C5 antibody or its antigen-binding fragment suitable for the fusion protein of the present invention can be any anti-C5 antibody or its antigen-binding fragment.

[0350] In some embodiments, the anti-C5 antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that specifically binds to C5, such as the anti-C5 antibody or its antigen-binding fragment disclosed in CN113754763A, or Eculizumab, Ravulizumab, Pozelimab, Crovalimab, Tesidolumab, or their antigen-binding fragments. In some embodiments, the anti-C5 antibody or its antigen-binding fragment comprises 1, 2, 3, 4, 5, or 6 CDRs of an antibody that specifically binds to C5, such as the anti-C5 antibody disclosed in CN113754763A, Eculizumab, Ravulizumab, Pozelimab, Crovalimab, or Tesidolumab. In some embodiments, the anti-C5 antibody or its antigen-binding fragment comprises the 1st, 2nd, and 3rd heavy chain variable regions (CDRs), namely HCDR1, HCDR2, and HCDR3, of antibodies that specifically bind to C5, such as the anti-C5 antibody disclosed in CN113754763A, Eculizumab, Ravulizumab, Pozelimab, Tesidolumab, or Crovalimab. In some embodiments, the anti-C5 antibody or its antigen-binding fragment comprises the 1st, 2nd, and 3rd light chain variable regions (CDRs), namely LCDR1, LCDR2, and LCDR3, of antibodies that specifically bind to C5, such as the anti-C5 antibody disclosed in CN113754763A, Eculizumab, Ravulizumab, Pozelimab, Tesidolumab, or Crovalimab. In some embodiments, the anti-C5 antibody or its antigen-binding fragment comprises three heavy chain variable regions (CDRs) and three light chain variable regions (CDRs) of antibodies that specifically bind to C5, such as the anti-C5 antibody disclosed in CN113754763A, Eculizumab, Ravulizumab, Pozelimab, Tesidolumab, or Crovalimab. In some embodiments, the anti-C5 antibody or its antigen-binding fragment comprises the heavy chain variable region of a known antibody that specifically binds to C5, such as the anti-C5 antibody disclosed in CN113754763A, Eculizumab, Ravulizumab, Pozelimab, Tesidolumab, or Crovalimab.In some embodiments, the anti-C5 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that specifically binds to C5, such as the anti-C5 antibody disclosed in CN113754763A, Eculizumab, Ravulizumab, Pozelimab, Tesidolumab, or Crovalimab. In some embodiments, the anti-C5 antibody or its antigen-binding fragment comprises a heavy chain of an antibody that specifically binds to C5, such as the anti-C5 antibody disclosed in CN113754763A, Eculizumab, Ravulizumab, Pozelimab, Tesidolumab, or Crovalimab. In some embodiments, the anti-C5 antibody or its antigen-binding fragment comprises a light chain of an antibody that specifically binds to C5, such as the anti-C5 antibody disclosed in CN113754763A, Eculizumab, Ravulizumab, Pozelimab, Tesidolumab, or Crovalimab. In some embodiments, the anti-C5 antibody or its antigen-binding fragment comprises the heavy and light chains of an antibody that specifically binds to C5, such as the anti-C5 antibody disclosed in CN113754763A, Eculizumab, Ravulizumab, Pozelimab, Tesidolumab, or Crovalimab.

[0351] In some implementations, the anti-C5 antibody is the anti-C5 antibody described in Part III.

[0352] In some embodiments, the anti-C5 antibody or its antigen-binding fragment or fusion protein includes an Fc region. In some embodiments, the fusion protein includes a dimerized Fc region formed by dimerization of the Fc region. In some embodiments, the first and second Fc regions are identical. In other embodiments, the first and second Fc regions are different, and they pair and heterodimerize. In some embodiments, the Fc region of the fusion protein is linked at its C-terminus to the hybrid protein of the present invention (e.g., its N-terminus), for example, the first Fc region and / or the second Fc region are linked to the hybrid protein of the present invention via or not via a linker.

[0353] The Fc region fragment suitable for anti-C5 antibodies or fusion proteins can be any antibody Fc region. The Fc region can include native sequence Fc regions and variant Fc regions. Native sequence Fc domains encompass a wide range of 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, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). For example, the Fc region of the antibody of the present invention can contain two or three constant domains, namely a CH2 domain, a CH3 domain, and optionally a CH4 domain. In some embodiments, the antibody Fc region may also have an IgG hinge region or a partial IgG hinge region at the N-terminus, for example, an IgG1 hinge region or a partial IgG1 hinge region. Mutations may be contained in said hinge region. In some embodiments, the hinge region may be EPKSS or EPKSC.

[0354] Preferably, the Fc region of the antibody or fusion protein of the present invention comprises CH2-CH3 from the N-terminus to the C-terminus, or comprises a 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 present invention is a human IgG Fc, such as human IgG1 Fc, human IgG2 Fc, human IgG3 or human IgG4 Fc, such as the human IgG2 or human IgG4 Fc region, or a human IgG2 / IgG4 hybrid Fc region (e.g., part of CH2 comes from IgG2, and part of CH2 and CH3 come from IgG4, such as the Fc region of the C5 antibody drugs Soliris and Ultomiris).

[0355] The Fc region in the antibody or fusion protein of the present invention can be mutated to obtain the desired properties. Mutations of the Fc region are known in the art.

[0356] In one embodiment, the Fc region contains a mutation, such as S228P, that increases antibody stability, particularly the stability of IgG4 type antibodies.

[0357] In one embodiment, the Fc region is modified to enhance its effector function (e.g., complement activation function). In another embodiment, the effector function has been reduced or eliminated relative to the wild-type Fc region. In yet another embodiment, the effector function is reduced or eliminated by means selected from: using a naturally occurring Fc isotype with reduced or eliminated effector function, and Fc region modification.

[0358] In a preferred embodiment, the Fc region has reduced effector functions mediated by the Fc region, such as reduced or eliminated ADCC, ADCP, or CDC effector functions, for example, containing mutations that achieve the above functions.

[0359] As those skilled in the art will understand, in accordance with the intended use of the antibody molecules of the present invention, the antibody molecules of the present invention may also include modifications in the Fc domain to alter the binding affinity for one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor.

[0360] In some embodiments, the Fc region contains a mutation that reduces binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in this invention has an L234A / L235A mutation (LALA mutation) that reduces binding to the Fcγ receptor.

[0361] In one embodiment, the Fc region contains a mutation that prolongs the antibody's half-life (e.g., in vivo half-life), for example, the Fc region contains a mutation that increases binding to the FcRn receptor. For instance, in some embodiments, the Fc region used in this invention has mutations that increase binding to the FcRn receptor, such as one or more of the following: YTE mutation (M252Y / S254T / T256E), LA mutation (M428L / N434A), or LS mutation (M428L / N434S). In some embodiments, the Fc region used in this invention has LA, YTE, and LS mutations that decrease binding to the Fcγ receptor.

[0362] In one embodiment, the Fc region contains or is composed of an amino acid sequence SEQ ID NO:59 or an amino acid sequence having at least 90% identity with it, such as 95%, 96%, 97%, 99% or higher.

[0363] In some embodiments, the Fc region containing the mutation that increases binding to the Fcγ receptor contains an amino acid sequence that has at least 90% identity with SEQ ID NO:59, such as 95%, 96%, 97%, 99% or higher, and contains M428L and N434A.

[0364] In some embodiments, the Fc region suitable for use in this invention comprises an amino acid sequence having at least 90% identity with SEQ ID NO:59, for example 95%, 96%, 97%, 99% or higher identity and containing S228P, M428L and N434A mutations.

[0365] In cases where the Fc regions differ, mutations that facilitate heterodimerization can be included in the Fc regions of the fusion protein of the present invention. In one embodiment, mutations are introduced in the CH3 regions of the two Fc regions. Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 regions of the first and second Fc regions are engineered in a complementary manner such that each CH3 region (or the heavy chain containing it) can no longer homodimerize with itself but is forced to heterodimerize with other complementary engineered CH3 regions (so that the first and second CH3 regions heterodimerize and no homodimer is formed between the two first CH3 regions or the two second CH3 regions). Preferably, based on the knob-in-Hole technique, corresponding knot and holo mutations are introduced in the first monomeric Fc region and the second monomeric Fc region, respectively. This technique is illustrated, for example, Merchant, AM, et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681.

[0366] In some embodiments of the present invention, the present invention provides a fusion protein comprising a full-length anti-C5 antibody and a hybrid protein, wherein the hybrid protein is linked to the anti-C5 antibody (via or not via a linker).

[0367] In some embodiments of the invention, the invention provides a fusion protein comprising a full-length anti-C5 antibody and a hybrid protein, wherein the hybrid protein is linked to the anti-C5 antibody heavy chain at its C-terminus or N-terminus (via or without a linker).

[0368] In some embodiments of the present invention, a fusion protein is provided comprising a full-length anti-C5 antibody and a hybrid protein, wherein...

[0369] The anti-C5 antibody is linked 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 (via or without a linker).

[0370] The light chain of the anti-C5 antibody constitutes the light chain of the fusion protein;

[0371] For example, the connector is selected from one or more glycine (G)n, GS, G n S, G n S n 、(G n S) n Or (GSG) n Or (G4S) n , where n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7, for example, the connector is G, GSG or G4S.

[0372] In some implementations, the fusion protein comprises two heavy chains and two light chains.

[0373] In some embodiments, the heavy chain of the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 54, 55, 56, 57 or 58, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence, or is composed of the sequence.

[0374] In some embodiments, the light chain of the fusion protein comprises the amino acid sequence shown 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 with the amino acid sequence, or is composed of the sequence.

[0375] In some embodiments, the fusion protein comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:54, 55, 56, 57 or 58, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence, or is composed of the sequence, and the light chain comprises the amino acid sequence shown 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 with the amino acid sequence, or is composed of the sequence.

[0376] 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 shown in SEQ ID NO:54, 55, 56, 57 or 58, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:50.

[0377] In some embodiments, the fusion protein of the present invention further comprises a signal peptide (e.g., at its N-terminus, such as the N-terminus of the heavy chain), such as the amino acid sequence shown in SEQ ID NO:17.

[0378] In one aspect, the present invention also provides an immunoconjugate comprising the molecules of the present invention (anti-C5 antibody or its antigen-binding fragment or its fusion protein), wherein the molecules of the present invention are conjugated with other active agents (e.g., anti-hemolytic drugs) or labels to form the immunoconjugate.

[0379] V. Polynucleotides, vectors, and hosts

[0380] This invention provides nucleic acids encoding any of the molecules of this invention described above (anti-C5 antibodies or their antigen-binding fragments or fusion proteins thereof). A vector comprising said nucleic acid is also provided. In one embodiment, the vector is an expression vector (e.g., a pCDNA vector, such as pCDNA3.1). A host cell comprising said nucleic acid or said vector is also provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells, such as 293FT or Expi293 cells). In yet another embodiment, the host cell is prokaryotic.

[0381] In one aspect, the present invention provides a nucleic acid encoding any of the above-mentioned anti-C5 antibodies or their antigen-binding fragments or their fusion proteins.

[0382] To facilitate production and purification, anti-C5 antibodies, their antigen-binding fragments, or their fusion proteins can be fused to the N-terminus with a secretory signal peptide and / or a tagged peptide that facilitates purification, such as a hexahistine tag or biotin label.

[0383] As will be apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0384] In some embodiments, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO:39, 44, 45, 50, 54-58, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO:39, 44, 45, 50, 54-58.

[0385] The nucleic acid sequence encoding the molecule of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification.

[0386] In one embodiment, one or more vectors comprising the nucleic acids of the present invention are provided. In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector. The vector includes, but is not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC). In a preferred embodiment, the expression vector is pCDNA, such as pCDNA3.1.

[0387] In one embodiment, a host cell comprising one or more of the polynucleotides of the present invention is provided. In some embodiments, a host cell comprising the expression vector of the present invention is provided. As used herein, the term "host cell" refers to any kind of cell system that can be engineered to produce the antibody molecules of the present invention. Host cells suitable for replicating and supporting the expression of the antibody molecules of the present invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors as needed, and large quantities of vector-containing cells can be cultured for inoculation into large-scale fermenters to obtain sufficient quantities of the molecules of the present invention for clinical applications. 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 (HEK 293 or 293F cells or 293FT cells or Expi293 cells), young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, and myeloma cell lines such as YO, NSO, P3X63, and Sp2 / O. Suitable mammalian host cell lines for antibody production are known in the art. In a preferred embodiment, the host cell is a CHO or HEK293 cell or 293FT cell or Expi293 cell.

[0388] V. Production and purification of the molecules of this invention

[0389] In another aspect, the present invention provides a method for producing the molecule of the present invention (anti-C5 antibody or its antigen-binding fragment or its fusion protein), the method comprising: culturing a host cell containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the molecule; optionally further comprising assembling the polypeptide chain to produce the molecule under conditions suitable for assembling the polypeptide chain into the molecule.

[0390] For recombinant production, a polynucleotide encoding the polypeptide chain of the molecule of the present invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YACs). Once an expression vector containing one or more polynucleotides of the present invention has been prepared for expression, the expression vector can be transfected or introduced into suitable host cells. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

[0391] The molecules prepared as described herein can be purified using known existing 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 also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art.

[0392] The purity of the molecules of the present invention can 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 activity of the antibody molecules provided herein can be identified, screened, or characterized by a variety of assays known in the art.

[0393] VI. Determination Method

[0394] The molecules (anti-C5 antibodies or their antigen-binding fragments or their fusion proteins) provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art.

[0395] The binding or dissociation properties of the molecules of this invention with human complement C5 protein can be determined by methods known in the art, such as ELISA, Western blotting, ForteBio, or the exemplary methods disclosed in Example 5 of this document.

[0396] The hemolytic inhibitory effect of the molecules of this invention can be measured by methods known in the art, such as in vitro and / or cellular experiments. For example, a hemolysis assay, such as the method shown in Example 4, can be used to detect the hemolytic inhibitory effect of the molecules on the classical complement immune pathway and / or the alternative complement immune pathway.

[0397] VII. Pharmaceutical compositions, drug combinations, and kits

[0398] In one aspect, the present invention provides compositions, such as pharmaceutical compositions, drugs, or formulations, said compositions comprising molecules of the present invention (e.g., anti-C5 antibodies or their antigen-binding fragments or fusion proteins thereof, or immunoconjugates, etc.).

[0399] In one embodiment, the composition further comprises pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0400] As used herein, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. For information on the use and applications of pharmaceutical excipients, see “Handbook of Pharmaceutical Excipients”, 8th edition, R.C. Rowe, P.J. Seskey and S.C. Wen, Pharmaceutical Press, London, Chicago.

[0401] The compositions, pharmaceuticals, or formulations of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injections), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0402] The compositions, pharmaceuticals, or formulations of the present invention may also contain other therapeutic agents in addition to one or more molecules of the present invention, said other therapeutic agents being required for the specific indication being treated and preferably not adversely affecting each other's activity. Thus, in one embodiment, the composition, formulation, or pharmaceutical, for example, a pharmaceutical composition, comprises one or more molecules of the present invention, and a combination of one or more other therapeutic agents.

[0403] The compositions, drugs, or formulations of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injections), powders or suspensions, liposomes, and suppositories. The compositions, drugs, or formulations of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion). Preferred forms depend on the intended mode of administration and therapeutic use.

[0404] The present invention also provides pharmaceutical combinations or pharmaceutical combination products comprising the molecules of the present invention (anti-C5 antibody or its antigen-binding fragment or its fusion protein or its immunoconjugate). Optionally, the pharmaceutical combinations or pharmaceutical combination products further comprise one or more other therapeutic agents (e.g., antihemolytic agents).

[0405] The present invention also provides a complete set of pillboxes containing the aforementioned drug combination, for example, the complete set of pillboxes comprising, within the same package:

[0406] - A first container containing a pharmaceutical composition comprising the molecules of the present invention (anti-C5 antibody or its antigen-binding fragment or its fusion protein or its immunoconjugate);

[0407] - Optionally, it also includes a second container containing a pharmaceutical composition comprising one or more other therapeutic agents (e.g., an antihemolytic agent) (in some embodiments, the two or more other therapeutic agents are in the same container or in separate containers).

[0408] VIII. Applications and Methods

[0409] In one aspect, the present invention provides a method for preventing or treating complement system-related diseases or conditions in subjects, including administering to subjects an effective amount of the molecules of the present invention (e.g., anti-C5 antibodies or their antigen-binding fragments or their fusion proteins or their immunoconjugates, etc.), or compositions, drugs or preparations containing them.

[0410] In some embodiments, the complement system-related disease is caused by abnormal activation or dysregulation of the complement system. In some embodiments, the abnormal activation or dysregulation of the complement system is due to, for example, microbial infection or an increase in autoantibodies, or due to a decrease, absence, dysfunction, or functional impairment or blockage of complement regulatory proteins. In some embodiments, treatment of the disease will benefit from inhibiting the activity of the complement system.

[0411] In some embodiments, the complement system-related disease or condition is a complement C5 or C3-related disease or condition. In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels) of complement C5 or C3 protein or C5a or C3a protein (e.g., compared to healthy subjects). In some embodiments, the subject's blood or blood cells have (e.g., elevated levels, such as nucleic acid or protein levels) of complement C5 or C3 protein or C5a or C3a protein (e.g., compared to healthy subjects' blood or blood cells). In some embodiments, treatment of the disease would benefit from suppressing the nucleic acid or protein levels of complement C5 or C3 protein or C5a or C3a protein.

[0412] In some implementations, the complement-related disease or condition may be a disease requiring hemolysis inhibition, such as a disease requiring inhibition of hemolysis via the classical complement immune pathway and / or the alternative complement immune pathway.

[0413] In some implementations, the complement system-related disease or condition is one that requires inhibition of C3b generation or deposition activity.

[0414] In some embodiments, the molecules of the present invention or compositions or drugs or preparations containing them delay the onset of the disease and / or symptoms associated with the disease.

[0415] In some embodiments, the molecules of the present invention or compositions or pharmaceuticals or formulations comprising them may also be administered in combination with one or more other therapies, such as treatments and / or other therapeutic agents, for the purposes described herein, such as for the prevention and / or treatment of the related diseases or conditions mentioned herein.

[0416] The administration route of the molecules or compositions containing them, or drugs or preparations of the present invention is according to known methods, such as injection or infusion.

[0417] In other respects, the present invention provides the use of the molecules of the present invention or compositions comprising them in the production or preparation of medicaments for the purposes described herein, such as for the prevention or treatment of complement system-related diseases or conditions mentioned herein.

[0418] IV. Diagnosis and Testing

[0419] In some implementations, the molecules provided herein (e.g., anti-C5 antibodies or their antigen-binding fragments or their fusion proteins or their immunoconjugates, etc.) can be used to detect the presence of complement C5 in biological samples.

[0420] When used herein, the term "detection" includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, ELISA assays, and PCR techniques (e.g., RT-PCR). In some embodiments, the biological sample is a bodily fluid.

[0421] In some embodiments, the method includes contacting a biological sample with a molecule as described herein under conditions that allow it to bind to complement C5, and detecting whether a complex is formed between the molecule and complement C5, wherein the formation of the complex indicates the presence of complement C5. This method can be in vitro or in vivo. In one embodiment, the molecules of the present invention are used to select subjects suitable for treatment with an inhibitor of complement C5 (e.g., an antibody against complement C5, such as the anti-C5 antibody of the present invention or its antigen-binding fragment or its fusion protein or its immunoconjugate, etc.), for example, where complement C5 is a biomarker for selecting the subject.

[0422] In some embodiments, labeled molecules of the invention are provided (e.g., anti-C5 antibodies or antigen-binding fragments thereof or their fusion proteins or immunoconjugates thereof). Labeling includes, but is not limited to, labels or portions that are directly detected (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.

[0423] In some implementations, the label is a marker such as biotin or a His tag.

[0424] In some embodiments provided herein, the sample is obtained prior to treatment with the molecules of the present invention or compositions, pharmaceuticals, or formulations comprising them. In some embodiments, the sample is obtained prior to treatment with other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

[0425] In some implementations, complement C5 is tested before treatment, for example, before initiating treatment or before a treatment interval.

[0426] In some embodiments, a method for treating the disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) for the presence of complement C5, thereby determining a complement C5 value; comparing the complement C5 value with a control value (e.g., a value in a normal individual); and if the complement C5 value is greater than the control value, administering to the subject a therapeutically effective amount of a molecule of the present invention, or a composition, drug, or preparation thereof, optionally in combination with one or more other therapies, thereby treating the disease. Example

[0427] Example 1. Construction of hybrid protein molecules

[0428] To construct protein expression vectors for the heterozygous molecules and prior art complement regulatory molecules involved in this invention, conventional molecular cloning techniques were employed. Using synthesized gene DNA (Suzhou Genewiz Biotechnology / Shanghai Sangon Biotech) as a template, the DNA encoding each protein was subcloned into the pCDNA3.1 expression vector (purchased from BioWind). After the plasmids were verified to be correct by sequencing, they were used for transient protein expression. Each encoded protein had a signal peptide (MGWSCIILFLVATATGVHS) added to its N-terminus and a 6xHis tag (GHHHHHH) added to its C-terminus to facilitate subsequent secretory expression in mammalian cells and nickel column purification.

[0429] The amino acid sequences of each protein are as follows:

[0430] >CR13m (human CR1 CCP1-3 or SCR1-3, containing N29K, S37Y, G79D and D109N mutations, sequence derived from patent US9988611_B2): SEQ ID NO:34

[0431] QCNAPEWLPFARPTNLTDEFEFPIGTYLKYECRPGYYGRPFSIICLKNSVWTGAKDRCRRKSCRNPPDPVN

[0432] GMVHVIKDIQFGSQIKYSCTKGYRLIGSSSATCIISGNTVIWDNETPICDRIPCGLPPTITNGDFISTNRENFH

[0433] YGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCGHHHHHHH

[0434] >D24 (human DAF / CD55 CCP2-4 or SCR2-4, sequence reference Hui-fen Zhang et al. THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 276, No. 29, Issue of July 20, pp. 27290-27295, 2001): SEQ ID NO: 35

[0435]

[0436] >FH15 (human CFH CCP1-5 or SCR1-5, containing the V62I mutation, sequence reference Masha Fridkis-Hareli et al. Blood. 2011 Oct 27; 118(17):4705-4713 and Agustín Tortajada et al. Hum Mol Genet. 2009 September 15; 18(18):3452-3461):SEQ ID NO:36

[0437] EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPGHHHHHH

[0438] >F1D34-1( Human CFH CCP1 or SCR1 -human DAF / CD55 CCP3-4 or SCR3-4):SEQ ID NO: 22

[0439] EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGGHHHHHH

[0440] >F1D34-2i(human CFH CCP1 or SCR1 -human DAF / CD55 CCP3-4 or SCR3-4, comprising the V62I mutation):SEQ ID NO: 24

[0441] EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQKKSCPNPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGGHHHHHH

[0442] >CR15D34( Human CR1, CCP15, or SCR15 -human DAF / CD55 CCP3-4 or SCR3-4):SEQ ID NO: 37

[0443]

[0444] >FH13 (human CFH CCP1-3 or SCR1-3, containing the V62I mutation, with two extra amino acids at the C-terminus for linking the His purification tag): SEQ ID NO:60

[0445] EDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNIIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISGHHHHHH

[0446] >D14 (human DAF / CD55 CCP1-4 or SCR1-4, no leader sequence, two extra amino acids at the C-terminus for linking the His purification tag): SEQ ID NO:61

[0447] DCGLPPDVPNAQPALEGRTSFPEDTVITYKCEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQNYFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCP NPGEIRNGQIDVPGGILFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGIIQGERDHYGYRQSVTYACNKGFTMIGEHSIYCTVNNDEGEWSGPPPECRGKSGHHHHHH

[0448] Example 2. Expression and purification of heterozygous proteins

[0449] After amplification with DH5α strain (Yisheng Biotechnology), the plasmid was prepared using the NucleoBond Xtra Midi Plus (MACHEREY-NAGEL, see product manual) plasmid extraction kit. The prepared plasmid was then transfected into Expi293 cells (Thermo Fisher Scientific) with PEI (PolyScience) for transient protein expression (method referenced). V., et al. BMC Biotechnol 13, 52, 2013.). The harvested supernatant was purified by affinity chromatography using a pre-packed nickel column (GE Lifesciences, HisTrap HP, see product manual for details). The sample was first washed with 20 mM imidazole buffer, then eluted with 500 mM imidazole buffer, and concentrated using ultrafiltration tubes. The protein sample was replaced with PBS buffer using Ultra (Merck Millipore). The replaced protein sample was then filtered through a 0.2 μm filter for sterilization. Protein quantification was performed using the NanoDrop (Thermofisher) A280 method based on the theoretical extinction coefficient.

[0450] Example 3. Protein purity detection

[0451] SDS-PAGE

[0452] Take 2.5 μg of protein and add it separately to loading buffers (Sangon Biotech) containing and without reducing agent. For samples with loading buffer containing reducing agent, boil at 95°C for 5 minutes to fully denature the protein. For samples without loading buffer, do not perform heat treatment. Use a 10% Precast-GLgel Tris-Glycine precast gel (Sangon Biotech) for electrophoresis. Electrophoresis was performed using the Tetra electrophoresis system (BioRad). After electrophoresis, the gel was stained with 0.1% Coomassie brilliant blue and destained with ethanol-glacial acetic acid solution.

[0453] SDS-PAGE electrophoresis results showed that, except for a slightly diffused band of protein D14, the bands of the other proteins were relatively clear, indicating that the purity of each protein was good under both reducing and non-reducing conditions (e.g., Figure 2 (As shown). In addition, due to differences in protein reduction, denaturation and modification states, as well as electrophoretic conditions, there is a certain difference between the electrophoretic molecular weight and the theoretical molecular weight.

[0454] SEC-HPLC

[0455] Protein purity analysis was performed using size exclusion chromatography (SEC). Purified protein samples in PBS were applied to a 300 × 4.6 mm, 5 μm column (TOSOH) on a TSKgel Super SW3000 HPLC instrument (DIONEX). SEC was performed using a U3000 HPLC instrument with isocratic elution at a flow rate of 0.25 mL / min. All proteins were detected using UV at 280 nm and 214 nm. Component analysis was performed using the instrument's built-in software.

[0456] The analysis results showed that the monomeric purities of the hybrid proteins F1D34-1 and F1D34-2i in solution reached 92% and 95%, respectively. Figure 3 (A / B) The monomers have good purity.

[0457] Example 4. Detection of complement inhibition activity

[0458] 4.1 Detection of Complement Classical Pathway (CP) Inhibitory Activity

[0459] This experiment investigated the inhibitory effects of different concentrations of the target proteins on hemolysin-mediated hemolysis of sheep erythrocytes induced by complement activity in normal human serum, and analyzed and compared the inhibitory activity of each protein on complement CP.

[0460] The experimental procedure is briefly described as follows:

[0461] (1) Protein gradient dilution: The protein was prepared into a 12000 nM solution using GVB++ buffer (gelatin florfenicol buffer containing Ca2+ and Mg2+, catalog number: 25-02080, Tian Enze), and then serially diluted 4 times, for a total of 7 gradients.

[0462] (2) Serum dilution: Normal human serum NHS (Shanghai Ruibai Biotechnology) was taken out of the -70℃ freezer, thawed naturally at 4℃, and then diluted to 4% with GVB++ buffer.

[0463] (3) Activation of sheep red blood cells: Sheep red blood cells (Nanjing Senbeijia Biotechnology) were washed with GVB++ buffer until the supernatant was clear, and then resuspended with GVB++ buffer. Hemolysin (BM351Y, Beijing Bosi) was added to the red blood cell suspension at a ratio of 1:4000. The sheep red blood cells were activated by incubation at 4℃ for 15 minutes. After activation, the sheep red blood cells were washed twice with GVB++ and then resuspended with GVB++ buffer.

[0464] (4) Incubation: Mix 25 μL of diluted protein and serum in a 96-well plate, then add 50 μL of the red blood cell suspension obtained in step (3) to make the initial concentration of protein 3000 nM and the final concentration of serum 1%. After thorough mixing, incubate in a 37°C constant temperature incubator for 1 hour, and set up a negative control (containing only serum) and a positive control (containing only serum and red blood cells).

[0465] (5) Termination: After incubation, add 100 μL of 20 mM EDTA-GVB buffer (GVBE) to each well to terminate the reaction;

[0466] (6) Reading: Centrifuge the 96-well plate at 3000 rpm for 5 minutes, take 100 μL of supernatant into a new 96-well flat plate, detect the absorbance at OD405 nm in a multi-functional plate reader, and save the data;

[0467] (7) Data processing: Substitute the obtained OD405nm readings into the following formula to calculate the erythrocyte hemolysis inhibition rate. The calculation formula is as follows:

[0468] Hemolysis inhibition rate (%) = (Positive control reading - Experimental group reading) / (Positive control reading - Negative control reading) * 100%

[0469] Then, a four-parameter fitting plot was performed with the final protein concentration on the x-axis and the hemolysis inhibition rate on the y-axis to calculate the IC50 value of the protein. The results are shown below. Figure 4 .

[0470] The results of the CP hemolytic inhibition activity assay showed that the CFH-DAF hybrid proteins F1D34-1 and F1D34-2i both exhibited potent CP inhibitory activity. Their activity was comparable to that of the CR1 activity-enhancing fragment CR13m and the DAF fragment D24, significantly superior to the CFH fragment FH15 and the DAF fragment D14, and far superior to the CR1-DAF hybrid protein CR15D34, which showed only weak CP inhibitory activity. The CFH fragment FH13, however, showed virtually no activity (e.g., CR15D34 exhibited only weak CP inhibitory activity). Figure 4 (As shown). This demonstrates that the hybrid protein obtained through domain hybridization exhibits excellent CP inhibitory activity.

[0471] 4.2 Detection of inhibitory activity of the alternative complement pathway (AP)

[0472] This experiment measured the degree of inhibition of rabbit erythrocyte hemolysis caused by complement activity in normal human serum by different concentrations of the test protein, and analyzed and compared the inhibitory activity of each protein on complement AP.

[0473] The experimental procedure is briefly described as follows:

[0474] (1) Protein gradient dilution: The protein was prepared into a 28000 nM solution using GVBMG buffer (gelatin floret buffer containing Mg2+ and EGTA, catalog number: 25-02090, Tian Enze), and then subjected to a 4-fold gradient dilution, for a total of 7 gradients.

[0475] (2) Dilution of serum: Normal human serum NHS (Shanghai Ruibai Biotechnology) was taken out of the -70℃ freezer, thawed naturally at 4℃, and then diluted to 60% with GVBMG;

[0476] (3) Rabbit red blood cell preparation: Wash rabbit red blood cells (Nanjing Senbeijia Biotechnology) with GVBMG until the supernatant is clear, and then resuspend the red blood cells in GVBMG;

[0477] (4) Incubation: Mix 25 μL of diluted protein and serum in a 96-well plate, then add 50 μL of the red blood cell suspension obtained in step (3) to make the initial concentration of protein 7000 nM and the final concentration of serum 15%. After thorough mixing, incubate in a 37°C constant temperature incubator for 1 hour, and set up a negative control (containing only serum) and a positive control (containing only serum and red blood cells).

[0478] (5) Termination: After incubation, add 100 μL of 10 mM EDTA-GVB buffer (GVBE) to each well to terminate the reaction;

[0479] (6) Reading: Centrifuge the 96-well plate at 3000 rpm for 5 minutes, take 100 μL of supernatant into a new 96-well flat plate, detect the absorbance at OD405 nm in a multi-functional plate reader, and save the data;

[0480] (7) Data processing: Substitute the obtained OD405nm readings into the following formula to calculate the erythrocyte hemolysis inhibition rate. The calculation formula is as follows:

[0481] Hemolysis inhibition rate (%) = (Positive control reading - Experimental group reading) / (Positive control reading - Negative control reading) * 100%

[0482] Then, a four-parameter fitting plot was performed with the final protein concentration on the x-axis and the hemolysis inhibition rate on the y-axis to calculate the antibody's IC50 value. The results are shown below. Figure 5 .

[0483] The results of the AP hemolytic inhibition activity assay showed that the CFH-DAF hybrid proteins F1D34-1 and F1D34-2i both exhibited potent AP inhibitory activity, superior to CFH fragments FH15 and FH13 and DAF fragments D24 and D14, and also superior to the CR1-DAF hybrid protein CR15D34 and the CR1 activity-enhancing fragment CR13m (e.g., Figure 5 (As shown). This indicates that the hybrid protein obtained through domain hybridization has superior AP inhibitory activity.

[0484] 4.3 Detection of complement C3b deposition inhibition activity

[0485] This experiment investigated the inhibition of C3b deposition on the surface of rabbit erythrocytes by different complement regulatory proteins, and analyzed and compared the inhibitory activity of each protein on C3b deposition.

[0486] The experimental procedure is briefly described as follows:

[0487] (1) Protein gradient dilution: The protein was prepared into a 1400 nM solution using GVBMG buffer (gelatin floret buffer containing Mg2+ and EGTA, catalog number: 25-02090, Tian Enze), and then serially diluted 3 times, for a total of 7 gradients.

[0488] (2) Serum dilution: To prevent complement activation from causing hemolysis and rupture of rabbit erythrocytes, which would prevent accurate detection of C3b deposition on the cell surface, normal human serum with C5 removed was used for the experiment. C5-Dpl NHS (Complement Technology) was taken out of the -70°C freezer, thawed naturally at 4°C, and then diluted to 40% with GVBMG;

[0489] (3) Rabbit erythrocyte preparation: 4% rabbit erythrocytes (Nanjing Senbega Biotechnology) were washed with GVBMG until the supernatant was clear, and then resuspended in GVBMG to a cell density of 1x10⁻⁶. 7 cells / mL;

[0490] (4) Incubation: Take 25 μL of diluted protein and serum and mix them in a 96-well plate. Then add 50 μL of the red blood cell suspension obtained in step (3) to make the initial concentration of protein 350 nM and the final concentration of serum 10%. After mixing thoroughly, incubate in a 37°C constant temperature incubator for 1 hour. Set up a negative control (containing only complement-inactivated serum and red blood cells) and a positive control (containing only normal serum and red blood cells).

[0491] (5) Termination: After incubation, add 100 μL of 10 mM EDTA-GVB buffer (GVBE) to each well to terminate the reaction;

[0492] (6) Detection: After incubating the cells, wash them three times with PBS and then add fluorescently labeled anti-human C3b / iC3b antibody for staining (APC anti-complement C3b / iC3b Antibody, Biolegend). After incubating at 4°C for 30 minutes, wash the cells three times again. Use a flow cytometer (Cytoflex, Beckman) to detect the APC fluorescence MFI (mean fluorescence intensity) of each sample and save the data.

[0493] (7) Data processing: The deposition rate of C3b on the surface of erythrocytes was calculated using the following formula:

[0494] Deposition rate (%) = (Experimental group MFI value / Positive control MFI value) * 100%

[0495] Then, a four-parameter fitting plot was generated with the final protein concentration on the x-axis and the deposition rate on the y-axis to calculate the IC50 value of the antibody. The results are shown below. Figure 6 .

[0496] Experimental results on C3b deposition showed that the CFH-DAF hybrid proteins F1D34-1 and F1D34-2i exhibited potent C3b deposition inhibitory activity, superior to CFH fragments FH15 and FH13 and DAF fragments D24 and D14, and also superior to the CR1-DAF hybrid protein CR15D34 and the CR1 activity-enhancing fragment CR13m (e.g., Figure 6 (As shown). This indicates that the hybrid protein obtained through domain hybridization has superior C3b deposition inhibition activity.

[0497] Example 5: Preparation and activity of anti-C5 antibody

[0498] 5.1 Preparation of anti-C5 antibody

[0499] The anti-human C5 antibody was obtained through mouse immunization and hybridoma screening, and then humanized. Its sequence and preparation method can be found in CN113754763A(16H46L39am).

[0500] 5.2 pH-dependent modification of anti-C5 antibody

[0501] Existing reports indicate that molecules with pH-dependent dissociation properties can rapidly dissociate from target molecules within endosomes, thereby reducing target-mediated clearance (TMDD), prolonging the in vivo half-life, and rapidly obtaining pH-dependent binding molecules 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.).

[0502] After screening for "histidine substitution" in the antibody CDR region, the C5 antibody phAb with pH-dependent dissociation characteristics was finally obtained. The sequence is shown in Table 1.

[0503] The pH-dependent dissociation characteristics of antibodies were detected using a ForteBio Octet K2 assay. The detection procedure is briefly described below:

[0504] Antibody was loaded into a neutral pH buffer at a concentration of 10 μg / mL using an ANTI-HUMAN IgG FC sensor (FORTEBIO, 18-5060). After baseline, C5 antigen (Complement Technology, A120) was bound at a concentration of 10 μg / mL. Dissociation was then performed in pH 7.4 and pH 5.4 buffers, respectively. The pH 5.4 buffer can be obtained by adjusting the pH using phosphate buffer or citrate buffer. The parameters were set as follows: loading 180 s, baseline 60 s, association 180 s, dissociation 500 s. Detailed instructions can be found in the ForteBio Octet user manual.

[0505] Test results as follows Figure 7 As shown, compared to pH 7.4 buffer, the modified C5 antibody phAb dissociates from C5 more quickly in pH 5.4 buffer.

[0506] 5.3 pH-dependent in vivo activity assay of C5 antibody

[0507] To verify the complement-inhibiting activity of pH-dependent C5 antibodies in animals, human C5 transgenic mice were used for testing. Specifically, serum was collected from human C5 transgenic mice (Southern Model Animal Center), and the human C5 content in the serum was detected using ELISA. Based on the human C5 content in the serum, 1 mg of C5 antibody was administered via tail vein injection at a rate of 105 μg / mL C5. Here, phAb is the pH-dependent C5 antibody of this application, and Ab is 16H46L39am disclosed in CN113754763A, both using the same human IgG4 constant region sequence (with S228P and LA mutations (M428L / N434A)). Serum was collected before and after injection (1 hour, 3 days, 6 days, 12 days, 15 days, 20 days, and 23 days), and serum complement activity was detected according to the following procedure:

[0508] (1) Take 1 mL of chicken red blood cells (SBJ-RBC-C003, Nanjing Senbeijia Biotechnology Co., Ltd.) and wash them 3 times with GVB++ (gelatin-flora buffer containing Ca2+ and Mg2+, catalog number: 25-02080, Tian Enze). After resuspending, add anti-chicken red blood cell antibody (1 ug / mL, anti-cRBCs, catalog number: 203-4139, Rockland) and incubate at 4℃ for 15 minutes to activate the chicken red blood cells. Centrifuge, wash 3 times with GVB++, and resuspend the red blood cells in 1 mL of GVB++.

[0509] (2) Human serum with C5-depleted NHS (CompTech) at a final concentration of 10% was mixed with transgenic mouse serum samples at a final concentration of 6.25%, and both diluents were GVB++. Control 1 contained only serum (ODctrl1) and GVB++, and Control 2 was the serum sample before antibody injection (ODctrl2), with the degree of hemolysis serving as a 100% hemolysis control.

[0510] (3) Transfer 30 μL of activated chicken erythrocytes into serum and incubate at 37°C for 1 h. After centrifugation, aspirate 80 μL of the supernatant and read the OD405 using a multi-functional plate reader.

[0511] Hemolysis rate (%) = (OD sample - ODctrl1) / (ODctrl2 - ODctrl1) x 100

[0512] Test results as follows Figure 8 As shown, based on the test results, the pH-dependent C5 antibody phAb has a more durable complement-inhibiting activity.

[0513] Table 1. Anti-C5 antibody sequences

[0514]

[0515]

[0516] Example 6: Construction, expression, purification, and purity analysis of fusion protein molecules

[0517] 6.1 Molecular Construction

[0518] To construct antibody fusion proteins with stronger complement-inhibiting activity, the C-terminus of the anti-C5 antibody heavy chain is linked to the N-terminus of the hybrid protein, with or without a linker peptide (e.g., ...). Figure 9 As shown), a series of protein molecules were designed and constructed as follows:

[0519] Table 2: Construction of fusion protein molecules

[0520]

[0521]

[0522] The light chain of the fusion protein is the light chain of the anti-C5 antibody (SEQ ID NO:50), and the heavy chain sequence of the fusion protein is as follows:

[0523] Table 3: Heavy chain sequence of fusion protein

[0524]

[0525]

[0526]

[0527] The genes encoding the heavy and light chains of the fusion protein were synthesized by Suzhou Genewiz Biotechnology and Nanjing Qingke Biotechnology. For secretory expression, a signal peptide was added to the N-terminus of each fusion protein, with the sequence: MGWSCIILFLVATATGVHS (SEQ ID NO:17). The genes were cloned into a eukaryotic expression vector (pCDNA3.1, purchased from Miaoling Biotechnology) using standard molecular biology techniques. After sequencing verification, plasmids were prepared using a plasmid extraction kit (NucleoBond Xtra Midi Plus, MACHEY-NAGEL) (see manufacturer's instructions for specific procedures) for protein expression.

[0528] 6.2 Protein Expression, Purification and Purity Analysis

[0529] Protein expression was performed using transient transfection of Expi293 cells (purchased from Thermo Fisher). The prepared heavy and light chain plasmids were co-transfected into Expi293 cells using a PEI-MW40000 (PolySciences) to transiently express the desired protein. For PEI preparation and usage instructions, please refer to the PolySciences PEI MAX user manual. The Expi293 cell culture and transfection procedure can be found in the Thermo Fisher Expi293 expression system user manual. Cell supernatant was harvested by centrifugation 5-7 days after transient transfection and filtered through a 0.45 μm filter for protein purification.

[0530] Transiently expressed proteins were purified using a pre-packed Protein A column (GE Lifesciences). The procedure is described in the manufacturer's instructions. The harvested eluent protein sample was dialyzed or ultrafiltered to a PBS buffer system, and then filtered through a 0.22 μm filter for sterilization to obtain the protein sample for testing. NanoDrop was used. TM ThermoScientific spectrophotometers are used to quantify protein samples based on the theoretical extinction coefficient of fusion proteins.

[0531] Protein purity analysis was performed using size exclusion chromatography (SEC). The purified sample in PBS was applied to a 300 × 4.6 mm, 5 μm column (TOSOH) of a TSKgel SuperSW3000. SEC was performed using a U3000 HPLC instrument (DIONEX). All proteins were detected using UV at 280 nm and 214 nm. Elution was performed isocratic at a flow rate of 0.25 mL / min. The results showed that the fusion protein purified in one step using Protein A exhibited good monomeric purity in solution, with all monomers exceeding 90% (Table 4), indicating the physical homogeneity of the fusion protein as a monomeric protein in solution.

[0532] Table 4. SEC-HPLC purity of fusion proteins

[0533] DB395 96.9 1.5 1.5 DB476 98.6 1.4 ND DB620 96.8 2.1 1.1 DB621 96 2.1 1.9 DB623 90.2 8.0 1.8 DB517 98.1 1.7 0.2 DB624 90.3 7.9 1.8 DB625 90.8 7.4 1.8 DB626 91.4 6.8 1.8

[0534] Example 7: Complement Inhibition Activity of Fusion Protein

[0535] 7.1 Classical pathway (CP) hemolytic inhibition activity

[0536] This experiment investigated the inhibitory effects of different concentrations of the tested fusion proteins on hemolysis via the classical complement pathway induced by activated chicken erythrocytes in 15% normal human serum, and analyzed and compared the inhibitory activity of each fusion protein on hemolysis via the classical complement pathway.

[0537] The experimental procedure is briefly described as follows:

[0538] (1) Protein gradient dilution: Each fusion protein was prepared into a 600 nM solution using GVB++ buffer (gelatin florfenicol buffer containing Ca2+ and Mg2+, catalog number: 25-02080, Tian Enze), and then subjected to a 3-fold gradient dilution, for a total of 7 gradients.

[0539] (2) Serum dilution: Normal human serum NHS (Shanghai Ruibai Biotechnology) was taken out of the -70℃ freezer, thawed naturally at 4℃, and then diluted to 25% with GVB++ buffer.

[0540] (3) Activation of chicken red blood cells: Chicken red blood cells (SBJ-RBC-C003, Nanjing Senbeijia Biotechnology) were washed with GVB++ buffer until the supernatant was clear, and then resuspended with an equal volume of GVB++ buffer. Then, 0.2 μL of anti-chicken red blood cell antibody (203-4139, Rockland) was added to each 1 ml of chicken red blood cells. The chicken red blood cells were activated by incubation at 4℃ for 15 minutes. After activation, the chicken red blood cells were washed twice with GVB++ and then resuspended with GVB++ buffer.

[0541] (4) Incubation: Mix 25 μL of diluted protein and serum in a 96-well plate, then add 50 μL of the red blood cell suspension obtained in step (3) to make the initial concentration of protein 150 nM and the final concentration of serum 6.25%. After thorough mixing, incubate in a 37°C constant temperature incubator for 1 hour, and set up a negative control (containing inactivated serum) and a positive control (containing only serum and red blood cells);

[0542] (5) Termination: After incubation, add 100 μL of 20 mM EDTA-GVB buffer (GVBE) to each well to terminate the reaction;

[0543] (6) Reading: Centrifuge the 96-well plate at 3000 rpm for 5 minutes, take 100 μL of supernatant into a new 96-well flat plate, detect the absorbance at OD405 nm in a multi-functional plate reader, and save the data;

[0544] (7) Data processing: Substitute the obtained OD405nm readings into the following formula to calculate the erythrocyte hemolysis inhibition rate. The calculation formula is as follows:

[0545] Hemolysis inhibition rate (%) = (Positive control reading - Experimental group reading) / (Positive control reading - Negative control reading) * 100%

[0546] Then, a four-parameter fitting plot was performed with the final protein concentration on the x-axis and the hemolysis inhibition rate on the y-axis to calculate the IC50 value of the protein. The results are shown below. Figure 10 .

[0547] The results of the chicken erythrocyte hemolysis assay showed that the F1D34 series fusion proteins (DB621, DB623, DB624, DB625, and DB626) exhibited good hemolytic inhibitory activity with little difference among the groups, and the IC50 for hemolysis inhibition reached approximately 5 nM. However, compared with the C15D34 fusion protein DB476 and the FH1-5 fusion protein DB395, they showed certain advantages. The experimental results are as follows... Figure 10 As shown.

[0548] 7.2 Alternative Pathway (AP) Hemolytic Inhibition Activity

[0549] The method for detecting the complement AP inhibitory activity of the fusion protein is the same as in 4.2.

[0550] The fusion protein was serially diluted 3-fold starting from 350 nM, resulting in a total of 7 concentrations.

[0551] Experimental results showed that all fusion proteins exhibited good AP inhibitory activity, and all achieved complete inhibition at high concentrations, overcoming the problem that single C5 antibodies could not completely inhibit AP activity (e.g., Eculizumab, see...). Figure 13B, or see In Vitro Combination Studies of ACH-4471 with Eculizumab to Assess a Potential “Switch” Treatment Approach for Paroxysmal Nocturnal Hemoglobinuria. Blood, Volume 130, Supplement 1, 2017, Page 2198), shows the activity advantage of the fusion protein.

[0552] On the other hand, the inhibitory activities of the F1D34 series fusion proteins were basically consistent, comparable to those of the D24 fusion protein, and slightly better than those of the FH1-5 fusion protein, as shown in the experimental results. Figure 11 As shown.

[0553] 7.3C3b deposition inhibition activity

[0554] The experimental method was the same as in 4.3, except that the final protein concentration was serially diluted 2-fold from 700 nM to a total of 7 concentrations.

[0555] According to the test results, the F1D34 fusion protein showed significantly better activity in inhibiting deposition on the surface of C3b cells than the C15D34 and D2-4 fusion proteins, and was also superior to the FH1-5 fusion protein. The experimental results are as follows: Figure 12 As shown.

[0556] Example 8: Comparison of complement inhibition activities of fusion proteins

[0557] To further demonstrate the activity advantages of the fusion protein of this invention, the activities of the existing complement inhibitory protein C5 monoclonal antibody Eculizumab and the bifunctional C5 antibody FH1-5 fusion protein were compared. The Eculizumab sequence was derived from Recommended INN list R49 (2003) and expressed in our laboratory (using the same method as above) or purchased from Alexion (Soliris); the bifunctional C5 antibody FH1-5 fusion protein FMEH-IgG4PLA-FH1-5 sequence was derived from patent WO2020219922A1 and expressed in our laboratory (using the same method as above). The protein purity was detected by SEC-HPLC, and the monomer purity was above 95%.

[0558] The methods for detecting complement CP and AP inhibitory activity are the same as in 4.1 and 4.2, except that:

[0559] For CP inhibition assay, the final serum concentration was 5%, and the final concentration of the fusion protein was serially diluted 4-fold starting from 150 nM, for a total of 7 concentrations; for AP inhibition assay, the final serum concentration was 10%, and the final concentration of the fusion protein was serially diluted 3-fold starting from 300 nM, for a total of 7 concentrations.

[0560] The test results showed that the F1D34 fusion protein phAb-GSG-F1D34-2i had stronger activity in inhibiting CP hemolysis in sheep erythrocytes, significantly better than eculizumab and FMEH-IgG4PLA-FH1-5, while eculizumab was superior to FMEH-IgG4PLA-FH1-5 (e.g., Figure 13 (As shown in A); On the other hand, for the inhibition of AP hemolysis in rabbit erythrocytes, the F1D34 fusion protein phAb-GSG-F1D34-2i was slightly better than FMEH-IgG4PLA-FH1-5, both were superior to Eculizumab, and both overcame the problem of insufficient AP hemolysis inhibition by Eculizumab (e.g. Figure 13 (as shown in B).

[0561] Comparison of C3b deposition inhibition activities (Wieslab method)

[0562] use The Complement System Alternative Pathway kit (Svar, COMPLAP330RUO) replaces the final C5b-9 (MAC) detection antibody with a C3b antibody (prepared in-house, sequence see SEQ ID NO:1 and 5 in US2012 / 0128674 A1) to detect the deposition of C3b generated after complement system activation at the bottom of the ELISA plate, and the effect of adding antibody or fusion protein on C3b generation or deposition on the bottom surface of the plate.

[0563] The detection procedure is briefly described as follows: Dilute normal human serum (NHS, Shanghai Ruibai Biotechnology) and the protein sample to be tested using the buffer provided in the kit. Take 50 μL of the diluted NHS and the protein sample to be tested (phAb-GSG-F1D34-2i, Eculizumab, and FMEH-IgG4PLA-FH1-5), mix well, and add to the ELISA plate provided in the kit. The final concentration of serum is 5%, and the final concentrations of the protein sample to be tested are 900, 300, 100, 33.33, 11.11, and 3.7 nM. At the same time, set Blank, PC, and NC according to the instructions. After incubation at 37°C for 1 hour, the sample was washed three times with wash solution. A final concentration of 1 μg / mL of mouse C3b antibody was added, and the sample was incubated at 37°C for 1 hour. After washing three times with wash solution, Goat Anti-Mouse IgG-Fc Secondary Antibody (HRP) (1:2000 dilution, Sinopharm, SSA006) was added, and the sample was washed three times with wash solution. The substrate was then added for color development, and the process was terminated. The absorbance at OD405nm was measured using a microplate reader, and the inhibition rate was calculated.

[0564] %complement inhibition:[1-(Sample-NC) / (PC-NC)]x100

[0565] Experimental results showed that the F1D34 fusion protein phAb-GSG-F1D34-2i had stronger activity, significantly better than FMEH-IgG4PLA-FH1-5, while Eculizumab had virtually no C3b deposition inhibitory activity (e.g., Figure 14 (As shown).

[0566] sequence list

[0567]

[0568]

[0569]

[0570]

[0571]

[0572]

Claims

1. An anti-C5 antibody or its antigen-binding fragment, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region VL comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 are the three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO: 39; and LCDR1, LCDR2, and LCDR3 are the three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO: 45; wherein the CDRs are defined using the Kabat, AbM, Chothia, Contact, or IMGT schemes as defined below. 。 2. An anti-C5 antibody or its antigen-binding fragment, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH comprises HCDR1 composed of the amino acid sequence shown in SEQ ID NO:40, HCDR2 composed of the amino acid sequence shown in SEQ ID NO:41, and HCDR3 composed of the amino acid sequence shown in SEQ ID NO:42; and the light chain variable region VL comprises LCDR1 composed of the amino acid sequence shown in SEQ ID NO:46, LCDR2 composed of the amino acid sequence shown in SEQ ID NO:47, and LCDR3 composed of the amino acid sequence shown in SEQ ID NO:

48.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region VH, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

39.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

39.

5. The antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region is composed of the amino acid sequence of SEQ ID NO:

39.

6. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a light chain variable region VL, wherein the light chain variable region comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

45.

7. The antibody of claim 6 or its antigen-binding fragment, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

45.

8. The antibody of claim 6 or its antigen-binding fragment, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

45.

9. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:39; and the light chain variable region comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:

45.

10. The antibody or antigen-binding fragment of claim 9, comprising a heavy chain variable region VH and a light chain variable region VL, wherein VH comprises an amino acid sequence as shown in SEQ ID NO:39, and VL comprises an amino acid sequence as shown in SEQ ID NO:

45.

11. The antibody or antigen-binding fragment thereof of claim 9, comprising a heavy chain variable region VH and a light chain variable region VL, wherein VH consists of an amino acid sequence as shown in SEQ ID NO:39, and VL consists of an amino acid sequence as shown in SEQ ID NO:

45.

12. The antibody or antigen-binding fragment thereof of any one of claims 1, 2, 4, 5, 7, 8, 10 and 11, further comprising the heavy chain constant region HC.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the antibody heavy chain constant region HC is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4.

14. The antibody or antigen-binding fragment thereof according to claim 12, wherein the antibody heavy chain constant region HC is the heavy chain constant region of IgG2 or IgG4, or a heavy chain constant region of IgG2 / IgG4 hybrid type.

15. The antibody or antigen-binding fragment thereof of claim 12, wherein the heavy chain constant region (i) Contains an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 43; or (ii) An amino acid sequence having one or more, but no more than 10, amino acid substitutions compared to the amino acid sequence of SEQ ID NO:

43.

16. The antibody of claim 15 or an antigen-binding fragment thereof, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

43.

17. The antibody of claim 16 or an antigen-binding fragment thereof, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

43.

18. The antibody of claim 12 or an antigen-binding fragment thereof, wherein the heavy chain constant region comprises a mutation that increases binding to the FcRn receptor and / or a mutation that increases antibody stability.

19. The antibody of claim 18 or its antigen-binding fragment thereof, wherein the mutation that increases binding to the FcRn receptor is one or more of the following: YTE mutation M252Y / S254T / T256E, LA mutation M428L / N434A, or LS mutation M428L / N434S.

20. The antibody of claim 19 or the antigen-binding fragment thereof, wherein the mutation that increases binding to the FcRn receptor is the LA mutation M428L / N434A.

21. The antibody of claim 18 or its antigen-binding fragment, wherein the mutation that increases antibody stability is S228P.

22. The antibody or antigen-binding fragment thereof of any one of claims 1, 2, 4, 5, 7, 8, 10 and 11, comprising a light chain constant region.

23. The antibody or antigen-binding fragment thereof of claim 22, wherein the light chain constant region is a lambda or kappa light chain constant region.

24. The antibody or antigen-binding fragment thereof of claim 22, wherein the light chain constant region (i) Contains an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 49; or (ii) An amino acid sequence comprising one or more, but not more than 10, amino acid substitutions compared to the amino acid sequence of SEQ ID NO:

49.

25. The antibody of claim 24 or an antigen-binding fragment thereof, wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO:

49.

26. The antibody of claim 22 or an antigen-binding fragment thereof, wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO:

49.

27. The antibody or antigen-binding fragment thereof of any one of claims 1, 2, 4, 5, 7, 8, 10, and 11, comprising a heavy chain, wherein the heavy chain... (i) Contains an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 44; or (ii) An amino acid sequence comprising one or more, but not more than 10, amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 44, wherein the amino acid change does not occur in the CDR region.

28. The antibody of claim 27 or an antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

44.

29. The antibody of claim 27 or an antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

44.

30. The antibody or antigen-binding fragment thereof of any one of claims 1, 2, 4, 5, 7, 8, 10, and 11, comprising a light chain, wherein the light chain... (i) Contains an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 50; or (ii) An amino acid sequence comprising one or more, but not more than 10, amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 50, wherein the amino acid change does not occur in the CDR region.

31. The antibody of claim 30 or an antigen-binding fragment thereof, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

50.

32. The antibody of claim 30 or an antigen-binding fragment thereof, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

50.

33. The antibody or antigen-binding fragment thereof of any one of claims 1, 2, 4, 5, 7, 8, 10 and 11, comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO:44; and the light chain comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO:

50.

34. The antibody of claim 33 or an antigen-binding fragment thereof, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 44, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

50.

35. The antibody of claim 33 or its antigen-binding fragment, comprising a heavy chain and a light chain, wherein the heavy chain consists of the amino acid sequence shown in SEQ ID NO: 44, and the light chain consists of the amino acid sequence shown in SEQ ID NO:

50.

36. The antibody or antigen-binding fragment thereof of any one of claims 1, 2, 4, 5, 7, 8, 10 and 11, wherein the antibody is a humanized antibody or a chimeric antibody.

37. The antibody or antigen-binding fragment thereof of any one of claims 1, 2, 4, 5, 7, 8, 10 and 11, wherein the antibody is a monoclonal antibody.

38. The antibody or antigen-binding fragment thereof of any one of claims 2-11, wherein the antigen-binding fragment is an antibody fragment selected from: Fab, Fab', Fab'-SH, Fv, scFv, or (Fab')2.

39. A fusion protein comprising an anti-C5 antibody or an antigen-binding fragment thereof and a hybrid protein, wherein the hybrid protein comprises or is composed of the following: (i) Human complement factor H, i.e., CCP1 of CFH (ii) Human decay acceleration factors are CCP3 and CCP4 of DAF, where CCP3 and CCP4 of DAF are directly connected together to form CCP3-4; The antibody or its antigen-binding fragment is linked to the hybrid protein via or without a linker; The anti-C5 antibody or its antigen-binding fragment thereof is the antibody or its antigen-binding fragment as described in any one of claims 1-38; The anti-C5 antibody or its antigen-binding fragment comprises an Fc region, wherein the Fc region is linked at its C-terminus to the N-terminus of a heterozygous protein, with or without a linker; wherein (1) CCP1 of human CFH contains or is composed of the amino acid sequence of human CFH protein from position 19 to 82, and CCP3 of human DAF contains or is composed of the amino acid sequence of human DAF protein from position 161 to 222, and CCP4 of human DAF contains or is composed of the amino acid sequence of human DAF protein from position 223 to 285. (2) The CCP1 of human CFH contains, or is composed of, the amino acid sequence of the human CFH protein from positions 19 to 84, and the CCP3 of human DAF contains, or is composed of, the amino acid sequence of the human DAF protein from positions 163 to 222, and The CCP4 of human DAF consists of or is composed of the amino acid sequence from position 223 to 285 of the human DAF protein; (3) The CCP1 of human CFH contains, or is composed of, the amino acid sequence of human CFH protein from positions 19 to 82, and the CCP3-4 of human DAF contains, or is composed of, the amino acid sequence of human DAF protein from positions 161 to 285; or (4) CCP1 of human CFH contains or is composed of the amino acid sequence of human CFH protein from position 19 to 84, and CCP3-4 of human DAF contains or is composed of the amino acid sequence of human DAF protein from position 163 to 285. The amino acid positions of the human CFH protein correspond to the amino acid position numbers shown in SEQ ID NO:3, and the amino acid positions of the human DAF protein correspond to the amino acid position numbers shown in SEQ ID NO:

1.

40. The fusion protein of claim 39, comprising a full-length anti-C5 antibody and a hybrid protein, wherein... The anti-C5 antibody is linked to the N-terminus of the hybrid protein at the C-terminus of its Fc region to form the heavy chain of the fusion protein, with or without a linker. The light chain of the anti-C5 antibody constitutes the light chain of the fusion protein.

41. The fusion protein of claim 40, wherein the fusion protein comprises two heavy chains and two light chains.

42. The fusion protein of any one of claims 39-41, wherein the human CFH CCP1 has a V62I mutation.

43. The fusion protein of any one of claims 39-41, wherein the human CFH protein is a natural human CFH protein or a protein comprising thereof. (i) The amino acid sequence shown in SEQ ID NO:3 or 5, (ii) The amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO:4 or 6; Or it may consist of the amino acid sequence shown in (i) or (ii).

44. The fusion protein of any one of claims 39-41, wherein the human DAF protein is a natural human DAF protein or a subset thereof. (i) The amino acid sequence shown in SEQ ID NO:1; (ii) The amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO:2; Or it may consist of the amino acid sequence shown in (i) or (ii).

45. The fusion protein according to any one of claims 39-41, wherein The human CFH CCP1 contains, or is composed of, the amino acid sequence of SEQ ID NO: 12, 13, 14 or 15; The CCP3 of the human DAF contains, or is composed of, the amino acid sequence of SEQ ID NO: 7 or 8; the CCP4 of the human DAF contains, or is composed of, the amino acid sequence of SEQ ID NO: 9; and / or The CCP3-4 of the human DAF contains, or is composed of, the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:

11.

46. ​​The fusion protein of any one of claims 39-41, wherein the fusion protein comprises a signal peptide.

47. The fusion protein of any one of claims 39-41, wherein the fusion protein comprises a signal peptide at its N-terminus.

48. The fusion protein of claim 47, wherein the signal peptide is a secretory signal peptide.

49. The fusion protein of claim 48, wherein the secretory signal peptide comprises the amino acid sequence shown in SEQ ID NO:

17.

50. The fusion protein of any one of claims 39-41, wherein the hybrid protein comprises the amino acid sequence of any one of SEQ ID NO:18-33.

51. The fusion protein of claim 50, wherein the hybrid protein comprises the amino acid sequence of any one of SEQ ID NO:18-33.

52. The fusion protein of any one of claims 39-41, wherein the linker is selected from one or more glycines (G)n, GS, GnS, GnSn, (GnS)n, (GSG)n or (G4S)n, wherein n is an integer of 2, 3, 4, 5, 6 or 7.

53. The fusion protein of any one of claims 39-41, wherein the linker is G, GSG, or G4S.

54. The fusion protein of claim 40, wherein the heavy chain of the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 54, 55, 56, 57 or 58; and the light chain of the fusion protein comprises the amino acid sequence shown in SEQ ID NO:

50.

55. The fusion protein of claim 40, wherein the heavy chain of the fusion protein consists of the amino acid sequence shown in SEQ ID NO: 54, 55, 56, 57 or 58; and the light chain of the fusion protein consists of the amino acid sequence shown in SEQ ID NO:

50.

56. A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described in any one of claims 1-38, or a fusion protein as described in any one of claims 39-55.

57. An expression vector comprising the nucleic acid molecule of claim 56.

58. The expression vector of claim 57, wherein the expression vector is pCDNA3.

1.

59. A host cell comprising the nucleic acid molecule of claim 56 or the expression vector of claim 57 or 58.

60. The host cell of claim 59, wherein the host cell is prokaryotic or eukaryotic.

61. The host cell of claim 59, wherein the host cell is a CHO cell or a 293 cell.

62. The host cell of claim 61, wherein the 293 cell is an Expi293 cell.

63. A method for preparing an antibody or antigen-binding fragment thereof as described in any one of claims 1-38, or a fusion protein as described in any one of claims 39-55, the method comprising culturing a host cell as described in any one of claims 59-62 under conditions suitable for expression of the antibody or fusion protein.

64. The method of claim 63, wherein the method further comprises isolating the protein from the host cell or the host cell culture medium, and / or purifying the protein.

65. An immunoconjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-38, or a fusion protein as described in any one of claims 39-55, and a label.

66. A pharmaceutical composition, drug, or formulation comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-38, or the fusion protein as described in any one of claims 39-55.

67. The pharmaceutical composition, drug, or formulation of claim 66, further comprising pharmaceutical excipients.

68. Drug combination products, which include The antibody or antigen-binding fragment thereof as described in any one of claims 1-38, or the fusion protein as described in any one of claims 39-55; and Other treatments.

69. A non-diagnostic or non-therapeutic method for detecting the presence of complement C5 in a biological sample, comprising contacting the biological sample with an antibody or antigen-binding fragment thereof as described in any one of claims 1-38, or a fusion protein as described in any one of claims 39-55, under conditions that allow it to bind 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 the complex indicates the presence of complement C5.

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