Anti-c5 and HTRA1 bispecific antibody and use thereof

Abstract

The present invention relates to a novel anti-C5 antibody, a novel HTRA1 antibody, and a bispecific antibody that specifically binds C5 and HTRA1 and is constructed on the basis of the anti-C5 antibody and the HTRA1 antibody. In particular, the present invention relates to an anti-C5 antibody that specifically binds different epitopes of C5 or a bispecific antibody that specifically binds different epitopes of C5 and HTRA1. The present invention further relates to a nucleic acid encoding the antibody, an expression vector comprising the nucleic acid, and a host cell comprising the nucleic acid or the expression vector. The present invention further relates to a pharmaceutical composition or pharmaceutical combination comprising the antibody. The present invention further relates to a method for and use in treating a disease on the basis of the antibody.

Description

Anti-C5 and HTRA1 bispecific antibodies and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202311675539.2 and application date of December 7, 2023, and the Chinese patent application with application number 20241228514.6 and application date of February 29, 2024, and claims the priority of the said Chinese patent applications. The entire contents of the said Chinese patent applications are hereby introduced into this application as a reference.

[0003] The present invention relates to a novel anti-C5 antibody and a novel HTRA1 antibody, as well as bispecific antibodies constructed based thereon that specifically bind to C5 and HTRA1. In particular, the present invention relates to an anti-C5 antibody that specifically binds to different epitopes of C5 or a bispecific antibody that specifically binds to different epitopes of C5 and HTRA1. The present invention also relates to nucleic acids encoding the antibodies, expression vectors comprising the nucleic acids, and host cells comprising the nucleic acids or expression vectors. The present invention also relates to pharmaceutical compositions or drug combinations comprising the antibodies, and to methods and uses for treating diseases based on the antibodies.

[0004] Background of the Invention

[0005] The complement system is an important component of the human innate immunity. When activated, it causes target cell lysis and promotes phagocytosis through opsonization. Complement is activated through three major pathways via a series of proteolytic steps: the classical pathway, which is usually activated by immune complexes; the alternative pathway, which can be induced by unprotected cell surfaces; and the mannose-binding lectin pathway. All three pathways of the complement cascade converge on the proteolytic cleavage of the complement component 5 (C5) protein. The cleavage of complement component 5 (C5) results in the production of fragments C5a and C5b, a process that is crucial for the activation of the complement cascade. C5a can produce pleiotropic physiological responses by binding to its receptor. C5a is a potent proinflammatory mediator that induces chemotactic migration, enhances cell adhesion, stimulates the oxidative burst, and induces the release of multiple inflammatory mediators (such as histamine or cytokines). C5b mediates the formation of the membrane attack complex (MAC, or C5b-9), which leads to cell lysis in the late stages of complement-dependent cytotoxicity (CDC). Furthermore, in nucleated cells that are resistant to cytolysis by C5b-9, sublytic amounts of C5b-9 can cause cell activation, leading to cell proliferation, production of proinflammatory mediators, and production of extracellular matrix.

[0006] The serine protease HtrA1 serine peptidase 1 (HtrA1) (PRSS11; PA cluster (Clan PA), family 51) belongs to an evolutionarily conserved family of HtrA proteins. In humans, HtrA1, HtrA3, and HtrA4 share the same domain architecture: an N-terminal IGFBP-like component and a Kazal-like component, a protease domain with a trypsin-like fold, and a C-terminal PDZ domain. The physiological relevance of HtrA1 has been reliably established by the identification of human loss-of-function mutations that cause familial ischemic cerebral small-vessel disease. The molecular mechanism involves defective TGFβ inhibition by HtrA1, which leads to increased TGFβ signaling. Dysregulated TGFβ signaling through aberrant HtrA1 expression may also contribute to arthritic diseases, in conjunction with HtrA1-mediated degradation of various extracellular matrix components or indirectly through upregulation of matrix metalloproteinases. Additionally, human genetic studies have identified a strong correlation between the development of age-related macular degeneration (AMD) and SNPs in the HtrA1 promoter region that result in increased HtrA1 transcript levels.

[0007] Existing technologies have developed antibodies targeting C5, such as eculizumab. However, in vitro hemolysis inhibition assays have shown that while eculizumab exhibits good inhibition of classical complement pathway hemolysis, its activity in blocking the alternative complement pathway (AP) is insufficient, resulting in its efficacy failing to fully meet patient needs. For example, some PNH patients treated with eculizumab (Soliris) still require blood transfusions due to extravascular hemolysis.

[0008] The prior art has also developed a combination of different C5 antibodies (WO2019118556A1), and has also developed antibodies that specifically bind to HTRA1 (WO2017075212A1).

[0009] However, the existing anti-C5 antibodies are still insufficient in blocking the complement pathway, and the therapeutic effect needs to be improved. At the same time, combination therapy requires multiple injections, which also brings a burden to patients.

[0010] Therefore, there is a need in the art to develop therapeutic agents that have stronger blocking activity, better therapeutic effects, and can simplify administration. Summary of the Invention

[0011] In one aspect, the present invention develops novel VHH antibodies that specifically bind to C5, and heavy chain antibodies comprising the same. The present invention also develops novel VHH antibodies that specifically bind to HTRA1, and heavy chain antibodies comprising the same.

[0012] In another aspect, the present invention develops an anti-C5 antibody that specifically binds to a different epitope of C5 based on an anti-C5 VHH.

[0013] In another aspect, the present invention develops a multispecific binding protein based on anti-C5 VHH and anti-HTRA1 VHH, such as a bispecific antibody, which specifically binds to C5 and HTRA1, in particular, it specifically binds to different epitopes of C5 and simultaneously specifically binds to HTRA1.

[0014] In one embodiment, the bispecific antibody of the present invention has one or more of the following characteristics:

[0015] (1) Simultaneously block human C5 and human HTRA1;

[0016] (2) having a stronger complement pathway blocking pathway, such as being able to completely block the alternative complement pathway;

[0017] (3) Demonstrated superior therapeutic efficacy compared to single-target drugs in the sodium iodate-induced geographic atrophy model;

[0018] (4) A single administration can achieve both anti-C5 and anti-HTRA1 effects.

[0019] In some embodiments, the present invention relates to the following specific embodiments:

[0020] 1. A VHH antibody that specifically binds to C5, comprising

[0021] three complementarity determining regions (CDRs) contained in the VHH represented by any one of SEQ ID NO: 23, 17 or 22,

[0022] Preferably, the CDR1 sequence is according to the AbM definition, CDR2 is according to the Kabat definition, and CDR3 is according to the AbM definition.

[0023] 2. The VHH antibody specifically binding to C5 according to embodiment 1, comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0024] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10;

[0025] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; or

[0026] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 9, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 10.

[0027] 3. The VHH antibody specifically binding to C5 according to embodiment 1, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region

[0028] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 23, 17 or 22; or

[0029] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 23, 17, or 22.

[0030] 4. A VHH antibody that specifically binds to C5, comprising

[0031] three complementarity determining regions (CDRs) contained in the VHH represented by any one of SEQ ID NO: 20, 18, 19 or 21,

[0032] Preferably, the CDR1 sequence is according to the AbM definition, CDR2 is according to the Kabat definition, and CDR3 is according to the AbM definition.

[0033] 5. The VHH antibody specifically binding to C5 according to embodiment 4, comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0034] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6;

[0035] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 30; or

[0036] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5, 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 30 or 6.

[0037] 6. The VHH antibody specifically binding to C5 according to embodiment 4, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region

[0038] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 20, 18, 19 or 21; or

[0039] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 20, 18, 19, or 21.

[0040] 7. A heavy chain antibody that specifically binds to C5, comprising the VHH antibody of any one of embodiments 1-6.

[0041] 8. The heavy chain antibody that specifically binds to C5 according to embodiment 7, which comprises or consists of the VHH antibody that specifically binds to C5 according to any one of embodiments 1 to 6 linked to an antibody constant region or Fc region.

[0042] 9. A VHH antibody that specifically binds to HTRA1, comprising

[0043] The three complementarity determining regions (CDRs) contained in the VHH shown in SEQ ID NO: 25 or 24,

[0044] Preferably, the CDR1 sequence is according to the AbM definition, CDR2 is according to the Kabat definition, and CDR3 is according to the AbM definition.

[0045] 10. The VHH antibody specifically binding to HTRA1 according to embodiment 9, comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0046] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 14, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 15, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 16; or

[0047] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13; or

[0048] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11 or 14, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12 or 15, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13 or 16.

[0049] 11. The VHH antibody specifically binding to HTRA1 according to embodiment 9, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region

[0050] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 25 or 24; or

[0051] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 25 or 24.

[0052] 12. A heavy chain antibody that specifically binds to HTRA1, comprising the VHH antibody that specifically binds to HTRA1 according to any one of embodiments 9-11.

[0053] 13. The heavy chain antibody that specifically binds to HTRA1 according to embodiment 12, comprising or consisting of the VHH antibody that specifically binds to HTRA1 according to any one of embodiments 9 to 11 linked to an antibody constant region or Fc region.

[0054] 14. The heavy chain antibody that specifically binds to C5 according to embodiment 8 or the heavy chain antibody that specifically binds to HTRA1 according to embodiment 13, wherein the antibody constant region or Fc region is derived from human IgG1, human IgG2, human IgG3 or human IgG4.

[0055] 15. The VHH antibody that specifically binds to C5 according to any one of embodiments 1-6, or the heavy chain antibody that specifically binds to C5 according to embodiment 7 or 8, or the VHH antibody that specifically binds to HTRA1 according to any one of embodiments 9-11, or the heavy chain antibody that specifically binds to HTRA1 according to embodiment 12 or 13, or the heavy chain antibody that specifically binds to C5 or the heavy chain antibody that specifically binds to HTRA1 according to embodiment 14, wherein the antibody is a chimeric antibody or a humanized antibody.

[0056] 16. An antibody that specifically binds to C5, comprising two or three or more antigen binding regions that specifically bind to C5, wherein the antigen binding regions bind to different epitopes and respectively comprise the VHH antibody that specifically binds to C5 of any one of claims 1-6 and 15, or the heavy chain antibody that specifically binds to C5 of any one of embodiments 7-8 and 14-15.

[0057] 17. The antibody that specifically binds to C5 according to embodiment 16, comprising one or two VHH antibodies that specifically bind to C5 or heavy chain antibodies comprising said VHH antibodies according to any one of embodiments 1-3, and one or two VHH antibodies that specifically bind to C5 or heavy chain antibodies comprising said VHH antibodies according to any one of embodiments 4-6; preferably, it comprises one or two VHH antibodies that specifically bind to C5 according to any one of embodiments 4-6, and one VHH antibody that specifically binds to C5 according to any one of embodiments 1-3, preferably, the VHH antibodies or heavy chain antibodies are connected in series, preferably via a linker;

[0058] Preferably, the antibody comprises or consists of a chain comprising, from N-terminus to C-terminus:

[0059] (1) the VHH antibody that specifically binds to C5 according to any one of embodiments 4 to 6 or the VHH antibody that specifically binds to C5 according to any one of embodiments 1 to 3,

[0060] (2) the VHH antibody that specifically binds to C5 according to any one of embodiments 4-6 - the VHH antibody that specifically binds to C5 according to any one of embodiments 4-6 - the VHH antibody that specifically binds to C5 according to any one of embodiments 1-3, or

[0061] (3) the VHH antibody that specifically binds to C5 according to any one of embodiments 4-6 - the VHH antibody that specifically binds to C5 according to any one of embodiments 1-3 - the VHH antibody that specifically binds to C5 according to any one of embodiments 4-6;

[0062] Preferably, the VHHs are connected via a linker.

[0063] 18. A multispecific antibody comprising one or more antigen-binding regions that specifically bind to C5 and one or more other antigen-binding regions, wherein the antigen-binding region that specifically binds to C5 comprises the VHH antibody that specifically binds to C5 of any one of embodiments 1-6 and 15, or the heavy chain antibody that specifically binds to C5 of any one of embodiments 7-8 and 14-15. Preferably, the multispecific antibody is a bispecific antibody.

[0064] 19. The multispecific antibody of embodiment 18, further comprising one or more antigen-binding regions that specifically bind to HTRA1, for example, the antigen-binding region that specifically binds to HTRA1 comprises the VHH antibody that specifically binds to HTRA1 of any one of embodiments 9-11 and 15, or the heavy chain antibody that specifically binds to HTRA1 of any one of embodiments 12-15.

[0065] 20. A multispecific antibody comprising an antigen binding region that specifically binds to HTRA1 and one or more other antigen binding regions, wherein the antigen binding region that specifically binds to HTRA1 comprises the VHH antibody that specifically binds to HTRA1 of any one of embodiments 9-11 and 15, or the heavy chain antibody that specifically binds to HTRA1 of any one of embodiments 12-15. Preferably, the multispecific antibody is a bispecific antibody.

[0066] 21. The multispecific antibody of embodiment 20, further comprising one or more antigen binding regions that specifically bind to C5, wherein the antigen binding region that specifically binds to C5 comprises the VHH antibody that specifically binds to C5 of any one of embodiments 1-6 and 15, or the heavy chain antibody that specifically binds to C5 of any one of embodiments 7-8 and 14-15.

[0067] 22. A bispecific antibody comprising one or more antigen-binding regions that specifically bind to C5 and one or more antigen-binding regions that specifically bind to HTRA1, wherein the antigen-binding regions that specifically bind to C5 bind to different epitopes on C5.

[0068] 23. The bispecific antibody of embodiment 22, comprising two antigen-binding regions that specifically bind to C5 and one antigen-binding region that specifically binds to HTRA1, wherein the two antigen-binding regions that specifically bind to C5 bind to different epitopes on C5.

[0069] 24. The bispecific antibody of embodiment 23, wherein the two antigen-binding regions that specifically bind to C5 are VHHs that specifically bind to different epitopes of C5, and the antigen-binding region that specifically binds to HTRA1 is a VHH that specifically binds to HTRA1, wherein the three VHHs are connected in series, optionally connected by a linker.

[0070] 25. The bispecific antibody of embodiment 24, wherein the linker comprises (G) n , where n=5-10, for example, 5, 6, 7, 8, 9, 10.

[0071] 26. The bispecific antibody of embodiment 22 or 23, wherein the VHHs are connected via N-terminus to N-terminus, C-terminus to C-terminus, or N-terminus to C-terminus, optionally via a linker.

[0072] 27. The bispecific antibody of any one of embodiments 24-26, wherein one antigen binding region that specifically binds to C5 comprises or consists of the VHH of any one of embodiments 1-3, and the other antigen binding region that specifically binds to C5 comprises or consists of the VHH of any one of embodiments 4-6, or the antigen binding region that specifically binds to HTRA1 comprises or consists of the VHH of any one of embodiments 9-11.

[0073] 28. The bispecific antibody of any one of embodiments 24-26, wherein one antigen binding region that specifically binds to C5 comprises or consists of the VHH of any one of embodiments 1-3, the other antigen binding region that specifically binds to C5 comprises or consists of the VHH of any one of embodiments 4-6, and the antigen binding region that specifically binds to HTRA1 comprises or consists of the VHH of any one of embodiments 9-11.

[0074] 29. The bispecific antibody of any one of embodiments 24-28, comprising the following chains, each comprising, from N-terminus to C-terminus:

[0075] VHH1 that specifically binds to C5 - VHH2 that specifically binds to C5 - VHH3 that specifically binds to HTRA1,

[0076] The VHHs are connected by a linker;

[0077] wherein VHH1 comprises or consists of the VHH of any one of embodiments 4-6, VHH1 comprises or consists of the VHH of any one of embodiments 1-3, and VHH3 comprises or consists of the VHH of any one of embodiments 9-11.

[0078] 30. The bispecific antibody of embodiment 29, comprising or consisting of the following chain, wherein the chain comprises the amino acid sequence shown in SEQ ID NO:27, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of the amino acid sequence.

[0079] 31. A nucleic acid molecule encoding the VHH antibody that specifically binds to C5 of any one of embodiments 1-6 and 15, or the heavy chain antibody that specifically binds to C5 of embodiment 7, 8, or 15, or the VHH antibody that specifically binds to HTRA1 of any one of embodiments 9-11, or the heavy chain antibody that specifically binds to HTRA1 of embodiment 12, 13, or 15, or the heavy chain antibody that specifically binds to C5 or the heavy chain antibody that specifically binds to HTRA1 of any one of embodiments 14 or 15; or the multispecific antibody of any one of embodiments 17-20, or the bispecific antibody of any one of embodiments 21-28.

[0080] 32. An expression vector comprising the nucleic acid molecule of embodiment 31, preferably, the expression vector is pCDNA3.1.

[0081] 33. A host cell comprising the nucleic acid molecule of embodiment 31 or the expression vector of embodiment 32. Preferably, the host cell is prokaryotic or eukaryotic, such as a CHO cell or a 293 cell, such as an Expi293 cell.

[0082] 34. A method for preparing the antibody of any one of embodiments 1-30, comprising culturing the host cell of embodiment 33 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.

[0083] 35. An immunoconjugate comprising the antibody of any one of embodiments 1-30, and one or more other active ingredients.

[0084] 36. A pharmaceutical composition, medicament or formulation comprising the antibody of any one of embodiments 1-30 or the immunoconjugate of embodiment 35, and optionally a pharmaceutically acceptable excipient.

[0085] 37. Drug combination products containing

[0086] The antibody of any one of embodiments 1-30 or the immunoconjugate of embodiment 35; and

[0087] Other therapeutic agents.

[0088] 38. A method for preventing or treating an ocular disease or condition in a subject, comprising administering to the subject an effective amount of the antibody of any one of embodiments 1-30; or the immunoconjugate of embodiment 35; or the pharmaceutical composition or formulation of embodiment 36; or the pharmaceutical combination product of embodiment 37.

[0089] 39. The method of embodiment 38, wherein the disease or condition is a disease or condition in which the subject has (e.g., elevated levels, such as nucleic acid or protein levels) complement C5 protein and / or HTRA1 protein (e.g., compared to healthy subjects) or a sample, such as a body fluid, of the subject has (e.g., elevated levels, such as nucleic acid or protein levels) complement C5 protein and / or HTRA1 protein (e.g., compared to body fluids of healthy subjects).

[0090] 40. A method for detecting the presence of complement C5 and / or HTRA1 in a biological sample, comprising contacting the biological sample with the antibody of any one of embodiments 1-30 under conditions that allow the antibody to bind to complement C5 and / or HTRA1, and detecting whether a complex is formed between the antibody and complement C5 and / or HTRA1, wherein the formation of the complex indicates the presence of complement C5. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] FIG1 shows the results of an experiment in which anti-C5 VHH antibodies blocked hemolysis induced by the classical complement pathway.

[0092] FIG2 shows the results of an experiment in which anti-C5 VHH antibodies blocked hemolysis induced by the alternative complement pathway.

[0093] FIG3 shows the results of anti-C5 VHH antibodies blocking the production of C5a in the classical complement pathway (C5a ELISA).

[0094] Figure 4 shows the results of the anti-C5 VHH antibody bin assay.

[0095] FIG5 shows the results of anti-HTRA1 VHH antibody blocking the H2-Opt assay.

[0096] FIG6 shows the results of an anti-HTRA1 VHH antibody blocking Elastase assay.

[0097] FIG7 shows the results of experiments in which humanized anti-C5 VHH antibodies block hemolysis induced by the classical complement pathway and the alternative complement pathway.

[0098] Figure 8 shows the results of an affinity matured anti-HTRA1 VHH antibody blocking H2-Opt assay.

[0099] Figure 9 shows the results of an Elastase blocking assay with affinity matured anti-HTRA1 VHH antibodies.

[0100] Figure 10 shows the results of the hemolysis experiment induced by the classical complement pathway (Figure 10A) and the hemolysis experiment induced by the alternative complement pathway (Figure 10B) by different anti-C5 VHH antibody combinations, as well as the results of the hemolysis experiment induced by the classical complement pathway (Figure 10C) and the hemolysis experiment induced by the alternative complement pathway (Figure 10D) by dual-epitope anti-C5 antibodies.

[0101] FIG11 shows an exemplary structural diagram of a bispecific antibody.

[0102] FIG12 shows the results of an experiment in which bispecific antibody molecules blocked hemolysis induced by the classical complement pathway.

[0103] FIG13 shows the results of an experiment in which bispecific antibody molecules blocked hemolysis induced by the alternative complement pathway.

[0104] FIG14 shows the results of the bispecific antibody molecule blocking the H2-Opt assay.

[0105] FIG15 shows the results of the Elastase blocking assay using bispecific antibody molecules.

[0106] FIG16 shows the results of an in vivo efficacy test of bispecific antibody molecules in blocking geographic atrophy induced by sodium iodate.

[0107] Detailed Description of the Invention

[0108] I. Definition

[0109] It should be understood that the present invention is not limited to the specific methods, protocols, examples and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which will only be limited by the appended claims.

[0110] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0111] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0112] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5%, 4%, 3%, 2% or 1% less than the specified numerical value and an upper limit that is 5%, 4%, 3%, 2% or 1% greater than the specified numerical value.

[0113] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0114] When "first" and "second" are mentioned herein, it is only to distinguish the two domains or two chains, but does not indicate the positions of the two domains in any way.

[0115] As used herein, the terms "comprising" or "including" are intended 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 indicated, the context of consisting of the stated elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0116] As used herein, the term "HTRA1" refers to the serine protease HtrA1 serine peptidase 1 (UniProtKB / Swiss-Prot: Q92743). The terms "anti-HTRA1 antibody," "antibody that specifically binds to HTRA1," or "antibody that specifically binds to HTRA1" refer to antibodies that bind to HTRA1 with sufficient affinity. In one embodiment, the anti-HTRA1 antibodies of the present invention specifically bind to HTRA1 protein or have high binding affinity for proteins expressing HTRA1.

[0117] As used herein, the terms "C5," "C5 protein," or "complement C5" are used interchangeably and refer to the complement component C5 protein (UniProtKB / Swiss-Prot: P01031) of the complement system. The terms "anti-C5 antibody," "antibody that specifically binds to C5," or "antibody that specifically binds to C5" refer to antibodies that bind to C5 with sufficient affinity. In one embodiment, the anti-C5 antibodies of the present invention specifically bind to the C5 protein or have high binding affinity for proteins expressing C5.

[0118] In some aspects, the anti-C5 antibodies described herein also encompass anti-C5 antibodies that bind to different epitopes. In some aspects, the anti-C5 antibodies described herein also encompass bispecific or multispecific antibodies that bind to C5 and other antigens. In some aspects, the anti-HTRA1 antibodies described herein also encompass bispecific or multispecific antibodies that bind to HTRA1 and other antigens. In some aspects, the anti-C5 antibodies or anti-HTRA1 antibodies described herein also encompass bispecific antibodies that specifically bind to both C5 and HTRA1.

[0119] General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0120] The bispecific antibodies of the present invention may include a linker. As used herein, the term "linker" refers to any molecule that enables the direct connection of the different parts of a multispecific antibody. Examples of covalently linked linkers between different parts of a multispecific antibody include peptide linkers and non-protein polymers, including but not limited to copolymers of polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes or polyethylene glycol, polypropylene glycol. In some embodiments, the term "peptide linker" according to the present invention refers to an amino acid sequence, wherein the sequence links the amino acid sequences of the various parts of a multispecific antibody together. Preferably, the peptide linker has a length that is sufficient to connect two entities in a manner that allows them to maintain their conformations relative to each other so as not to hinder desired activity. The peptide linker may primarily include or may not primarily include the following amino acid residues: Gly, Ser, Ala or Thr. Useful linkers include glycine-serine polymers, including, for example, (G)n (SEQ ID NO: 38), (GS)n (SEQ ID NO: 39), (GSGGS)n (SEQ ID NO: 40), (GGGGS)n (SEQ ID NO: 41), (GGGS)n (SEQ ID NO: 42), and (GGGGS)nG (SEQ ID NO: 43), where n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Exemplary linkers include, for example, the sequence set forth in SEQ ID NO: 26.

[0121] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) in the Fc region may or may not exist. Two Fc regions can achieve dimerization to form a dimeric Fc, and two different Fc heterodimerizations form heterodimeric Fc. In this article, the terms "Fc region", "Fc portion" and "dimeric Fc (e.g., heterodimeric Fc)" do not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin and heavy chain constant region CH1 and light chain constant region CL, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases. In one embodiment, a human IgG heavy chain Fc region extends from Asp221, or from Cys226, or from Asp231, to the carboxyl-terminus of the heavy chain.

[0122] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0123] The term "binding molecule" refers to any molecule capable of specifically binding to a target, such as an antibody or antigen-binding fragment or fusion protein thereof.

[0124] The terms "VHH" or "VHH antibody" are used interchangeably herein and generally refer to an antibody that comprises or consists of only one heavy chain variable region and has antigen binding activity. VHHs generally comprise three CDRs in four highly conserved framework regions and generally have the following structure: FR1-CDR-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; CDR1 to CDR3 refer to complementarity determining regions 1-3. The CDR sequences in the VHH variable region can be determined according to any of the CDR definition schemes described in the "Definitions" section, and preferably the boundaries of the three CDRs in the variable region sequence can be defined by IMGT. VHHs generally include only heavy chain variable domains derived from heavy chain antibodies lacking light chains, also known as nanobodies. The VHHs used in the present invention are preferably from camelids, such as alpacas, or are humanized or sequence-optimized forms thereof (e.g., affinity mature forms to increase binding affinity). In some embodiments, a VHH of the invention is a monovalent, monospecific polypeptide molecule that consists of, or consists essentially of, a single heavy chain variable region (eg, the heavy chain variable region of a heavy chain antibody).

[0125] The single domain antibodies or VHHs of the present invention may also be contained in a larger polypeptide / protein. Examples of polypeptides / proteins containing the VHHs of the present invention include, but are not limited to, heavy chain antibodies (HcAbs) or multispecific antibodies or fusion proteins.

[0126] The "heavy chain antibody" described in the present invention refers to an antibody without a light chain, for example, its N-segment to C-segment may include VH-Fc or VH-CH2-CH3 or VH-hinge region-CH2-CH3, or may include VH-CH1-CH2-CH3. The heavy chain antibody of the present invention may also encompass homodimers, such as heavy chain dimer antibodies without light chains. The heavy chain antibody may include VH from a standard antibody or VH from a single domain antibody. For example, the VH in a heavy chain antibody may be VHH. In some embodiments, the heavy chain antibody of the present invention may be a heavy chain antibody having a framework region and / or a heavy chain constant region derived from a camelid (llama, camel, especially alpaca), a humanized form thereof or a sequence-optimized form thereof (affinity matured form), or a fragment thereof (e.g., a fragment comprising at least a portion of the constant region). The heavy chain antibody of the present invention also encompasses an antibody formed by fusing a heavy chain variable region or VHH with an Fc region (e.g., a human IgG Fc region, such as a human IgG1 or IgG4 Fc region).

[0127] When "VHH" is mentioned in the context of a heavy chain antibody or a multispecific antibody or a fusion protein, it should be understood that it is part of a multispecific antibody and not as a separate molecule.

[0128] The term "target" refers to the object to which a binding molecule is directed. A target can be an antigen, a ligand, or a receptor. The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. Those skilled in the art will appreciate that any macromolecule, including essentially all proteins or peptides, can serve as an antigen. In addition, 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 an antibody molecule. Where the binding molecule of the present invention relates to a target binding region derived from an antibody, "target" and "antigen" are used interchangeably.

[0129] As used herein, the term "target binding region" refers to the portion of a binding molecule, such as a multispecific binding molecule or a bispecific binding molecule, that binds a specific target or antigen. The target binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target binding region may or may not have a tertiary structure independent of the rest of the binding molecule and may or may not bind to its target as a separate entity. The target binding region can also be a receptor or a ligand, or a domain of a receptor that is capable of binding a ligand. In the case of multispecific antibodies or bispecific antibodies, the "target binding region" is also referred to as an "antigen binding region."

[0130] As used herein, the term "antigen binding region" refers to any portion of an antibody or antigen binding fragment thereof, such as a multispecific antibody or bispecific antibody, that binds to a specific target or antigen. The antigen binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such an antigen binding region may or may not have a tertiary structure independent of the remainder of the multispecific antibody or bispecific antibody and may or may not bind to its antigen / epitope as a separate entity. Where the binding molecule of the present invention relates to a target binding region derived from an antibody, "target binding region" and "antigen binding region" can be used interchangeably. In one embodiment, the antigen binding region of a multispecific antibody molecule for use in the present invention may comprise only a VH, such as from a VHH, such as a VHH.

[0131] The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, e.g., a bispecific binding molecule, i.e., the molecule comprises at least a first target binding region and a second target binding region, wherein the first target binding region binds to one target or antigen and the second target binding region binds to another antigen or target. The multispecific binding molecules according to the present invention also encompass multispecific molecules comprising multiple target binding regions / binding sites, such as trispecific binding molecules. In some embodiments, the multispecific binding molecules of the present invention are multispecific antibodies. In some embodiments, the bispecific binding molecules of the present invention are bispecific antibodies. In some embodiments, the multispecific binding molecules of the present invention are fusion proteins, which may, for example, comprise a target binding region from an antibody and a target binding region from a receptor or ligand.

[0132] As used herein, the term "monospecific" refers to a polypeptide / protein molecule having one or more target binding regions, each of which binds to the same site or structure of the same target or the same epitope of the same antigen. In some aspects, the monospecificity refers to different target binding regions binding to the same antigen.

[0133] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding regions, each of which binds to a different antigen. A multispecific antibody is an antibody that has binding specificity for at least two different antigens. In one embodiment, provided herein is a bispecific antibody that has binding specificity for a first antigen and a second antigen.

[0134] When referring to the "first antigen binding region" in a multispecific antibody or a bispecific antibody, it refers to the binding region that binds to the first antigen, and is not intended to limit the number of such antigen binding regions contained in the antibody. For example, a multispecific antibody or a bispecific antibody may contain one or more first antigen binding regions. For example, a bispecific antibody contains a first antigen binding region and a second antigen binding region, but may contain one or more first antigen binding regions and one or more second antigen binding regions. In some embodiments of the present invention, the binding regions that bind to the same antigen may be the same or different. For example, the binding regions that bind to the same antigen may bind to different epitopes and thus be different. In one embodiment, the bispecific antibody molecule of the present invention comprises two first antigen binding regions that specifically bind to different epitopes of C5 and one second antigen binding region that specifically binds to HTRA1.

[0135] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions.

[0136] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and North CDR definitions based on affinity propagation clustering using a large number of crystal structures. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention). In one embodiment, the HCDRs in the VHH antibodies of the present invention can be defined according to any one of the above rules or a combination of two or more rules.

[0137] In one embodiment, the HCDRs in the VHH antibodies of the invention are defined according to the following rules:

[0138] HCDR1 was defined according to AbM rules;

[0139] HCDR2 is defined according to the Kabat convention; and

[0140] HCDR3 was defined according to the AbM rules.

[0141] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a camelid VHH antibody) while being less immunogenic 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 portions of the antibody with their human counterparts (i.e., replacing portions of the variable region that do not participate in binding with corresponding portions of a human antibody).

[0142] As used herein, the terms "anti," "binding," or "specific binding" mean that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0143] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ) indicates that the dissociation constant is the dissociation rate constant and the association rate constant (K dis and K on ). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0144] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom.

[0145] 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, the individual is a human.

[0146] The term "treat," ..."

[0147] The term "preventing" includes the inhibition of the onset or development of a disease or condition or symptoms of a particular disease or condition.

[0148] As used herein, the term "therapeutic agent" encompasses any active substance or agent that is effective in preventing or treating an ocular disease or disorder, such as in the treatment of an ocular disease.

[0149] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (such as tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0150] The term "drug combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit and a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody of the present invention, and (ii) other therapeutic agent) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in a separate entity, wherein such administration provides two or more active agents with a preventive or therapeutically effective level in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0151] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0152] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0153] "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 a solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from fresh, frozen and / or preserved; a body fluid, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid.

[0154] II. Anti-C5 Antibodies

[0155] In one aspect, the present invention provides a C5 antibody having a higher binding affinity to C5. In some embodiments, the C5 antibody of the present invention is suitable for constructing the antigen binding region in the multispecific antibody molecule of the present invention.

[0156] In some embodiments, the anti-C5 antibodies of the present invention bind to C5 (e.g., human C5 or cynomolgus monkey C5) with higher affinity. In some embodiments, the anti-C5 antibodies of the present invention can bind to human C5 and / or cynomolgus monkey C5 with high affinity, e.g., its K D In some embodiments, the binding K of an anti-C5 antibody of the invention to human C5 and / or cynomolgus C5 is less than or equal to about 2 nM, such as less than or equal to about 5, 4, 3, 2, 1.5, 1, 0.5, 0.4, 0.3, 0.25, 0.2, or 0.15 nM. D In some embodiments, the binding K of the anti-C5 antibodies of the invention to human C5 and / or cynomolgus monkey C5 is greater than about 0.05 nM or about 0.1 nM. D The value is between any two numerical ranges mentioned above. In some embodiments, the binding affinity of the anti-C5 antibody of the present invention is determined by biomembrane interferometry.

[0157] In some embodiments, the anti-C5 antibodies of the present invention are capable of blocking the classical complement pathway, for example, blocking its induced hemolysis.

[0158] In some embodiments, the anti-C5 antibodies of the invention are capable of blocking the alternative complement pathway, for example, blocking its induced hemolysis.

[0159] In some embodiments, the anti-C5 antibodies of the present invention can block the interaction between C5 and C5 convertase, thereby blocking the formation of C5a, such as blocking the generation of C5a in the classical complement pathway.

[0160] In some embodiments, the anti-C5 antibodies of the present invention are capable of completely inhibiting the biological activity of C5.

[0161] In some embodiments, the anti-C5 antibody of the present invention is a single domain antibody, particularly a VHH antibody. In some embodiments, the anti-C5 antibody of the present invention is a VHH-His antibody with a His tag (e.g., 6-His) at the N-terminus or C-terminus, such as a VHH-6His antibody.

[0162] In some embodiments, the anti-C5 single-domain antibody of the present invention is a VHH antibody comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region generally has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; VHH CDR1 to VHH CDR3 refer to complementarity determining regions 1 to 3. The CDR sequences in the VHH variable region can be determined according to any CDR definition scheme described in the "Definitions" section. Preferably, the boundaries of the three CDRs in the VHH sequence can be defined as follows: CDR1 is defined according to the AbM definition, CDR2 is defined according to the Kabat definition, and CDR3 is defined by the AbM definition.

[0163] In some embodiments, the anti-C5 VHH antibody of the present invention comprises

[0164] (i) three complementarity determining regions (CDRs) contained in the VH represented by any one of SEQ ID NOs: 17-23, or

[0165] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions relative to the sequence of (i);

[0166] Preferably, the CDR1 sequence is according to the AbM definition, CDR2 is according to the Kabat definition, and CDR3 is according to the AbM definition.

[0167] In some embodiments, the anti-C5 VHH antibody of the present invention comprises or consists of a heavy chain variable region comprising

[0168] (i) three complementarity determining regions (CDRs) contained in the VH set forth in any one of SEQ ID NOs: 17-23, or

[0169] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions relative to the sequence of (i);

[0170] Preferably, the CDR1 sequence is according to the AbM definition, the CDR2 sequence is according to the Kabat definition, and the CDR3 sequence is according to the AbM definition.

[0171] In some embodiments, the anti-C5 VHH antibodies of the present invention comprise complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3. In some embodiments, the anti-C5 VHH antibodies of the present invention comprise or consist of a heavy chain variable region comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3.

[0172] In some embodiments, the VHH CDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 1 or 4, or the VHH CDR1 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 1 or 4.

[0173] In some embodiments, the VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, 5, 7, 8 or 9, or the VHH CDR2 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: 2, 5, 7, 8 or 9.

[0174] In some embodiments, the VHH CDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 3, 6, 10, or 30, or the VHH CDR3 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 3, 6, 10, or 30.

[0175] In one embodiment, the anti-C5 VHH antibody of the present invention comprises the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0176] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5, 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 30; or

[0177] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 30; or

[0178] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6; or

[0179] (iv) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 9, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 10; or

[0180] (v) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; or

[0181] (vi) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10.

[0182] In one embodiment, an anti-C5 VHH antibody of the invention comprises or consists of a heavy chain variable region, wherein said heavy chain variable region comprises complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0183] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5, 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 30; or

[0184] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 30; or

[0185] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6; or

[0186] (iv) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 9, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 10; or

[0187] (v) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; or

[0188] (vi) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10.

[0189] In some embodiments, the anti-C5 VHH antibody of the present invention comprises or consists of a heavy chain variable region, wherein the heavy chain variable region

[0190] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 17-23; or

[0191] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 17-23; or

[0192] (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 shown in any one of SEQ ID NOs: 17 to 23, preferably, the amino acid changes do not occur in the CDR regions.

[0193] In some embodiments, the anti-C5 VHH antibody of the present invention comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 17-23.

[0194] In one embodiment, the anti-C5 VHH antibody of the present invention is a humanized antibody. Humanization can be achieved by replacing one or more amino acid residues, especially framework region sequences, in a non-human natural VHH sequence (e.g., a VHH sequence from camelids or alpacas immunization) with residues at corresponding positions in the heavy chain VH of a conventional human antibody. Methods for humanizing VHHs are well known in the art, such as the method described in Example 3. Typically, humanizing substitutions are performed in a manner that maintains the favorable binding properties of single-domain antibodies. Tests for determining the biological properties of humanized single-domain antibodies, such as binding affinity, are well known in the art to determine and select suitable humanized residue mutations or combinations of mutations.

[0195] In some embodiments, the humanized single domain antibody of the present invention can be obtained by a method comprising the following steps:

[0196] Determine the CDR loop structure of the parent single domain antibody (e.g., a camelid VHH antibody from a phage display library screen):

[0197] ① Determine the CDR loop structure;

[0198] ② Find the closest homologous sequence for each V / J region of VHH in the human germline sequence database;

[0199] ③ Construct the CDR region of VHH onto the human framework region;

[0200] ④ Using sequence and structural features, determine the amino acid positions in the framework region that maintain CDR function;

[0201] ⑤ Perform back mutation at the sequence position determined to be important;

[0202] ⑥Optimize amino acids at risk sites.

[0203] In one aspect of the invention, the invention also provides an anti-C5 antibody comprising two or more VHHs of the invention, such as tandem VHH antibodies, wherein different VHH antibodies bind to different epitopes on C5, such as tandemly connected via or without a linker.

[0204] In one embodiment, the different VHHs are linked via N-terminus to N-terminus, C-terminus to C-terminus, or N-terminus to C-terminus.

[0205] In one embodiment, an anti-C5 antibody of the invention comprises two or three VHHs binding to different epitopes connected in tandem via a linker.

[0206] In some embodiments, an anti-C5 antibody of the invention comprises or consists of a chain comprising, from N-terminus to C-terminus:

[0207] (1) VHH1 described herein that specifically binds to C5 - VHH2 described herein that specifically binds to C5,

[0208] (2) VHH1 described herein that specifically binds to C5 - VHH1 described herein that specifically binds to C5 - VHH2 described herein that specifically binds to C5, or

[0209] (3) VHH1 specifically binding to C5 described herein - VHH2 specifically binding to C5 described herein - VHH1 specifically binding to C5 described herein,

[0210] Optionally, the VHHs are connected via a linker.

[0211] In some embodiments, the VHH1 that specifically binds to C5 and the VHH2 that specifically binds to C5 are anti-C5 VHHs described herein that bind to different epitopes.

[0212] (i) comprises the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5, 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 30;

[0213] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 30; or

[0214] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6;

[0215] or

[0216] (ii) comprises a heavy chain variable region comprising, or consisting of, an amino acid sequence as set forth in any one of SEQ ID NOs: 18-21, or an amino acid sequence having at least 90% identity thereto;

[0217] and specifically binds to C5 VHH2

[0218] (i) comprises the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0219] (a) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 9, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 10; or

[0220] (b) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; or

[0221] (c) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10.

[0222] (ii) comprises a heavy chain variable region comprising, or consisting of, the amino acid sequence shown in any one of SEQ ID NOs: 17, 22, or 23, or an amino acid sequence that is at least 90% identical thereto;

[0223] Or vice versa.

[0224] In some embodiments, the VHH1 that specifically binds to C5 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the VHH2 that specifically binds to C5 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23.

[0225] In some embodiments, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO:26.

[0226] In some embodiments, the anti-C5 antibody of the present invention comprises a chain comprising, or consisting of, the amino acid sequence shown in SEQ ID NO: 34, 35 or 36, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0227] In some embodiments, the anti-C5 antibody of the present invention comprises the amino acid sequence shown in SEQ ID NO:34, 35 or 36, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of the amino acid sequence.

[0228] III. Anti-HTRA1 Antibodies

[0229] In one aspect, the present invention provides an HTRA1 antibody having a higher binding affinity to HTRA1. In some embodiments, the HTRA1 antibody of the present invention is suitable for constructing an antigen-binding region in a multispecific antibody molecule.

[0230] In some embodiments, the anti-HTRA1 antibodies of the present invention bind to HTRA1 (e.g., human HTRA1 or cynomolgus monkey HTRA1) with higher affinity. In some embodiments, the anti-HTRA1 antibodies of the present invention can bind to human HTRA1 and / or cynomolgus monkey HTRA1 with high affinity, e.g., its K D The binding K of the anti-HTRA1 antibodies of the present invention to human HTRA1 and / or cynomolgus monkey HTRA1 is less than or equal to about 3 nM, such as less than or equal to about 5, 4, 3, 2, 1 or 0.9 nM. D In some embodiments, the binding K of the anti-HTRA1 antibodies of the present invention to human HTRA1 and / or cynomolgus monkey HTRA1 is greater than about 0.1 nM or about 0.5 nM. D The value is between any two numerical ranges mentioned above. In some embodiments, the binding affinity of the anti-C5 antibody of the present invention is determined by biomembrane interferometry.

[0231] In some embodiments, the anti-HTRA1 antibodies of the present invention can inhibit the activity of HTRA1, such as inhibiting its serine protease or elastase activity. In some embodiments, the anti-HTRA1 antibodies of the present invention can block the activity of HTRA1 in cleaving H2-Opt. In some embodiments, the anti-HTRA1 antibodies of the present invention can block the activity of HTRA1 in cleaving elastin.

[0232] In some embodiments, the anti-HTRA1 antibodies of the present invention are single domain antibodies, particularly VHH antibodies.

[0233] In some embodiments, the anti-HTRA1 single-domain antibody of the present invention is a VHH antibody comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region generally has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; VHH CDR1 to VHH CDR3 refer to complementarity determining regions 1 to 3. The CDR sequences in the VHH variable region can be determined according to any CDR definition scheme described in the "Definitions" section, and preferably, the boundaries of the three CDRs in the VHH sequence can be defined by IMGT.

[0234] In some embodiments, the anti-HTRA1 VHH antibody of the present invention comprises

[0235] (i) three complementarity determining regions (CDRs) contained in the VH represented by SEQ ID NO: 24 or 25, or

[0236] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions relative to the sequence of (i);

[0237] Preferably, the CDR1 sequence is according to the AbM definition, CDR2 is according to the Kabat definition, and CDR3 is according to the AbM definition.

[0238] In some embodiments, the anti-HTRA1 VHH antibody of the present invention comprises or consists of a heavy chain variable region comprising

[0239] (i) three complementarity determining regions (CDRs) contained in the VH represented by SEQ ID NO: 24 or 25, or

[0240] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions relative to the sequence of (i);

[0241] Preferably, the CDR1 sequence is according to the AbM definition, CDR2 is according to the Kabat definition, and CDR3 is according to the AbM definition.

[0242] In some embodiments, the anti-HTRA1 VHH antibodies of the present invention comprise complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3. In some embodiments, the anti-HTRA1 VHH antibodies of the present invention comprise or consist of a heavy chain variable region comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3.

[0243] In some embodiments, the VHH CDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 11 or 14, or the VHH CDR1 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 11 or 14.

[0244] In some embodiments, the VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12 or 15, or the VHH CDR2 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: 12 or 15.

[0245] In some embodiments, the VHH CDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 13 or 16, or the VHH CDR3 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 13 or 16.

[0246] In one embodiment, the anti-HTRA1 VHH antibody of the present invention comprises the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0247] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13; or

[0248] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 14, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 15, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 16; or

[0249] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11 or 14, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12 or 15, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13 or 16.

[0250] In one embodiment, the anti-HTRA1 VHH antibody of the invention comprises or consists of a heavy chain variable region, wherein said heavy chain variable region comprises complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0251] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13; or

[0252] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 14, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 15, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 16;

[0253] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11 or 14, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12 or 15, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13 or 16.

[0254] In some embodiments, the anti-HTRA1 VHH antibody of the present invention comprises or consists of a heavy chain variable region, wherein the heavy chain variable region

[0255] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 24 or 25; or

[0256] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 24 or 25; or

[0257] (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 selected from SEQ ID NO: 24 or 25, preferably, the amino acid changes do not occur in the CDR region.

[0258] In some embodiments, the anti-HTRA1 VHH antibody of the present invention comprises or consists of an amino acid sequence selected from SEQ ID NO: 24 or 25.

[0259] In one embodiment, the anti-HTRA1 VHH antibody of the present invention is a humanized antibody. Humanization can be achieved by replacing one or more amino acid residues, especially framework regions, in a non-human natural VHH sequence (e.g., a VHH sequence from camelids or alpacas immunization) with residues at corresponding positions in the heavy chain VH of a conventional human antibody. Methods for humanizing VHHs are well known in the art, such as the method described in Example 3. Generally, humanizing substitutions are performed in a manner that maintains the favorable binding properties of single-domain antibodies. Assays for determining the biological properties of humanized single-domain antibodies, such as binding affinity, are well known in the art to determine and select suitable humanized residue mutations or combinations of mutations.

[0260] In some embodiments, the humanized single domain antibody of the present invention can be obtained by a method comprising the following steps:

[0261] Determine the CDR loop structure of the parent single domain antibody (e.g., a camelid VHH antibody from a phage display library screen):

[0262] ① Determine the CDR loop structure;

[0263] ② Find the closest homologous sequence for each V / J region of VHH in the human germline sequence database;

[0264] ③ Construct the CDR region of VHH onto the human framework region;

[0265] ④ Using sequence and structural features, determine the amino acid positions in the framework region that maintain CDR function;

[0266] ⑤ Perform back mutation at the sequence position determined to be important;

[0267] ⑥Optimize amino acids at risk sites.

[0268] IV. Anti-C5 Heavy Chain Antibody or Anti-HTRA1 Heavy Chain Antibody

[0269] In another aspect of the present invention, the present invention also provides a heavy chain antibody comprising the heavy chain variable region of the VHH antibody of the present invention.

[0270] In some embodiments, a single domain antibody or VHH of the present invention (e.g., a camel-derived VHH or a humanized form thereof) can be linked to a constant region of a human antibody or a portion thereof, such as an Fc region, to produce a heavy chain antibody comprising a VHH-constant region or VHH-CH1-Fc or VHH-Fc. In one embodiment, the heavy chain antibody comprises a VHH antibody of the present invention and an Fc region at its C-terminus. In some embodiments, VHH and Fc are linked by a hinge region or a portion thereof, such as a hinge region from IgG (e.g., a hinge region of IgG1, 2, 3, or 4) or a portion thereof.

[0271] In one aspect of the invention, the anti-C5 antibody of the invention is an anti-C5 heavy chain antibody. In some embodiments, the anti-C5 heavy chain antibody of the invention comprises an anti-C5 VHH as defined herein, or a heavy chain variable region thereof, and a heavy chain constant region or an Fc region of a heavy chain constant region.

[0272] In one aspect of the invention, the anti-HTRA1 antibody of the invention is an anti-HTRA1 heavy chain antibody. In some embodiments, the anti-HTRA1 heavy chain antibody of the invention comprises an anti-HTRA1 VHH as defined herein, or a heavy chain variable region thereof, and a heavy chain constant region or an Fc region of a heavy chain constant region.

[0273] In some embodiments, the heavy chain antibody comprises a constant region from a human or non-human primate (eg, cynomolgus monkey) antibody, such as a constant region from human IgG1, human IgG2, human IgG3, or human IgG4.

[0274] In some embodiments, the heavy chain antibody comprises an Fc portion from a human or non-human primate (e.g., cynomolgus monkey). In yet another embodiment, the heavy chain antibody comprises a human IgG Fc region, e.g., a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region, preferably a human IgG1 or human IgG4 Fc region.

[0275] In one embodiment, the heavy chain antibody according to the present invention can dimerize with another polypeptide chain (e.g., the same or different heavy chain antibody) comprising an Fc region through the Fc region. Therefore, in one embodiment, the present invention also provides a homologous or heterologous multimeric protein comprising a heavy chain antibody of the present invention. In a preferred embodiment, the protein preferably comprises a heavy chain antibody formed by pairing two identical heavy chain antibody chains.

[0276] In some embodiments, the anti-C5 antibodies of the present invention comprise a heavy chain comprising a heavy chain variable region and an Fc region. In some embodiments, the anti-C5 antibodies of the present invention comprise or consist of a heavy chain comprising or consisting of a heavy chain variable region and an Fc region of an anti-C5 VHH of the present invention.

[0277] In some embodiments, the anti-HTRA1 antibodies of the present invention comprise a heavy chain comprising a heavy chain variable region and an Fc region. In some embodiments, the anti-HTRA1 antibodies of the present invention comprise or consist of a heavy chain comprising or consisting of a heavy chain variable region and an Fc region of an anti-HTRA1 VHH of the present invention.

[0278] V. Multispecific Antibodies

[0279] In some embodiments, the present invention provides a multispecific antibody, such as a bispecific antibody, that specifically binds to C5, and optionally to other antigens such as HTRA1. In some embodiments, the multispecific antibody comprises an antigen-binding region that specifically binds to C5 (e.g., an anti-C5 VHH antibody or heavy chain antibody of the present invention), such as one or more antigen-binding regions that specifically bind to C5, and optionally to other antigen-binding regions, such as an antigen-binding region that specifically binds to HTRA1.

[0280] In some embodiments, the present invention provides a multispecific antibody, such as a bispecific antibody, that specifically binds to HTRA1, and optionally to other antigens such as C5. In some embodiments, the multispecific antibody comprises an antigen-binding region that specifically binds to HTRA1 (e.g., an anti-HTRA1 VHH antibody or heavy chain antibody of the present invention), such as one or more antigen-binding regions that specifically bind to HTRA1, and optionally to other antigen-binding regions, such as an antigen-binding region that specifically binds to C5.

[0281] In some embodiments, the present invention provides a multispecific antibody, such as a bispecific antibody, that specifically binds to C5 and HTRA1, and optionally other antigens.

[0282] Therefore, one aspect of the present invention relates to a bispecific antibody comprising at least two different antigen-binding regions that specifically bind to C5, and an antigen-binding region that specifically binds to HTRA1.

[0283] In some embodiments, the antigen binding region that specifically binds to C5 is from an anti-C5 antibody, e.g., an anti-C5 antibody described herein, e.g., an anti-C5 VHH described herein. In some embodiments, the two different antigen binding regions that specifically bind to C5 bind to different epitopes of C5. In some embodiments, the two different antigen binding regions that specifically bind to C5 are two different anti-C5 VHHs described herein.

[0284] In some embodiments, the antigen binding region that specifically binds HTRA1 is from an anti-HTRA1 antibody, eg, from an anti-HTRA1 antibody described herein, eg, an anti-HTRA1 VHH described herein.

[0285] In some embodiments, the bispecific antibodies of the present invention may comprise one or more antigen-binding regions that specifically bind to HTRA1. In one embodiment, the bispecific antibody comprises one antigen-binding region that specifically binds to HTRA1. In one embodiment, the bispecific antibody comprises two antigen-binding regions that specifically bind to C5 and bind to different C5 epitopes, and one antigen-binding region that specifically binds to HTRA1.

[0286] In some embodiments, the bispecific antibodies of the present invention may comprise two different anti-C5 VHHs and one anti-HTRA1 VHH, wherein the three VHHs are linked in series. In some embodiments, the VHHs of the bispecific antibodies of the present invention are linked by a linker or not. In some embodiments, the VHHs of the bispecific antibodies of the present invention are all linked by a linker.

[0287] In some embodiments, in the bispecific antibodies of the invention, the N-terminus of one VHH is linked to the C-terminus of another VHH, or the N-terminus of one VHH is linked to the N-terminus of another VHH, or the C-terminus of one VHH is linked to the C-terminus of another VHH.

[0288] In some embodiments, the bispecific antibody of the invention comprises or consists of a chain comprising, from N-terminus to C-terminus:

[0289] VHH1 that specifically binds to C5 - VHH2 that specifically binds to C5 - VHH that specifically binds to HTRA1,

[0290] The VHHs are connected with or without a linker.

[0291] In some embodiments, the VHH1 that specifically binds to C5 and the VHH2 that specifically binds to C5 are anti-C5 VHHs described herein that bind to different epitopes. In some embodiments, the VHH that specifically binds to HTRA1 is an anti-HTRA1 VHH described herein.

[0292] In some embodiments, the bispecific antibody of the invention comprises or consists of a chain comprising, from N-terminus to C-terminus:

[0293] VHH1 described herein that specifically binds to C5 - VHH2 described herein that specifically binds to C5 - VHH3 described herein that specifically binds to HTRA1,

[0294] The VHHs are connected by linkers.

[0295] In some embodiments, the VHH1 that specifically binds to C5 and the VHH2 that specifically binds to C5 are anti-C5 VHHs described herein that bind to different epitopes.

[0296] In some embodiments, a VHH1 that specifically binds to C5

[0297] (i) comprises the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0298] (a) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5, 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 30; or

[0299] (b) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 30; or

[0300] (c) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6;

[0301] (ii) comprises a heavy chain variable region comprising, or consisting of, an amino acid sequence as set forth in any one of SEQ ID NOs: 18-21, or an amino acid sequence having at least 90% identity thereto;

[0302] and specifically binds to C5 VHH2

[0303] (i) comprises the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0304] (a) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 9, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 10; or

[0305] (b) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; or

[0306] (c) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10.

[0307] (ii) comprises a heavy chain variable region comprising, or consisting of, an amino acid sequence as set forth in any one of SEQ ID NOs: 17, 22, or 23, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0308] Or vice versa.

[0309] In some embodiments, a VHH3 that specifically binds to HTRA1

[0310] (i) comprises the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0311] (a) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13; or

[0312] (b) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 14, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 15, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 16; or

[0313] (c) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11 or 14, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12 or 15, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13 or 16;

[0314] (ii) comprises a heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 24 or 25, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0315] In some embodiments, the VHH1 that specifically binds to C5 comprises or consists of the amino acid sequence shown in SEQ ID NO:20.

[0316] In some embodiments, the VHH2 that specifically binds to C5 comprises or consists of the amino acid sequence shown in SEQ ID NO:23.

[0317] In some embodiments, the VHH3 that specifically binds to HTRA1 comprises or consists of the amino acid sequence shown in SEQ ID NO:25.

[0318] In some specific embodiments, VHH1 comprises or consists of the amino acid sequence shown in SEQ ID NO:20, VHH2 comprises or consists of the amino acid sequence shown in SEQ ID NO:23, and VHH3 comprises or consists of the amino acid sequence shown in SEQ ID NO:25.

[0319] In some embodiments, the linker comprises (G) n , wherein n=5-10, such as 5, 6, 7, 8, 9, 10. In some embodiments, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO: 26.

[0320] In some embodiments, the bispecific antibody of the invention comprises a chain comprising, or consisting of, the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0321] In some embodiments, the bispecific antibody of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0322] VI. Polynucleotides, Vectors, and Hosts

[0323] The present invention provides nucleic acids encoding any of the above antibody molecules of the present invention (anti-C5 antibodies or anti-HTRA1 antibodies or bispecific antibodies). A vector comprising the nucleic acid is also provided. In one embodiment, the vector is an expression vector (e.g., a pCDNA vector, e.g., pCDNA3.1). A host cell comprising the nucleic acid or the 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, e.g., HEK 293 or 293F cells or 293FT cells or Expi293 cells). In another embodiment, the host cell is prokaryotic.

[0324] In one aspect, the present invention provides nucleic acids encoding any of the above anti-C5 antibodies or anti-HTRA1 antibodies or bispecific antibodies.

[0325] To facilitate production and purification, anti-C5 antibodies or anti-HTRA1 antibodies or bispecific antibodies can be fused to a secretory signal peptide at the N-terminus or C-terminus (eg, the C-terminus), and / or a tag peptide that facilitates purification, such as a hexahistidine tag or a biotin tag.

[0326] As will be apparent to those skilled in the art, because of codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0327] In some embodiments, the nucleic acid of the invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 17-25 or 27 or 34, or a nucleic acid encoding an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 17-25 or 27 or 34.

[0328] Nucleic acid sequences encoding molecules 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.

[0329] VII. Vectors and Host Cells

[0330] In one embodiment, one or more vectors comprising nucleic acid 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. Vectors include but are not limited to viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector is pCDNA, such as pCDNA3.1.

[0331] In one embodiment, a host cell comprising one or more polynucleotides of the present invention is provided. In some embodiments, a host cell comprising an expression vector of the present invention is provided. 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 SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells or 293FT cells or Expi293 cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63 and Sp2 / 0, etc. Mammalian host cell lines suitable for producing antibodies are known in the art. In a preferred embodiment, the host cell is a CHO or HEK293 cell or a 293FT cell or an Expi293 cell.

[0332] VIII. Production and Purification of the Molecules of the Invention

[0333] In another aspect, the present invention provides a method for producing an antibody molecule (anti-C5 antibody or anti-HTRA1 antibody or bispecific antibody) of the present invention, the method comprising: culturing a host cell containing a protein encoding the polypeptide chain under conditions suitable for expression of the polypeptide chain of the molecule; optionally further comprising assembling the polypeptide chains to produce the molecule under conditions suitable for assembly of the polypeptide chains into the molecule.

[0334] For recombinant production, the 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 a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). Once an expression vector comprising one or more polynucleotides of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, liposome-based transfection or other conventional techniques.

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

[0336] The purity of the molecules of the 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 activities of the antibody molecules provided herein can be identified, screened or characterized by a variety of assays known in the art.

[0337] IX. Assay

[0338] The molecules provided herein (anti-C5 antibodies or anti-HTRA1 antibodies or bispecific antibodies) can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0339] The binding or dissociation properties of the anti-C5 antibodies or bispecific antibodies of the present invention to human complement C5 protein can be determined by methods known in the art, such as thin-layer interferometry (BLI), ELISA, Western blotting, ForteBio, etc., or the exemplary method disclosed in Example 4 herein.

[0340] The hemolysis-inhibiting effect of the anti-C5 antibodies or bispecific antibodies of the present invention can be measured by methods known in the art, such as in vitro assays and / or cell-based assays. For example, a hemolysis assay, such as the method described in Example 5 or 6, can be used to detect the hemolysis-inhibiting effect of the molecule on the classical complement immunity pathway and / or the alternative complement immunity pathway.

[0341] The effect of the anti-C5 antibody or bispecific antibody of the present invention on blocking the classical or alternative pathway of complement or the biological activity of blocking the production of C5a can be determined by methods known in the art, such as detecting the content of C5a in a hemolytic system induced by the classical or alternative pathway of complement, such as the method shown in Example 7.

[0342] The therapeutic effects of the anti-C5 antibody or bispecific antibody of the present invention can be determined by an in vivo efficacy experiment on geographic atrophy induced by sodium dodecyl sulfate, such as the method described in Example 16.

[0343] The effect of the anti-HTRA1 antibody or bispecific antibody of the present invention in blocking HTRA1 enzyme activity (eg, serine protease activity or elastase activity) can be determined by H2-Opt assay or Elastase assay, such as the method described in Example 9 or 10.

[0344] X. Immunoconjugates

[0345] In one aspect, the invention provides an immunoconjugate comprising a molecule of the invention (e.g., an anti-C5 antibody or an anti-HTRA1 antibody or a bispecific antibody) and one or more other active ingredients (e.g., an active ingredient from a drug or therapeutic agent for treating a disease of the invention, such as a small molecule that enhances the therapeutic effect of the molecule of the invention).

[0346] XI. Pharmaceutical Compositions

[0347] In one aspect, the invention provides a composition, eg, a pharmaceutical composition, medicament or formulation, comprising a molecule of the invention (eg, an anti-C5 antibody or an anti-HTRA1 antibody or a bispecific antibody, or an immunoconjugate, etc.).

[0348] In one embodiment, the composition further comprises pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0349] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents that are physiologically compatible. For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0350] The compositions or medicines or preparations of the present invention can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injections), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0351] The compositions, medicaments, or formulations of the invention may also comprise, in addition to one or more molecules of the invention, other therapeutic agents, as required for the particular indication being treated, which preferably do not adversely affect the activity of each other. Thus, in one embodiment, a composition, formulation, or medicament, e.g., a pharmaceutical composition, comprises one or more molecules of the invention, in combination with one or more other therapeutic agents.

[0352] The composition of the present invention or medicine or preparation can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injections), powders or suspensions, liposomes and suppositories. The composition of the present invention or medicine or preparation are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion). The preferred form depends on the mode of administration and therapeutic use of expectation.

[0353] XII. Pharmaceutical Combinations and Kits

[0354] The present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising a molecule of the present invention (anti-C5 antibody or anti-HTRA1 antibody or bispecific antibody or immunoconjugate thereof). Optionally, the pharmaceutical combination or pharmaceutical combination product further comprises one or more other therapeutic agents.

[0355] The present invention also provides a complete kit comprising the drug combination, for example, the complete kit comprises in the same package:

[0356] - a first container containing a pharmaceutical composition comprising a molecule of the invention (anti-C5 antibody or anti-HTRA1 antibody or bispecific antibody or immunoconjugate thereof);

[0357] - Optionally, a second container further comprising a pharmaceutical composition comprising one or more additional therapeutic agents (in some embodiments, the two or more additional therapeutic agents are in the same container, or in separate containers).

[0358] XIII. Use and Methods

[0359] In one aspect, the present invention provides a method for preventing or treating a disease or disorder related to the complement system and / or HTRA1 in a subject, comprising administering to the subject an effective amount of a molecule of the present invention (e.g., an anti-C5 antibody or an anti-HTRA1 antibody or a bispecific antibody or an immunoconjugate thereof, etc.), or a composition, a drug or a formulation comprising the same.

[0360] In some embodiments, the present invention provides a method for preventing or treating a complement system-related disease or disorder in a subject, comprising administering to the subject an effective amount of a molecule of the present invention (e.g., an anti-C5 antibody or bispecific antibody or immunoconjugate thereof, etc.), or a composition or drug or formulation comprising the same.

[0361] In some embodiments, the present invention provides a method for preventing or treating an HTRA1-related disease or disorder in a subject, comprising administering to the subject an effective amount of a molecule of the present invention (e.g., an anti-HTRA1 antibody or bispecific antibody or immunoconjugate thereof, etc.), or a composition, medicament or formulation comprising the same.

[0362] In one aspect, the present invention provides a method for preventing or treating a disease or disorder related to the complement system and HTRA1 in a subject, comprising administering to the subject an effective amount of a molecule of the present invention (e.g., an anti-C5 antibody or an anti-HTRA1 antibody or a bispecific antibody or an immunoconjugate thereof, etc.), or a composition or a drug or a formulation comprising the same.

[0363] In some embodiments, the complement system-related disease is caused by abnormal activation of the complement system or dysregulation of the complement system. In some embodiments, the treatment of the disease will benefit from inhibiting the activity of the complement system.

[0364] In some embodiments, the complement system-related disease or disorder is a complement C5-related disease or disorder. In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels) complement C5 or C5a protein (e.g., compared to healthy subjects). In some embodiments, the subject's biological sample has (e.g., elevated levels, such as nucleic acid or protein levels) complement C5 or C5a (e.g., compared to biological samples of healthy subjects). In some embodiments, the treatment of the complement system-related disease will benefit from inhibiting complement C5 or C5a at the nucleic acid or protein level, or from blocking the production of C5a.

[0365] In some embodiments, the complement-related disease or disorder can be a disease requiring inhibition of hemolysis, such as a disease requiring inhibition of hemolysis of the classical pathway of complement immunity and / or the alternative pathway of complement immunity.

[0366] In some embodiments, the HTRA1-associated disease or condition comprises a subject having (e.g., elevated levels, such as nucleic acid or protein levels) HTRA1 (e.g., compared to healthy subjects). In some embodiments, a biological sample of the subject comprises (e.g., elevated levels, such as nucleic acid or protein levels) HTRA1 (e.g., compared to biological samples of healthy subjects). In some embodiments, the HTRA1-associated disease or condition would benefit from treatment by inhibiting HTRA1 at the nucleic acid or protein level.

[0367] In some embodiments, the biological sample is a body fluid, such as vitreous humor or blood.

[0368] In some embodiments, the HTRA1-associated disease or disorder is a disease requiring inhibition of HTRA1 activity (eg, its serine protease activity and / or elastase activity).

[0369] In some embodiments, the disease or disorder is an ocular disease or disorder.

[0370] In some embodiments, a molecule of the invention or a composition or medicament or formulation or combination product comprising the same delays the onset of a disorder and / or symptoms associated with the disorder.

[0371] In some embodiments, the molecules of the invention, or compositions or medicaments or formulations comprising the same, can also be administered in combination with one or more other therapies, e.g., treatment modalities and / or other therapeutic agents, for the uses described herein, e.g., for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0372] Routes and methods of administration of the molecules of the invention or compositions or medicaments or formulations or combination products comprising the same are according to known methods, for example, injection or infusion, for example through the vitreous cavity, for example by intravitreal injection.

[0373] In other aspects, the invention provides the use of a molecule of the invention or a composition or combination product comprising the same in the manufacture or preparation of a medicament for the uses described herein, such as for preventing or treating the relevant diseases or conditions mentioned herein.

[0374] In other aspects, the present invention also provides molecules of the present invention (eg, anti-C5 antibodies or anti-HTRA1 antibodies or bispecific antibodies or immunoconjugates thereof, etc.), or compositions or drugs or preparations or combination products comprising the same, for use in therapy.

[0375] In other aspects, the present invention also provides molecules of the present invention (e.g., anti-C5 antibodies or anti-HTRA1 antibodies or bispecific antibodies or immunoconjugates thereof, etc.), or compositions or drugs or preparations or combination products comprising the same, which are used to treat the relevant diseases or disorders mentioned herein.

[0376] XIIII. Diagnosis and Testing

[0377] In certain embodiments, the molecules provided herein (eg, anti-C5 antibodies or anti-HTRA1 antibodies or bispecific antibodies or immunoconjugates thereof, etc.) can be used to detect the presence of complement C5 and / or HTRA1 in a biological sample.

[0378] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is a body fluid.

[0379] In certain embodiments, the method comprises contacting a biological sample with a molecule as described herein (e.g., an anti-C5 antibody or bispecific antibody or immunoconjugate thereof) 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. The method can be an in vitro or in vivo method. In one embodiment, the molecules of the invention are used to select subjects suitable for treatment with an inhibitor of complement C5 (e.g., an antibody against complement C5, such as an anti-C5 antibody or bispecific antibody or immunoconjugate thereof of the invention, etc.), for example, wherein complement C5 is a biomarker for selecting the subject.

[0380] In certain embodiments, the method comprises contacting a biological sample with a molecule as described herein (e.g., an anti-HTRA1 antibody or bispecific antibody or immunoconjugate thereof) under conditions that allow binding to HTRA1, and detecting whether a complex forms between the molecule and HTRA1, wherein the formation of the complex indicates the presence of complement HTRA1. The method can be an in vitro or in vivo method. In one embodiment, the molecule of the invention is used to select a subject suitable for treatment with an inhibitor against HTRA1 (e.g., an antibody against HTRA1, such as an anti-HTRA1 antibody or bispecific antibody or immunoconjugate thereof of the invention, etc.), for example, wherein HTRA1 is a biomarker for selecting the subject.

[0381] In certain embodiments, the method comprises contacting a biological sample with a molecule as described herein (e.g., a bispecific antibody or immunoconjugate thereof) under conditions that allow for binding to complement C5 and / or HTRA1, and detecting whether a complex is formed between the molecule and complement C5 or HTRA1, wherein the formation of the complex indicates the presence of complement C5 and / or HTRA1. The method can be an in vitro or in vivo method. In one embodiment, the molecule of the invention is used to select a subject suitable for treatment with an inhibitor of complement C5 and / or HTRA1 (e.g., an antibody against complement C5 and / or HTRA1, such as an anti-C5 antibody or anti-HTRA1 antibody or bispecific antibody or immunoconjugate thereof of the invention, etc.), for example, wherein complement C5 and / or HTRA1 is a biomarker for selecting the subject.

[0382] In some embodiments, the biological sample is a body fluid, such as vitreous humor or blood.

[0383] In certain embodiments, a labeled molecule of the invention (e.g., an anti-C5 antibody or anti-HTRA1 antibody or bispecific antibody of the invention or an immunoconjugate thereof, etc.) is provided. Labels include, but are not limited to, directly detected labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, such as enzymes or ligands, for example, by an enzymatic reaction or molecular interaction.

[0384] In some embodiments, the label is a marker such as biotin or a His tag.

[0385] In some embodiments provided herein, the sample is obtained prior to treatment with a molecule of the invention, or a composition, medicament, formulation, or combination product comprising the same. In some embodiments, the sample is obtained prior to treatment with another therapy. In some embodiments, the sample is obtained during treatment with another therapy, or after treatment with another therapy.

[0386] In some embodiments, complement C5 and / or HTRA1 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0387] In some embodiments, a method of treating a disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a subject sample) for the presence of complement C5 and / or HTRA1, thereby determining a complement C5 and / or HTRA1 value, comparing the complement C5 and / or HTRA1 value to a control value (e.g., a value in a normal individual), and if the complement C5 and / or HTRA1 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, medicament or formulation comprising the same, optionally in combination with one or more other therapies, thereby treating the disease.

[0388] Any or all features described above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are for illustrative purposes and should not be construed as constituting any limitation. Example

[0389] Example 1. Preparation and panning of C5 phage display library

[0390] 1.1 Two healthy adult alpacas (Chengdu Apec Company) were selected and 0.5 or 0.25 mg of recombinant protein antigen C5 (Beijing ACRO Company) was mixed with Freund's complete or incomplete adjuvant in a 1:1 ratio. The alpacas were immunized by multiple subcutaneous injections at the back for a total of five immunizations, with an immunization interval of 2-3 weeks.

[0391] 1.2 Collect 50 ml of alpaca peripheral blood and separate lymphocytes at a rate of 2.5×10 7 1 mL of Trizol reagent (Thermo Fisher Scientific) was added to each viable cell, and total RNA was extracted using chloroform / isopropanol precipitation. 10 μg of RNA was used as a template for reverse transcription using the PrimeScript Reverse Transcription Kit (Takara). The cDNA was used as a template for a first-round PCR reaction using the forward primer Alp-VhL and the reverse primer Alp-2b / 2cR to obtain the first-round PCR product. A second-round PCR reaction was performed using the first-round PCR product as a template using the forward primer Alp-VhF and the reverse primer Alp-JHR-SalI to obtain the second-round PCR product. The pC3-HF vector and the second-round PCR product were double-digested with SacI and SalI (Thermo Fisher Scientific), respectively. The digested products were ligated with T4 ligase (Thermo Fisher Scientific). The ligated products were then electroporated into TG1 competent cells to construct a VHH antibody immune library.

[0392] The above library culture was inoculated into 100 ml of YT-AG medium (Shanghai Sangon Co., Ltd.) and cultured until the logarithmic growth phase. M13KO7 helper phage was added for infection. The cells were resuspended in 2× YT-AK medium (Shanghai Sangon Co., Ltd.) and incubated overnight at 30°C and 200 rpm. The culture supernatant was collected and recombinant phage was prepared by PEG / NaCl precipitation.

[0393] 1.3 The recombinant phage was subjected to 2-3 rounds of panning using biotin-labeled antigen C5 (ACRO C5, biotin-labeled in-house). 30 μL Pierce streptavidin magnetic beads (Thermo) and 100 pmoL biotin-labeled antigen were added to each tube and incubated at room temperature for 30 minutes. 1x10 12 Incubate the cfu recombinant phage at room temperature for 1 hour. Add 1 ml of 0.1%-0.05% PBST to the resulting mixture and wash 10-15 times. Finally, add 0.5 ml of pH 2.5 glycine buffer to elute the antigen-bound recombinant phage. Infect TG1 cells in the logarithmic growth phase and plate them on YT-AG plates for overnight culture. Simultaneously, take a small amount of the infected bacterial solution and spread it on YT-AG plates for formation of single colonies for the Binding ELISA assay described in 1.4. The next day, scrape the bacterial lawn from the plate and prepare recombinant phage according to the recombinant phage preparation method described in 1.2 for the next round of panning experiments.

[0394] 1.4 Binding ELISA assay and clone sequencing.

[0395] Take the monoclonal plate from step 1.3 and inoculate a single colony into a 96-well plate containing YT-A. Cultivate until the OD600 is approximately 0.5. Add IPTG to a final concentration of 1 mM and induce expression overnight at 30°C. Add polymyxin to the culture supernatant and incubate at 37°C for 30 minutes. After centrifugation, the supernatant is used for binding ELISA analysis.

[0396] C5 antigen (ACRO, Beijing) was diluted to 0.5 μg / ml in PBS buffer and coated on a 96-well ELISA plate. The antigen-coated plate was washed three times with PBST, and 300 μL of blocking agent was added to each well. The plate was blocked at room temperature for 1 hour. The plate was washed three times with PBST, and 80 μL of blocking agent plus 20 μL of bacterial expression supernatant was added and incubated at room temperature for 1 hour.

[0397] Wash three times with PBST, add 100 μL / well of Anti-Flag / HRP secondary antibody (Sigma) diluted in blocking agent, and incubate at room temperature for 1 hour. Wash six times with PBST, add 100 μL / well of TMB colorimetric solution, and develop in the dark for 5-15 minutes. Then add 100 μL / well of stop solution. Read the plate using a microplate reader and measure the absorbance at OD450 nm. Select bacterial clones with a reading greater than 0.5 and send them to GeneWeiZ for sequencing.

[0398] Example 2. Panning of HTRA1 phage display library

[0399] 2.1 Two to three rounds of solid phase panning were performed using our company's own VHH synthetic library and the customized HTRA1 recombinant antigen (UniProtKB / Swiss-Prot: Q92743) from Beijing Sino. The specific experimental method was to add 30 pmoL HTRA1 recombinant antigen to each ELISA well and coat overnight at 4°C. The plate was washed three times with PBST and blocked with blocking solution at room temperature for 1 hour. 5x10 11 Incubate the cfu recombinant phage at room temperature for 1 hour. After incubation, wash 10-15 times with 0.1%-0.05% PBST, with each wash time of 3-5 minutes. Finally, elute the antigen-bound recombinant phage with pH 2.5 glycine buffer, infect TG1 in the logarithmic growth phase and spread on YT-AG plates for overnight culture. At the same time, take a small amount of the infected bacterial solution, spread it on the YT-AG plate after gradient dilution to form a single clone on the plate for Binding ELISA detection in 2.2. The next day, scrape the bacterial lawn from the plate and prepare the recombinant phage according to the recombinant phage preparation method in 1.2 for the next round of panning experiments.

[0400] 2.2 Binding ELISA detection of binding activity and clone sequencing.

[0401] Take the monoclonal plate from 2.1 and inoculate a single colony into a 96-well plate containing YT-A. Cultivate until the OD600 is approximately 0.5. Add IPTG to a final concentration of 1 mM and induce expression overnight at 30°C. Add polymyxin to the culture supernatant and incubate at 37°C for 30 minutes. After centrifugation, the supernatant is used for binding ELISA analysis.

[0402] HTRA1 antigen (custom-made by Beijing Sino) was diluted to 0.5 μg / ml in PBS buffer and coated on a 96-well ELISA plate. Refrigerate at 4°C overnight. Wash the plate three times with PBST, add 300 μL of blocking agent per well, and incubate at room temperature for 1 hour. Wash three times with PBST, add 80 μL of blocking agent plus 20 μL of the expression supernatant of the above-mentioned TG1 monoclonal clone, and incubate at room temperature for 1 hour.

[0403] Wash three times with PBST, add 100 μL / well of Anti-Flag / HRP secondary antibody (Sigma) diluted in blocking agent, and incubate at room temperature for 40 minutes. Wash six times with PBST, add 100 μL / well of TMB colorimetric solution, and develop for 5-15 minutes in the dark. Then, add 100 μL / well of stop solution. Read the plate using a microplate reader and measure the absorbance at OD450 nm. Select bacterial clones with a reading greater than 0.5 and send them to Jinweizhi for sequencing.

[0404] Example 3. Eukaryotic expression and purification of candidate antibodies

[0405] The present invention utilizes molecular biological techniques to amplify the VHH antibody sequence from the anti-C5 or HTRA1-positive phage obtained in Example 1 using PCR and insert it into pCDNA3.1 with a His tag (HHHHHH). The sequence is then transfected into HEK-293 cells (Thermo Fisher Scientific), expressed, and purified to obtain a VHH-His recombinant antibody with a 6XHis tag at the C-terminus. All independently validated VHH antibodies mentioned below are VHH-His recombinant antibodies.

[0406] The purified antibodies were screened using complement hemolysis assays (CH50, ACH50) or enzyme activity assays (H2-Opt assay, Elastase assay), and ultimately two anti-C5 VHH antibodies (1,293) and one anti-HTRA1 antibody SA239 were obtained.

[0407] The full-length amino acid sequences of the anti-C5 VHH antibody (1,293) and one anti-HTRA1 antibody (SA239) obtained in the present invention, as well as the specific sequence numbers, can be found in the sequence listing.

[0408] Example 4 Determination of the binding kinetics between the VHH antibody of the present invention and antigen using thin-layer biofilm interferometry

[0409] The equilibrium dissociation constant (KD) of the antibodies of the present invention binding to human C5 or HTRA1 was determined using thin-layer interferometry (BLI). The assay was performed using a ForteBio Octet Red96e instrument, and affinity determination was performed according to existing methods (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8).

[0410] Half an hour before the start of the experiment, according to the number of samples, an appropriate number of SA sensors (18-5019, Satorius) were taken and immersed in SD buffer (PBS 1×, BSA 0.1%, Tween-20 0.05%).

[0411] Take 100 μl of SD buffer, antibody, and biotin-labeled antigen (including human C5 and human HTRA1) and add them to a 96-well black polystyrene half-volume microplate (Greiner, 675076). Arrange the plate according to the sample position and select the sensor position. The instrument setting parameters are as follows: running steps: Baseline, Loading ~ 1nm, Baseline, Association and Dissociation; the running time of each step depends on the sample binding and dissociation speed, the rotation speed is 400 rpm, and the temperature is 30 ° C. Use ForteBio analysis software to analyze K D value.

[0412] In the experiments described in the above assays, the affinities of the antibodies are shown in Table 1:

[0413] Table 1. Affinity constants (equilibrium dissociation constants) of monovalent binding of C5 antigen and antibody detected by ForteBio

[0414] Table 2. Affinity constants (equilibrium dissociation constants) of bivalent HTRA1 antigen-antibody binding detected by ForteBio

[0415] In the above experiments, the monovalent KD values ​​of antibody 1,293 for human C5 were 1.75E-10M and 7.23E-10M, respectively; the monovalent affinity KD values ​​for cyno C5 were 2.19E-10M and 1.35E-09M, respectively; and the monovalent affinity KD values ​​for human C5 (R885H) were also 1.33E-10M and 1.83E-09M, respectively.

[0416] The KD of the bivalent affinity of antibody SA239 to human HTRA1 is 8.608E-10M.

[0417] Example 5 Anti-C5 VHH Antibody Blocks Hemolysis Induced by Classical Complement Pathway (CH50)

[0418] Sensitized sheep erythrocytes (immune complexes) activate the classical complement pathway, leading to the formation of transmembrane pores on the erythrocyte surface, allowing extracellular water to penetrate, causing erythrocyte swelling and hemolysis. In this experiment, a series of concentrations of anti-C5 VHH antibodies were fully reacted with a certain amount of complement, and then reacted with sensitized sheep erythrocytes to verify the blocking effect of anti-C5 VHH antibodies on the classical complement pathway.

[0419] Weigh 0.825g of anhydrous calcium chloride and 5g of magnesium chloride (hexahydrate) and dissolve them in 50mL of GVB buffer to obtain a 1000X calcium-magnesium solution. Store at 4°C. Add 500μL of 1000X calcium-magnesium solution to 500mL of GVB buffer to obtain gelatin-veronal (GVB) buffer. Dissolve one tube of human serum complement powder (Sigma) in 1mL of ultrapure water, then dilute with 9mL of gelatin-veronal buffer and store at 4°C. Pipette 10mL of sheep red blood cells (Kejing Bio) into a 50mL centrifuge tube, add 30mL of 1X PBS, centrifuge at 500g at 4°C for 10 minutes, and discard the supernatant. Repeat the washing process until the supernatant is clear. Add 5mL of 1X PBS to the centrifuged red blood cells and gently pipette to dilute 10,000-fold. Mix 10μL of the diluted red blood cells with 10μL of Trypan Blue Stain (0.4%) and count using a cell counter. Dilute the erythrocytes to 1 x 10E9 cells / mL with 1X PBS. Simultaneously, dilute the hemolysin 1000-fold with 1X PBS. Mix equal volumes of both and incubate in a 37°C CO2 shaker at 120 rpm for 45 minutes. Place on ice for another 45 minutes. Centrifuge the erythrocytes at 500g at 4°C for 10 minutes, discard the supernatant, add 1X PBS to 40 mL, centrifuge at 500g at 4°C for 10 minutes, discard the supernatant, and repeat the wash twice. Finally, resuspend the sensitized sheep erythrocytes in 5 mL of 1X PBS. Dilute the sensitized erythrocytes 10,000-fold. Mix 10 μL of the diluted erythrocytes with 10 μL of Trypan Blue Stain (0.4%) and count using a cell counter. Dilute the erythrocytes to 1 x 10E8 cells / mL with gelatin-veronal buffer and place on ice until ready for use. The antibodies to be tested (test samples: Control: IgG1 antibody; ARC1905 (Beixin Biotechnology); VHH antibody 1 and VHH antibody 293) were serially diluted in gelatin-veronal buffer, starting at 200 nM and followed by 3-fold serial dilutions. Human complement serum was diluted 5-fold in gelatin-veronal buffer.

[0420] 100 μL of sample and 50 μL of diluted human complement serum were added to each well;

[0421] For the positive control group (data not shown), 100 μL of gelatin veronal buffer and 50 μL of diluted human complement serum were added to each well;

[0422] For the negative control group (data not shown), each well was the same as the positive control group, except that no subsequent contact with sheep erythrocytes was performed. The cells were shaken at 37°C, 120 rpm, for 45 min.

[0423] Add sensitized sheep red blood cells (50 μL) at a concentration of 1 x 10 cells / mL to each well. Incubate at 37°C (120 rpm) for 30 minutes, pipette evenly, and return the plate to the shaker. Incubate at 37°C (120 rpm) for 30 minutes, pipette evenly, and centrifuge at 1000 g (15°C) for 3 minutes. Aspirate 100 μL of the supernatant into a 96-well plate and read the OD at 405 nm using a microplate reader.

[0424] The erythrocyte hemolysis inhibition rate was calculated as follows:

[0425] Inhibition of hemolysis (%) = [1-(test sample-average value of negative control group) / (average value of positive control group-average value of negative control group)]*100%

[0426] The blocking results of the two anti-C5 VHH antibodies obtained in the present invention are shown in FIG1 . Candidate molecules 1 and 29 antibodies are capable of blocking the classical complement pathway.

[0427] Example 6 Anti-C5 VHH Antibody Blocks Hemolysis Induced by the Alternative Complement Pathway (ACH50)

[0428] When the alternative complement pathway (AP) is activated, C1, C2, and C4 are not involved, while C3, C5-C9, and factors B, D, P, H, and I are. Using EGTA to chelate Ca2+ in the sample blocks C1, thereby preventing activation of the traditional complement pathway. Adding rabbit red blood cells, which can activate factor B, activates the alternative complement pathway, leading to hemolysis of the rabbit red blood cells. Based on this principle, this experiment reacted test samples at varying concentrations with complement and MgEGTA, then added rabbit red blood cells and observed hemolysis to verify the blocking effect of anti-C5 VHH antibodies on the alternative complement pathway.

[0429] Place 5 mL of rabbit peripheral blood (Suzhou Huqiao Biotechnology) in a 50 mL centrifuge tube, add 35 mL of PBS, and centrifuge at 400 g for 10 min at 4°C. Discard the supernatant and repeat the wash cycle until the supernatant is clear. Resuspend the rabbit erythrocytes in 5 mL of PBS and dilute 2000-fold. Mix 10 μL of the diluted rabbit erythrocytes with 10 μL of Trypan Blue Stain (0.4%) and count them using a hemocytometer under an inverted microscope. Place the rabbit erythrocytes on ice until ready to use. Take two tubes of human serum complement powder, add 4 mL of GVB buffer, and gently shake to completely dissolve. Serially dilute each test sample (test samples: Control: IgG1 antibody; ARC1905; VHH antibody 1 and VHH antibody 293) in GVB buffer, starting at 6000 nM and followed by a 3-fold serial dilution.

[0430] According to the arrangement order of the 96-well plate, 25 μL human serum complement, 75 μL sample, 40 μL GVB buffer, and 10 μL 0.1 mol / L MgEGTA were added to each well in sequence.

[0431] Positive control group (data not shown): 25 μL complement, 115 μL GVB, and 10 μL 0.1 mol / L MgEGTA per well;

[0432] Negative control group (data not shown): Identical to the positive control group, but without subsequent contact with rabbit erythrocytes. Incubate at 37°C on a shaker at 120 rpm for 45 min. Rabbit erythrocytes were diluted with GVB buffer to a concentration of 1 x 10 cells / mL and 50 μL was added to each well. A separate 200 μL of rabbit erythrocytes was pipetted into three empty wells.

[0433] Incubate at 37°C in a constant temperature shaker at 120 rpm for 30 minutes. Remove the tube and pipette it thoroughly. Return the tube to the constant temperature shaker at 37°C in a constant temperature shaker at 120 rpm for 30 minutes. Remove the tube and pipette it thoroughly. Centrifuge at 1000g for 3 minutes at 15°C. Transfer 50 μL of the supernatant containing only rabbit erythrocytes to the negative control group and mix thoroughly. Transfer 100 μL of the supernatant to a new 96-well plate and read the OD at 405 nm on a microplate reader.

[0434] The erythrocyte hemolysis inhibition rate was calculated as follows:

[0435] Inhibition of hemolysis (%) = [1-(test sample-average value of negative control group) / (average value of positive control group-average value of negative control group)]*100%

[0436] The blocking results of the two anti-C5 VHH antibodies obtained in the present invention are shown in FIG2 . The candidate molecule 1,293 antibody is able to block the alternative complement pathway.

[0437] Example 7 Anti-C5 VHH Antibodies Block the Production of C5a in the Classical Complement Pathway (C5a ELISA)

[0438] In the classical and alternative pathways of complement, C5 interacts with the C5 convertase to form C5a and C5b. C5b ultimately forms the MAC complex, which lyses cells, while C5a interacts with the C5a receptor to participate in biological activities. This study used the Human C5a ELISA Kit II (BD, Cat. No. 557965) to measure C5a levels in samples induced by the classical or alternative pathways of complement, verifying the biological activity of anti-C5 VHH antibodies in blocking C5a production.

[0439] Remove the test kit from the 4°C refrigerator and allow to stand at room temperature for half an hour. Remove the sample to be tested (supernatant obtained in Example 5: CH50 supernatant) and dilute the CH50 supernatant 2-fold with Standard / Sample Diluent. Add 50 μL of ELISA Diluent to the antibody-coated plate. Add 100 μL of sample to each well, seal the plate, and incubate at room temperature for 2 hours. Dilute 48 μL of Enzyme Concentrate with 12 mL of Detection Antibody. Wash three times with 300 μL of Wash Buffer per well, tapping the plate on a paper towel for the final wash to remove any remaining liquid. Add 100 μL of Working Detector to each well, seal the plate, and incubate at room temperature for 1 hour. Wash three times with 300 μL of Wash Buffer per well, tapping the plate on a paper towel for the final wash to remove any remaining liquid. Add 100 μL of TMB One-Step Substrate Reagent to each well, protect from light, and incubate at room temperature for 30 minutes. Add 50 μL of Stop Solution to each well, gently tap to mix, and protect from light. Fluorescence readings were performed using a multifunctional microplate reader with the following settings: Optical Configuration: Monochromator, Read Mode: ABS, Read Type: Endpoint, two wavelengths (450 nm and 620 nm), Plate Type: 96Well Standard clrbtm, Shake: Off, and Read Order: Row.

[0440] The blocking results of the two anti-C5 VHH antibodies obtained in the present invention are shown in FIG3 . The candidate molecule 1,293 antibody is able to block the production of C5a in the classical complement pathway.

[0441] Example 8 Anti-C5 VHH Antibody Binding Epitope Analysis

[0442] Biofilm thin-layer interferometry (BLI) was used to determine epitope differences in the antibodies of the present invention binding to human C5 (ACRO, Beijing) and to stratify the groups. The assay was performed using a ForteBio Octet Red96e instrument. Half an hour before the start of the experiment, an appropriate number of SA sensors (18-5019, Satorius) were placed in SD buffer (PBS 1×, 0.1% BSA, 0.05% Tween-20) based on the number of samples.

[0443] The sample to be tested is divided into the first Ab (for saturating the antigen) and the second Ab (for competitive binding). Take 100 μl of SD buffer, antibody, and biotin-labeled antigen (including human C5) and add them to a 96-well black polystyrene half-volume microplate (Greiner, 675076). Arrange the plate according to the sample position and select the sensor position. The instrument setting parameters are as follows: running steps: Baseline, Loading Antigen ~1nm, Baseline, loading first Ab to saturation, second Ab Association; the running time of each step depends on the sample binding and dissociation rate, the rotation speed is 1000 rpm, and the temperature is 30°C. The second Ab that competes with the first Ab for binding binds to a similar antigen epitope. The second Ab has no competition or partial competition with the first Ab, and the antigen binding epitope is different.

[0444] The binding epitope results of the two anti-C5 VHH antibodies obtained in the present invention are shown in FIG4 , with 1,293 candidate molecules, and the epitopes of the antibodies are different.

[0445] Example 9 Anti-HTRA1 VHH Antibody Blocks H2-Opt Assay

[0446] HTRA1 is a serine protease. The peptide Mca-IRRVSYSF(K-Dnp)K (abbreviated as H2-Opt) can be cleaved by HTRA1, producing a fluorescent product. The rate of product production can be measured using a microplate reader to indicate the amount of active HTRA1 in the system, thereby validating the ability of anti-HTRA1 VHH antibodies to block HTRA1 cleavage of H2-Opt.

[0447] Weigh 5.85 g of sodium chloride, 1.25 g of CHAPS, and 3.0 g of Tris base, dissolve them in 400 mL of ultrapure water, adjust to pH 8.0, and dilute to 500 mL to obtain Reaction buffer B. Filter with a 0.22 μm filter and store in a 4°C refrigerator. Dissolve 5 mg of H2-optimal substrate peptide (INNOVAGEN) in 300 μL of ultrapure water to obtain H2-Opt stock solution (10 mM), aliquot, and store in a -40°C refrigerator. Dilute the H2-Opt stock solution to 50 μM with Reaction buffer B to obtain H2-Opt working solution.

[0448] Dilute recombinant human HTRA1 to 2.016 μg / mL (40 nM) with Reaction buffer B to obtain HTRA1 enzyme working solution. Serially dilute the test sample with Reaction buffer B.

[0449] 60 μL of HTRA1 enzyme working solution and 20 μL of test samples (Control (IgG1), Galegenimab, and SA239) were added to a black low-adsorption U-bottom 96-well plate in sequence;

[0450] The positive control group (used for evaluation experiments, data not shown) consisted of 20 μL Reaction buffer B and 60 μL HTRA1 enzyme working solution;

[0451] The negative control group (used for evaluation experiments, data not shown) consisted of 80 μL Reaction buffer B.

[0452] Incubate on ice for 30 minutes. Add 40 μL of H2-Opt working solution to each well. Analyze at 30°C, with Ex / Em = 320 nm / 405 nm, and collect data every minute for 45 minutes. Export the raw data and the slope obtained from the linear fit.

[0453] The blocking result of an anti-HTRA1 VHH antibody obtained in the present invention is shown in FIG5 , and the candidate molecule SA239 antibody is able to block the activity of HTRA1 in cleaving H2-Opt.

[0454] Example 10 Anti-HTRA1 VHH Antibody Blocking Elastase Assay

[0455] HTRA1 is a serine protease that cleaves some extracellular matrix components and exhibits elastase activity when the substrate is elastin. Green Elastase Assay Kit (Anaspec, AS-72178) can detect elastase activity and can be used to verify the activity of anti-HTRA1 VHH antibodies in blocking the cleavage of elastin by HTRA1.

[0456] Transfer the reagents from -30°C to room temperature. Prepare 1X Assay buffer by adding 10mL of Component C to 10mL of ultrapure water. Dilute Component A (prepared immediately before use) with 1X Assay buffer 100X to obtain the Elastase substrate solution. Dilute recombinant human HTRA1 (i.e., the recombinant human HTRA1 antigen from Example 1) to 20μg / mL with 1X Assay buffer to obtain the HTRA1 working solution. Perform a serial dilution of the test sample using 1X Assay buffer.

[0457] 10 μL of test samples (Control (IgG1), Galegenimab, and SA239) and 40 μL of HTRA1 working solution were added to a black low-adsorption U-bottom 96-well plate in sequence;

[0458] The positive control group (used for evaluation experiments, data not shown) consisted of 10 μL 1X Assay buffer and 40 μL HTRA1 working solution;

[0459] A negative control (used for experimental evaluation and sample reaction rate calculation, data not shown) consisted of 50 μL of 1X assay buffer. Incubate on ice for 30 min. Add 50 μL of Elastase substrate solution to each well. Samples were collected every 2 min for 30 min using an E / E ratio of 490 nm to 520 nm. The slope of the linear fit was derived.

[0460] The sample reaction rate is calculated as follows:

[0461] Sample reaction rate (RFU / s) = average value of test sample - negative control group

[0462] The blocking result of an anti-HTRA1 VHH antibody obtained in the present invention is shown in FIG6 , and the candidate molecule SA239 antibody is able to block the activity of HTRA1 in cleaving elastin.

[0463] Example 11 Humanization of anti-C5 VHH antibody, activity detection, and protein expression and purification

[0464] The present invention utilizes molecular biological technology to obtain anti-C5 positive VHH antibody sequence, and utilizes the transient expression and purification thereof to obtain VHH antibody protein.

[0465] The amino acid sequences of the heavy chain variable regions of the two anti-C5 VHH antibodies (1 and 293 and LA44) obtained in the present invention, as well as their sequence numbers, are shown in the attached table.

[0466] Humanization of immune library antibodies 1 and 293

[0467] Antibody 1,293 obtained in Example 5 and Example 6 was humanized by the following steps:

[0468] ① Determine the CDR loop structure;

[0469] ② Find the closest homologous sequence for each V / J region of VHH in the human germline sequence database;

[0470] ③ Construct the CDR region of VHH onto the human framework region;

[0471] ④ Using sequence and structural features, determine the amino acid positions in the framework region that maintain CDR function;

[0472] ⑤ Perform back mutation at the sequence position determined to be important;

[0473] ⑥Optimize amino acids at risk sites.

[0474] The amino acid sequences of the three humanized antibodies obtained in the present invention are shown in the attached sequence listing.

[0475] The specific sequence numbers are shown in Table 3:

[0476] Table 3: Anti-C5 VHH antibodies and their CDR sequences

[0477] Protein expression

[0478] Expi-293 cells (Thermo Fisher Scientific) were passaged according to the required transfection volume, and the cell density was adjusted to 1.5 × 10 cells per day before transfection. 6 cells / ml. The cell density on the day of transfection was approximately 3×10 6cells / ml. Opti-MEM medium (Gibco, 11058021) at 1 / 10 the final volume was used as transfection buffer. The appropriate plasmid was added and mixed. The appropriate polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid (plasmid:PEI mass ratio was 1:3), mixed, and incubated at room temperature for 10 minutes. After adding Expi293 cells, the cells were incubated at 36.5°C in 8% CO2. After 24 hours, FEED (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone) at 2% of the transfection volume, a glucose solution at a final concentration of 4 g / L, and VPA (Gibco, catalog number: 11140-050) at a final concentration of 2 mM / L were added. The cells were incubated at 36.5°C, 120 rpm, and 8% CO2. After continuous cultivation for 6 days, the culture was collected and centrifuged at 4000 rpm for 30 minutes. The cell supernatant was filtered through a 0.45 μM filter membrane and purified by affinity chromatography.

[0479] The specific affinity chromatography purification steps are as follows: use Amsphere TM A3 (JSR Life Sciences, catalog number: 10000327-C05) affinity chromatography column was used. Before purification, the column and tubing were detoxified with 0.1 M NaOH for 1 hour. The tubing and column were then rinsed with distilled water. The column was equilibrated with 5 column volumes of 1× PBS (Gibco, catalog number: 10010023). The collected supernatant was passed through the column and then rinsed with 10 column volumes of 1× PBS to remove nonspecifically bound proteins. The column was then rinsed with 5 column volumes of elution buffer (100 mM glycine, pH 2.7). The eluate was collected and the pH of the protein was adjusted to 6.0 with 2 M Tris.

[0480] IgG1 control (Control) (heavy chain: SEQ ID NO: 31; light chain: SEQ ID NO: 37), Galegenimab (heavy chain: SEQ ID NO: 28; light chain SEQ ID NO: 29), Eculizumab (heavy chain: SEQ ID NO: 32;

[0481] Light chain: SEQ ID NO: 33) was also synthesized, expressed, and purified in a similar manner.

[0482] The humanized antibody detection method is as described in Example 5 and Example 6 ( FIG. 7 , Control: IgG1). The results show that the humanized antibody C5 obtained in the present invention can completely inhibit the biological activity of C5.

[0483] Example 12 Anti-HTRA1 VHH affinity maturation

[0484] Affinity maturation was performed by introducing random amino acid mutations into the antigen binding region (CDR) using the SA239 VHH gene as a template. A degenerate primer containing the NNK codon (Suzhou Jinweizhi Co., Ltd.) and a framework region specific primer (Suzhou Jinweizhi Co., Ltd.) were designed and synthesized. The antibody mutant gene library was amplified by overlap extension PCR (OE-PCR), and the PCR fragment and vector were digested, connected, and transformed into TG1 competent cells using the same method as in Example 1 to prepare a recombinant phage library. Three rounds of phage panning and monoclonal ELISA identification were performed. The candidate monoclonal clone was expressed in eukaryotic form by the method of Example 3 for VHH recombinant antibodies, and the purified antibodies were further screened using H2-Opt assay and Elastase assay (experimental methods and steps were the same as in Examples 9 and 10). Finally, a mutant with significantly improved function and affinity was selected, clone number CL4D8M (Figures 8 and 9, Control: IgG1).

[0485] The specific sequence numbers are shown in Table 4:

[0486] Table 4: Anti-HTRA1 VHH antibodies and their CDR sequences

[0487] Example 13 Experiment on blocking complement-induced hemolysis by combining anti-C5 VHH antibodies with different epitopes

[0488] In the anti-C5 VHH antibody screening, 1 and 293 were found to completely block CH50 (Figure 1), but not ACH50 (Figure 2). In this experiment, we combined anti-C5 VHH antibodies targeting different epitopes to verify their blocking effects on ACH50.

[0489] The detection method of this experiment is as described in Examples 5 and 6, wherein HZ293H5.4 is a humanized antibody of the 293 molecule. The results (Figures 10A and B) show that the combination of anti-C5 VHH antibodies with different epitopes, with a starting concentration of 600 nM 1 and 600 nM HZ293H5.4, 4-fold serial dilution (CH50 experiment), and a starting concentration of 6000 nM 1 and 6000 nM HZ293H5.4, 3-fold serial dilution (ACH50 experiment), can completely block the classical complement pathway (CH50) (Figure 10A) and the alternative complement pathway (ACH50) (Figure 10B), respectively.

[0490] Example 14 Experiment on blocking complement-induced hemolysis by dual-epitope anti-C5 antibodies

[0491] Using GGGGGGGGGGG (SEQ ID NO: 26) as a linker, the screened anti-C5 VHH antibodies were constructed into anti-C5 bi-epitope antibodies. In terms of molecular form, the present invention used two anti-C5 VHHs with different epitopes (hzAP02-1.H29 and HZ293H5.4DV) to construct bivalent and trivalent antibodies.

[0492] The structure and sequence of the constructed B1 antibody (bivalent) are as follows:

[0493] HZ293H5.4DV-hzAP02-1.H29 (SEQ ID NO: 34), in which the two VHHs are connected by a linker (SEQ ID NO: 26).

[0494] The structure and sequence of the constructed B3 antibody (trivalent) are as follows:

[0495] HZ293H5.4DV-HZ293H5.4DV-hzAP02-1.H29 (SEQ ID NO: 35), wherein each VHH is connected by a linker (SEQ ID NO: 26).

[0496] The structure and sequence of the constructed B4 antibody (trivalent) are as follows:

[0497] HZ293H5.4DV-hzAP02-1.H29-HZ293H5.4DV (SEQ ID NO: 36), wherein each VHH is connected by a linker (SEQ ID NO: 26).

[0498] The blocking effect was verified by complement-induced hemolysis assay.

[0499] The experimental detection methods are as described in Examples 5 and 6 in the main text (wherein ARC1905 is from Beixin Biotechnology). The results (Figures 10C and D) show that the dual-epitope anti-C5 VHH antibody can completely block both the classical complement pathway (Figure 10C) and the alternative complement pathway (Figure 10D).

[0500] Example 15 Optimization of the molecular format of the anti-C5 / HTRA1 bispecific antibody

[0501] Using GGGGGGGGGGG as a linker, the screened anti-C5 VHH antibody and anti-HTRA1 antibody were constructed into an anti-C5 / HTRA1 bispecific antibody. In terms of molecular form, the present invention uses two anti-C5 VHHs and one anti-HTRA1 VHH. Based on the two anti-C5 VHHs, the present invention has constructed two identical anti-C5 VHHs (anti-C5 VHH monoepitope bivalent format) and two different anti-C5 VHHs (anti-C5 VHH biepitope format).

[0502] Two anti-C5 VHH antibodies (different epitopes) and one anti-HTRA1 VHH antibody were constructed into a bispecific antibody, the molecular structure of which is shown in FIG11 .

[0503] The constructed IAR045-001 antibody structure and sequence are as follows:

[0504] C5 HZ293H5.4D-C5 hzAP02-1.H-HTRA1 CL4D8M (SEQ ID NO: 27), wherein each VHH is connected by a linker (SEQ ID NO: 26).

[0505] The binding kinetics of IAR045-001 molecules and antigens were determined by thin-layer interferometry.

[0506] The detection method of this experiment is as described in Example 4. Human Complement C5 (Cat. No. CO5-H52Ha), Monkey Complement C5 (Cat. No. CO5-C52Hx), Human Complement C5 (R885H) (Cat. No. CO5-H52Hx), Mouse Complement C5 (Cat. No. CO5-M52H4), Rabbit Complement C5 (Cat. No. CO5-R52H4), and Rat Complement C5 (Cat. No. CO5-R52H5) proteins were purchased from Acro. Dog Complement C5 (sequence reference UniProtKB: A0A8I3NVC1, Q19-S1680), Monkey HTRA1 (sequence reference NCBI: XP_045217639.1, Q24-P481), Mouse HTRA1 (sequence reference UniProtKB: Q9R118, P24-P480), Rat HTRA1 (sequence reference UniProtKB: Q9QZK5, L23-P480), Rabbit HTRA1 (sequence reference NCBI: XP_051680286.1, full length), and Dog HTRA1 (sequence reference UniProtKB: A0A8C0RQ32, Q30-P485) proteins were expressed in-house. Human HTRA1 (sequence reference UniProtKB: Q92743, Q23-P480) protein was custom-ordered from Sino Biotechnology. The affinity of the antibodies is shown in Table 5:

[0507] Table 5. Affinity constants (equilibrium dissociation constants) of IAR045-001 antibody antigen binding detected by ForteBio

[0508] In the above experiments, the bispecific antibody molecule IAR045-001 and the K D The value is less than 2.84E-10M, which is comparable to the K of human C5 (R885H) D The value is 5.59E-10M, which is similar to the K of monkey C5. D The KD value of the C5 protein with mouse C5 is 4.46E-09M, and the KD value of the C5 protein with rat C5 is 4.46E-09M. D The value is less than 3.44E-10M, which is comparable to the K of rabbit C5. D The value is 2.08E-10M, it does not bind to dog C5, and the K for human HTRA1 is D The value is 1.58E-09M, which is similar to the K of monkey HTRA1. D The value is 1.31E-08M, which is similar to the K of mouse HTRA1. D The value was 7.75E-08M, which did not bind to rat HTRA1 and had a K of 0.05 for rabbit HTRA1. D The value is 2.61E-09M, which is similar to the K of dog HTRA1. D The value is 2.03E-09M.

[0509] The blocking effect was verified by complement-induced hemolysis assay.

[0510] The experimental detection method is as described in Examples 5 and 6 (ARC1905 is from Beixin Bio, Eculizumab is synthesized as described above, and Control: IgG1). The results show that IAR045-001 can completely block both the classical complement pathway ( FIG. 12 ) and the alternative complement pathway ( FIG. 13 ).

[0511] Example 15: Bispecific antibody molecules block HTRA1 enzyme activity

[0512] The bispecific antibody molecule IAR045-001 contains an anti-HTRA1 VHH antibody, and its blocking effect on HTRA1 was verified by H2-Opt assay and Elastase assay.

[0513] The detection method of this experiment is the same as that of Example 9 and Example 10. The results show that IAR045-001 can block the activity of HTRA1 in cleaving H2-Opt ( FIG. 14 ) and the activity of HTRA1 in cleaving elastin ( FIG. 15 ).

[0514] Example 16. In vivo efficacy study of sodium iodate-induced geographic atrophy

[0515] The therapeutic effect of the anti-C5 / HTRA1 bispecific antibody of the present invention was evaluated using the sodium iodate-induced geographic atrophy model.

[0516] Experimental animals: hC5 mice; sex: male; age: 6-8 weeks; source: Shanghai Model Organisms Technology Co., Ltd.

[0517] In this study, sodium iodate was injected into the tail vein to induce damage to the pigment epithelial cells in the mouse retina, resulting in phenotypes such as thinning of the outer nuclear layer (ONL) and photoreceptor outer segment (POS) of the retina, thereby establishing a mouse model similar to the human geographic atrophy phenotype. Twenty-eight complement C5 humanized mice (hC5 mice) were selected and divided into five groups: a model control group (4 mice), an IgG1 group (6 mice), a Galegenimab group (6 mice), an ARC1905 (Beixin Bio) group (6 mice), and an IAR045-001 group (6 mice). Sodium iodate was administered on Day 0, while the model control group remained untreated. The test article was injected intravitreally on Day 0 and Day 7. Ocular tissue samples were collected on Day 10, and the ocular tissue was sectioned and stained with H&E to analyze the inhibitory effect of the test article on geographic atrophy. The experimental design is shown in Table 6.

[0518] Table 6: Experimental design table

[0519] H&E staining histopathological examination

[0520] Evaluation indicators:

[0521] (1) Thickness of the retinal outer nuclear layer

[0522] (2) Thickness of the retinal POS layer

[0523] (3) Integrity of the retinal pigment epithelium

[0524] RPE integrity statistics for a 40 μm length at a distance of 250 μm from the optic nerve: brown area / total area*100%

[0525] As shown in Figure 16, Figure 16A shows a representative H&E staining of eye tissue in each experimental group. The arrows in the figure mark the inner nuclear layer (INL), outer nuclear layer (ONL), photoreceptor outer segment (POS), retinal pigment epithelium (RPE) and choroid in the mouse retina. The upper left picture shows the retinal tissue of normal mice, with intact ONL, POS and RPE; the upper middle picture shows the retinal tissue of IgG1 group mice, with thinner ONL and POS layers and discontinuous RPE compared to normal mice; the upper right picture shows the retinal tissue of Galegenimab group mice, with thicker ONL and POS layers and more intact RPE compared to IgG1 group; the lower left picture shows the retinal tissue of ARC1905 group mice, with thicker ONL and POS layers and more intact RPE compared to IgG1 group; the lower middle picture shows the retinal tissue of IAR045-001 group mice, with thicker ONL and POS layers and more intact RPE compared to IgG1 group.

[0526] FIG16B is a statistical diagram of the thickness of the retinal POS layer of each group of mice. It can be seen that the POS thickness of the IAR045-001 group is thicker than that of the IgG1, ARC1905 and Galegenimab groups.

[0527] FIG16C is a statistical diagram of the thickness of the retinal ONL layer of each group of mice. It can be seen that the ONL thickness of the IAR045-001 group is also thicker than that of the IgG1, ARC1905, and Galegenimab groups.

[0528] FIG16D is a statistical diagram of the integrity of the retinal pigment epithelial cell layer of each group of mice. It can be seen that the pigment epithelial cell layer of the IAR045-001 group is more intact than that of the IgG1, ARC1905, and Galegenimab groups.

[0529] In summary, the antibody of the present invention can significantly restore the thinning of the ONL layer, thinning of the POS layer and the integrity of the pigment epithelial cells induced by sodium iodate, and the effect is better than ARC1905 and Galegenimab.

[0530] Sequence Listing

Claims

1. A VHH antibody that specifically binds to C5, comprising The three complementarity determining regions (CDRs) contained in the VHH shown in any one of SEQ ID NO: 23, 17 or 22, Preferably, the CDR1 sequence is according to the AbM definition, CDR2 is according to the Kabat definition, and CDR3 is according to the AbM definition.

2. The VHH antibody that specifically binds to C5 according to claim 1, comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein (i) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10; (ii) VHH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, and VHH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3; or (iii) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 2 or 9, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3 or 10.

3. The VHH antibody that specifically binds to C5 according to claim 1, which comprises or consists of a heavy chain variable region, wherein the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 23, 17 or 22; or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 23, 17 or 22.

4. A VHH antibody that specifically binds to C5, comprising three complementarity determining regions (CDRs) contained in the VHH shown in any one of SEQ ID NO: 20, 18, 19 or 21, Preferably, the CDR1 sequence is according to the AbM definition, CDR2 is according to the Kabat definition, and CDR3 is according to the AbM definition.

5. The VHH antibody that specifically binds to C5 according to claim 4, comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 8, VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6; (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO:4, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO:5, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:6 or 30; or (iii) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5, 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 30 or 6.

6. The VHH antibody that specifically binds to C5 according to claim 4, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 20, 18, 19 or 21; or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 20, 18, 19 or 21.

7. A heavy chain antibody that specifically binds to C5, comprising the VHH antibody according to any one of claims 1 to 6.

8. The heavy chain antibody that specifically binds to C5 according to claim 7, which comprises or consists of the VHH antibody that specifically binds to C5 according to any one of claims 1 to 6 linked to an antibody constant region or Fc region.

9. A VHH antibody that specifically binds to HTRA1, comprising The three complementarity determining regions (CDRs) contained in the VHH shown in SEQ ID NO: 25 or 24, Preferably, the CDR1 sequence is according to the AbM definition, CDR2 is according to the Kabat definition, and CDR3 is according to the AbM definition.

10. The VHH antibody that specifically binds to HTRA1 according to claim 9, comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein (i) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16; or (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 13; or (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11 or 14, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12 or 15, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 13 or 16.

11. The VHH antibody that specifically binds to HTRA1 according to claim 9, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 25 or 24; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 25 or 24. 12 . A heavy chain antibody that specifically binds to HTRA1, comprising the VHH antibody that specifically binds to HTRA1 according to claim 9 . 13 . The heavy chain antibody specifically binding to HTRA1 according to claim 12 , comprising or consisting of the VHH antibody specifically binding to HTRA1 according to any one of claims 9 to 11 linked to an antibody constant region or Fc region.

14. The heavy chain antibody that specifically binds to C5 according to claim 8 or the heavy chain antibody that specifically binds to HTRA1 according to claim 13, wherein The antibody constant region or Fc region is derived from human IgG1, human IgG2, human IgG3 or human IgG4.

15. The VHH antibody that specifically binds to C5 according to any one of claims 1 to 6, or the heavy chain antibody that specifically binds to C5 according to claim 7 or 8, or the VHH antibody that specifically binds to HTRA1 according to any one of claims 9 to 11, or the heavy chain antibody that specifically binds to HTRA1 according to claim 12 or 13, or the heavy chain antibody that specifically binds to C5 or the heavy chain antibody that specifically binds to HTRA1 according to claim 14, wherein the antibody is a chimeric antibody or a humanized antibody.

16. An antibody that specifically binds to C5, comprising two or three or more antigen binding regions that specifically bind to C5, wherein the antigen binding regions bind to different epitopes and respectively comprise the VHH antibody that specifically binds to C5 of any one of claims 1-6 and 15, or the heavy chain antibody that specifically binds to C5 of any one of claims 7-8 and 14-15.

17. The antibody that specifically binds to C5 according to claim 16, comprising 1 or 2 VHH antibodies that specifically bind to C5 according to any one of claims 1-3 or heavy chain antibodies comprising said VHH antibodies, and 1 or 2 VHH antibodies that specifically bind to C5 according to any one of claims 4-6 or heavy chain antibodies comprising said VHH antibodies; preferably, it comprises 1 or 2 VHH antibodies that specifically bind to C5 according to any one of claims 4-6, and 1 VHH antibody that specifically binds to C5 according to any one of claims 1-3, preferably, said VHH antibodies or heavy chain antibodies are connected in series, preferably via a linker; Preferably, the antibody comprises or consists of the following chain, which comprises, from N-terminus to C-terminus: (1) the VHH antibody that specifically binds to C5 according to any one of claims 4 to 6 or the VHH antibody that specifically binds to C5 according to any one of claims 1 to 3, (2) the VHH antibody that specifically binds to C5 according to any one of claims 4 to 6 - the VHH antibody that specifically binds to C5 according to any one of claims 4 to 6 - the VHH antibody that specifically binds to C5 according to any one of claims 1 to 3, or (3) the VHH antibody that specifically binds to C5 according to any one of claims 4 to 6 - the VHH antibody that specifically binds to C5 according to any one of claims 1 to 3 - the VHH antibody that specifically binds to C5 according to any one of claims 4 to 6; Preferably, the VHHs are connected via a linker.

18. A multispecific antibody, comprising one or more antigen binding regions that specifically bind to C5 and one or more other antigen binding regions, wherein the antigen binding region that specifically binds to C5 comprises the VHH antibody that specifically binds to C5 of any one of claims 1-6 and 15, or the heavy chain antibody that specifically binds to C5 of any one of claims 7-8 and 14-15, preferably, the multispecific antibody is a bispecific antibody.

19. The multispecific antibody of claim 18, further comprising one or more antigen binding regions that specifically bind to HTRA1, for example, the antigen binding region that specifically binds to HTRA1 comprises the VHH antibody that specifically binds to HTRA1 of any one of claims 9-11 and 15, or the heavy chain antibody that specifically binds to HTRA1 of any one of claims 12-15.

20. A multispecific antibody comprising an antigen binding region that specifically binds to HTRA1 and one or more other antigen binding regions, wherein the antigen binding region that specifically binds to HTRA1 comprises the VHH antibody that specifically binds to HTRA1 of any one of claims 9 to 11 and 15, or the heavy chain antibody that specifically binds to HTRA1 of any one of claims 12 to 15, preferably, the multispecific antibody is a bispecific antibody.

21. The multispecific antibody of claim 20, further comprising one or more antigen binding regions that specifically bind to C5, wherein the antigen binding region that specifically binds to C5 comprises the VHH antibody that specifically binds to C5 of any one of claims 1-6 and 15, or the heavy chain antibody that specifically binds to C5 of any one of claims 7-8 and 14-15.

22. A bispecific antibody comprising one or more antigen binding regions that specifically bind to C5 and one or more antigen binding regions that specifically bind to HTRA1, wherein the antigen binding regions that specifically bind to C5 bind to different epitopes on C5.

23. The bispecific antibody of claim 22, comprising two antigen binding regions that specifically bind to C5 and one antigen binding region that specifically binds to HTRA1, wherein the two antigen binding regions that specifically bind to C5 bind to different epitopes on C5.

24. The bispecific antibody of claim 23, wherein the two antigen binding regions that specifically bind to C5 are VHHs that specifically bind to different epitopes of C5, and the antigen binding region that specifically binds to HTRA1 is a VHH that specifically binds to HTRA1, wherein the three VHHs are connected in series, and the VHHs are optionally connected via a linker.

25. The bispecific antibody of claim 24, wherein the linker comprises (G) n , where n=5-10, for example 5, 6, 7, 8, 9, 10.

26. The bispecific antibody of claim 22 or 23, wherein the VHHs are connected via N-terminus to N-terminus, C-terminus to C-terminus, or N-terminus to C-terminus, optionally via a linker.

27. The bispecific antibody of any one of claims 24-26, wherein one antigen-binding region that specifically binds to C5 comprises or consists of the VHH of any one of claims 1-3, and the other antigen-binding region that specifically binds to C5 comprises or consists of the VHH of any one of claims 4-6, or The antigen binding region that specifically binds to HTRA1 comprises or consists of the VHH according to any one of claims 9 to 11.

28. The bispecific antibody of any one of claims 24-26, wherein one antigen-binding region that specifically binds to C5 comprises or consists of the VHH of any one of claims 1-3, and the other antigen-binding region that specifically binds to C5 comprises or consists of the VHH of any one of claims 4-6, and The antigen binding region that specifically binds to HTRA1 comprises or consists of the VHH according to any one of claims 9 to 11.

29. The bispecific antibody of any one of claims 24-28, comprising the following chains, which from N-terminus to C-terminus comprise: VHH1 that specifically binds to C5 - VHH2 that specifically binds to C5 - VHH3 that specifically binds to HTRA1, The VHHs are connected via a linker; wherein VHH1 comprises or consists of the VHH of any one of claims 4 to 6, VHH1 comprises or consists of the VHH of any one of claims 1 to 3, and VHH3 comprises or consists of the VHH according to any one of claims 9 to 11.

30. The bispecific antibody of claim 29, comprising or consisting of a chain, wherein the chain comprises or consists of an amino acid sequence as shown in SEQ ID NO: 27, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

31. A nucleic acid molecule encoding a VHH antibody that specifically binds to C5 according to any one of claims 1-6 and 15, or a heavy chain antibody that specifically binds to C5 according to claim 7 or 8 or 15, or a VHH antibody that specifically binds to HTRA1 according to any one of claims 9-11, or a heavy chain antibody that specifically binds to HTRA1 according to claim 12 or 13 or 15, or a heavy chain antibody that specifically binds to C5 or a heavy chain antibody that specifically binds to HTRA1 according to any one of claims 14 or 15; or a multispecific antibody according to any one of claims 17-20, or a bispecific antibody according to any one of claims 21-28.

32. An expression vector comprising the nucleic acid molecule of claim 31, preferably, the expression vector is pCDNA3.

1.

33. A host cell comprising the nucleic acid molecule of claim 31 or the expression vector of claim 32, preferably, the host cell is prokaryotic or eukaryotic, such as a CHO cell, a 293 cell, such as an Expi293 cell.

34. A method for preparing an antibody according to any one of claims 1 to 30, comprising cultivating the host cell of claim 33 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.

35. An immunoconjugate comprising the antibody of any one of claims 1 to 30, and one or more other active ingredients.

36. A pharmaceutical composition or drug or formulation comprising the antibody of any one of claims 1 to 30 or the immunoconjugate of claim 35, and optionally a pharmaceutically acceptable excipient.

37. A drug combination product comprising The antibody of any one of claims 1 to 30 or the immunoconjugate of claim 35; and Other Therapeutic Agents.

38. A method for preventing or treating an ocular disease or condition in a subject, comprising administering to the subject an effective amount of the antibody of any one of claims 1-30; or the immunoconjugate of claim 35; or the pharmaceutical composition or formulation of claim 36; or the pharmaceutical combination product of claim 37.

39. The method of claim 38, wherein the disease or disorder is a disease or disorder in which the subject has (e.g., elevated levels, such as nucleic acid or protein levels) complement C5 protein and / or HTRA1 protein (e.g., compared to healthy subjects) or a sample, such as a body fluid, of the subject has (e.g., elevated levels, such as nucleic acid or protein levels) complement C5 protein and / or HTRA1 protein (e.g., compared to body fluids of healthy subjects).

40. A method for detecting the presence of complement C5 and / or HTRA1 in a biological sample, comprising contacting the biological sample with the antibody of any one of claims 1 to 30 under conditions that allow it to bind to complement C5 and / or HTRA1, and detecting whether a complex is formed between the antibody and complement C5 and / or HTRA1, wherein the formation of the complex indicates the presence of complement C5.

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