WO2025119329A1 - Anti-c5 and htra1 bispecific antibody and use thereof - Google Patents

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

CROSS-REFERENCE TO RELATED APPLICATIONS

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

The present invention relates to a novel anti-C5 antibody and a novel HTRA1 antibody, as well as bispecific antibodies specifically binding to C5 and HTRA1 constructed based thereon. In particular, the present invention relates to an anti-C5 antibody specifically binding to different epitopes of C5 or a bispecific antibody specifically binding to different epitopes of C5 and HTRA1. The present invention also 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 also relates to a pharmaceutical composition or a drug combination comprising the antibody, and the present invention also relates to a method and use for treating a disease based on the antibody.

Background of the Invention

The complement system is an important component of the body's natural immunity. When activated, it causes target cell lysis and promotes phagocytosis through opsonization. Complement is activated through three main pathways through 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 focus on the proteolytic cleavage of the complement component 5 (C5) protein. The cleavage of complement component 5 (C5) leads to the production of fragments C5a and C5b, which is crucial in 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 oxidative bursts, 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 stage 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.

Serine protease HtrA1 Serine peptidase 1 (HtrA1) (PRSS11; 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 leading to increased TGFβ signaling. Dysregulated TGFβ signaling via aberrant HtrA1 expression may also contribute to arthritic disease in conjunction with HtrA1-mediated degradation of multiple extracellular matrix components or indirectly via 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.

The existing technology has developed antibodies against C5, such as eculizumab. However, in the in vitro hemolysis inhibition activity test, although eculizumab showed good classical pathway hemolysis inhibition effect, its activity in blocking the complement alternative pathway (AP) was insufficient, resulting in its efficacy not being able to fully meet the needs of patients. For example, some PNH patients who have undergone eculizumab (Soliris) treatment are still unable to get rid of blood transfusion dependence due to extravascular hemolysis.

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

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.

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

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.

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.

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.

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

(1) Blocking human C5 and human HTRA1 simultaneously;

(2) having a stronger complement pathway blocking pathway, for example, being able to completely block the complement alternative pathway;

(3) It showed superior therapeutic effects compared with single-target drugs in the sodium iodate-induced geographic atrophy model;

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

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

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

three complementarity determining regions (CDRs) contained in the VHH represented by 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 embodiment 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, and 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 shown in SEQ ID NO:1, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:2, and VHH CDR3 comprises or consists of the amino acid sequence shown 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 specifically binding to C5 according to embodiment 1, 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: 23, 17 or 22; or

(ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 23, 17 or 22.

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

three complementarity determining regions (CDRs) contained in the VHH represented by 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 embodiment 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 shown in SEQ ID NO:4, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:7 or 8, and 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 shown in SEQ ID NO:4, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:5, and VHH CDR3 comprises or consists of the amino acid sequence shown in 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 specifically binding to C5 according to embodiment 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 NO: 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, which comprises the VHH antibody described in any one of embodiments 1-6.

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 1-6 connected to an antibody constant region or Fc region.

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

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 embodiment 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 shown in SEQ ID NO:11, VHH CDR2 comprises or consists of the amino acid sequence shown in 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 shown in SEQ ID NO:11 or 14, VHH CDR2 comprises or consists of the amino acid sequence shown in 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 specifically binding to HTRA1 according to embodiment 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 the 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 any one of embodiments 9-11.

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

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.

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.

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.

17. The antibody specifically binding to C5 according to embodiment 16, comprising 1 or 2 VHH antibodies specifically binding to C5 or heavy chain antibodies comprising said VHH antibodies according to any one of embodiments 1-3, and 1 or 2 VHH antibodies specifically binding to C5 or heavy chain antibodies comprising said VHH antibodies according to any one of embodiments 4-6; preferably, it comprises 1 or 2 VHH antibodies specifically binding to C5 according to any one of embodiments 4-6, and 1 VHH antibody specifically binding to C5 according to any one of embodiments 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 embodiments 4 to 6 - the VHH antibody that specifically binds to C5 according to any one of embodiments 1 to 3,

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

(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;

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 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.

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.

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.

21. The multispecific antibody of embodiment 20, which further comprises 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.

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 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.

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, and optionally the VHHs are connected by linkers.

25. The bispecific antibody of embodiment 24, wherein the linker comprises (G) _n , wherein n=5-10, such as 5, 6, 7, 8, 9, 10.

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.

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.

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, another 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.

29. The bispecific antibody of any one of embodiments 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 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.

30. The bispecific antibody of embodiment 29 comprises or consists 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.

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

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

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 CHO cells, 293 cells, such as Expi293 cells.

34. A method for preparing the antibody of any one of embodiments 1-30, the method 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.

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

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

37. Drug combination products containing

The antibody of any one of embodiments 1-30 or the immunoconjugate of embodiment 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 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.

39. The method of embodiment 38, wherein the disease or condition refers to a disease or condition in which the subject has (e.g., elevated levels, such as nucleic acid or protein levels) complement C5 protein and/or HTRA1 protein (e.g., compared to healthy subjects) or a sample of the subject, such as a body fluid, 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 described in any one of embodiments 1-30 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of the anti-C5 VHH antibody blocking hemolysis induced by the classical complement pathway.

Figure 2 shows the results of the anti-C5 VHH antibody blocking the hemolysis induced by the alternative complement pathway.

Figure 3 shows the results of anti-C5 VHH antibodies blocking the production of C5a in the classical pathway of complement (C5a ELISA).

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

Figure 5 shows the results of anti-HTRA1 VHH antibody blocking the H2-Opt assay.

Figure 6 shows the results of the anti-HTRA1 VHH antibody blocking Elastase assay.

Figure 7 shows the results of the humanized anti-C5 VHH antibody blocking the hemolysis induced by the classical complement pathway and the hemolysis induced by the alternative complement pathway.

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

Figure 9 shows the results of the affinity matured anti-HTRA1 VHH antibody blocking Elastase assay.

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

FIG. 11 shows an exemplary structural schematic diagram of a bispecific antibody.

FIG. 12 shows the results of an experiment in which bispecific antibody molecules blocked hemolysis induced by the classical complement pathway.

FIG. 13 shows the results of an experiment in which bispecific antibody molecules blocked hemolysis induced by the alternative complement pathway.

Figure 14 shows the results of the H2-Opt assay blocked by the bispecific antibody molecule.

Figure 15 shows the results of the Elastase blocking assay using a bispecific antibody molecule.

FIG. 16 shows the results of an in vivo efficacy test of bispecific antibody molecules blocking sodium iodate-induced geographic atrophy.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

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.

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.

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.

The term "about" when used in conjunction with a numerical value is meant to encompass a range of numerical values 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.

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

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

As used herein, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise indicated, it also covers the situation consisting of the stated elements, integers or steps. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

The term "HTRA1" as used herein refers to the serine protease HtrA1 serine peptidase 1 (UniProtKB/Swiss-Prot: Q92743). The terms "anti-HTRA1 antibody" and "antibody that specifically binds to HTRA1" or "antibody that specifically binds to HTRA1" refer to antibodies that are capable of binding to HTRA1 with sufficient affinity. In one embodiment, the anti-HTRA1 antibody of the present invention is capable of specifically binding to HTRA1 protein, or has a high binding affinity to a protein that expresses HTRA1.

As used herein, the terms "C5" or "C5 protein" or "complement C5" are used interchangeably and refer to the complement component C5 protein of the complement system (UniProtKB/Swiss-Prot: P01031). The terms "anti-C5 antibody" and "antibody that specifically binds to C5" or "antibody that specifically binds to C5" refer to antibodies that are capable of binding to C5 with sufficient affinity. In one embodiment, the anti-C5 antibody of the present invention is capable of specifically binding to the C5 protein, or has a high binding affinity to a protein that expresses C5.

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 C5 and HTRA1 at the same time.

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

The bispecific antibodies of the present invention may include a linker. As used herein, the term "linker" refers to any molecule that enables 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, polyoxyalkylene 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 interfere with desired activity. The peptide linker may 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), wherein 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 shown in SEQ ID NO:26.

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 natural immunoglobulin "Fc domain" comprises two or three constant domains, i.e., a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a natural 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 comprises 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 specified herein, the amino acid residues in the Fc region or the 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) of the Fc region may or may not be present. Two Fc regions may dimerize to form a dimeric Fc, and two different Fcs heterodimerize to form a heterodimeric Fc. Herein, 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 as well as the heavy chain constant region CH1 and the 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.

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 operably linked. Such vectors are referred to herein as "expression vectors."

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

The term "VHH" or "VHH antibody" can be used interchangeably herein and generally refers to an antibody that contains or consists of only one heavy chain variable region and has the activity of binding to an antigen. VHH generally contains three CDRs and four highly conserved framework regions, and generally has 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 complementary determining regions 1-3. The CDR sequence in the VHH variable region can be determined according to any CDR definition scheme described in the "Definition" section, and preferably the boundaries of the three CDRs in the variable region sequence can be defined by IMGT. VHH generally includes only a heavy chain variable domain derived from a heavy chain antibody lacking a light chain, also known as a nanobody. The VHH used in the present invention is preferably from a camelid, such as an alpaca, or a humanized form or sequence optimized form thereof (e.g., an affinity mature form 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, a heavy chain variable region of a heavy chain antibody).

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

The "heavy chain antibody" described in the present invention refers to an antibody without a light chain, for example, it may contain VH-Fc or VH-CH2-CH3 or VH-hinge region-CH2-CH3 from the N segment to the C segment, or may contain VH-CH1-CH2-CH3. The heavy chain antibody of the present invention may also cover homodimers, such as heavy chain dimer antibodies without light chains. The heavy chain antibody may contain VH from a standard antibody or VH from a single domain antibody. For example, the VH in the 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 containing at least part of the constant region). The heavy chain antibody of the present invention also covers 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).

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

The term "target" refers to the object to which a binding molecule is directed. A target can be an antigen, or a ligand or a receptor. The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. The skilled person will appreciate that any macromolecule, including essentially all proteins or peptides, can be used 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. When the binding molecule of the present invention relates to a target binding region derived from an antibody, "target" and "antigen" can be used interchangeably.

The term "target binding region" as used herein 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 remainder 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".

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 a specific target or antigen. The antigen binding region may 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. When the binding molecule of the present invention relates to a target binding region derived from an antibody, "target binding region" and "antigen binding region" may be used interchangeably. In one embodiment, the antigen binding region for the multispecific antibody molecule of the present invention may comprise only VH, such as from VHH, such as VHH.

The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, such as 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.

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

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.

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 it 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.

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, wherein each domain comprises four conserved framework regions (FRs) and three complementarity determining regions.

"Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms a structurally determined loop ("hypervariable loop") and/or contains antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in 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 many well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of the antibody 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 the variability of antibody sequences (Kabat et al., Sequen ces of Proteins of Immunological Interest, 4th Edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the 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.

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

HCDR1 was defined according to AbM rules;

HCDR2 is defined according to the Kabat convention; and

HCDR3 was defined according to AbM rules.

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 rest of the antibody with their human counterparts (i.e., replacing the portion of the variable region that does not participate in binding with the corresponding portion of a human antibody).

As used herein, the terms "anti," "binding," or "specific binding" mean that the binding is selective for a 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).

"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 be generally represented by a dissociation constant ( _KD ), which is the ratio of the dissociation rate constant and the association rate constant ( _Kdis and _Kon , respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

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.

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.

The term "treat," ...

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

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 treating an ocular disease.

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

The term "pharmaceutical combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) antibodies of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without specific time limits, or at the same or different time intervals, in a separate entity, wherein such administration provides two or more active agents at a preventive or therapeutically effective level in the patient. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and/or time interval of the two or more active agents are 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 a separate formulation, which can be the same or different.

The term "pharmaceutical composition" refers to a composition that is in a form that permits 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.

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.

"Subject/patient/individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; body fluids, such as tears, vitreous fluid, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid.

II. Anti-C5 Antibodies

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 an antigen binding region in a multispecific antibody molecule of the present invention.

In some embodiments, the anti-C5 antibodies of the present invention bind to C5 (e.g., human C5 or cynomolgus monkey C5) with a 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, for example, its K _D value is less than or equal to about 2nM, for example, less than or equal to about 5, 4, 3, 2, 1.5, 1, 0.5, 0.4, 0.3, 0.25, 0.2 or 0.15nM. In some embodiments, the K _D value of the binding of the anti-C5 antibodies of the present invention to human C5 and/or cynomolgus monkey C5 is greater than about 0.05nM or about 0.1nM. In some embodiments, the K _D value of the binding of the anti-C5 antibodies of the present invention to human C5 and/or cynomolgus monkey C5 is between any two of the above numerical ranges. In some embodiments, the binding affinity of the anti-C5 antibodies of the present invention is determined by biomembrane interferometry.

In some embodiments, the anti-C5 antibodies of the invention are capable of blocking the classical complement pathway, for example, blocking hemolysis induced by it.

In some embodiments, the anti-C5 antibodies of the invention are capable of blocking the alternative complement pathway, for example, blocking hemolysis induced by it.

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

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

In some embodiments, the anti-C5 antibody of the present invention is a single domain antibody, in particular a VHH antibody. In some embodiments, the anti-C5 antibody of the present invention is a VHH-His antibody with a His tag (eg, 6-His) at the N-terminus or C-terminus, such as a VHH-6His antibody.

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, and 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 "Definition" section, and 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 AbM.

In some embodiments, the anti-C5 VHH antibody of the invention comprises

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

(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);

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.

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

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

(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);

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.

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 of the present invention comprises or consists of a heavy chain variable region comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3.

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

In some embodiments, VHH CDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 2, 5, 7, 8 or 9, or VHH CDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 2, 5, 7, 8 or 9.

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

In one embodiment, the anti-C5 VHH antibody of the present invention comprises 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:4, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:5 or 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6 or 30; or

(ii) 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, and VHH CDR3 comprises or consists of the amino acid sequence shown in 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:7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6; or

(iv) 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; or

(v) 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, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:3; or

(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.

In one embodiment, the anti-C5 VHH antibody of the invention comprises or consists of a heavy chain variable region, wherein the heavy chain variable region comprises 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:4, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:5 or 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6 or 30; or

(ii) 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, and VHH CDR3 comprises or consists of the amino acid sequence shown in 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:7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6; or

(iv) 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; or

(v) 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, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:3; or

(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.

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

(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

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

(iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from any one of SEQ ID NOs: 17-23, and preferably, the amino acid changes do not occur in the CDR region.

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 NO:17-23.

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 of the heavy chain VH of a conventional human antibody. Methods for humanizing VHH 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. 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.

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

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

① Determine the CDR loop structure;

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

③Construct the CDR region of VHH onto the human framework region;

④Use sequence and structural features to determine the amino acid positions in the framework region that maintain the CDR function;

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

⑥Optimize amino acids at risk sites.

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.

In one embodiment, the different VHHs are linked via N-terminus and N-terminus, C-terminus and C-terminus, or N-terminus and C-terminus.

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.

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

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

(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

(3) VHH1 described herein that specifically binds to C5 - VHH2 described herein that specifically binds to C5 - VHH1 described herein that specifically binds to C5,

Optionally, the VHHs are connected via a linker.

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.

(i) comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein 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 or 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6 or 30;

(ii) 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, and VHH CDR3 comprises or consists of the amino acid sequence shown in 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:7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6;

or

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

VHH2 that specifically binds to C5

(i) contains the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, where

(a) 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; or

(b) 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, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:3; or

(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.

(ii) comprising a heavy chain variable region comprising, or consisting of, an amino acid sequence as shown in any one of SEQ ID NO: 17, 22 or 23, or an amino acid sequence having at least 90% identity thereto;

Or vice versa.

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

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

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

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.

III. Anti-HTRA1 Antibodies

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.

In some embodiments, the anti-HTRA1 antibodies of the present invention bind to HTRA1 (e.g., human HTRA1 or cynomolgus monkey HTRA1) with a 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, for example, its K _D value is less than or equal to about 3nM, for example, less than or equal to about 5, 4, 3, 2, 1 or 0.9nM. In some embodiments, the K _D value of the binding of the anti-HTRA1 antibodies of the present invention to human HTRA1 and/or cynomolgus monkey HTRA1 is greater than about 0.1nM or about 0.5nM. In some embodiments, the K _D value of the binding of the anti-HTRA1 antibodies of the present invention to human HTRA1 and/or cynomolgus monkey HTRA1 is between any two of the above numerical ranges. In some embodiments, the binding affinity of the anti-C5 antibodies of the present invention is determined by biomembrane interferometry.

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

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

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, and 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 sequence in the VHH variable region can be determined according to any CDR definition scheme described in the "Definition" section, and preferably, the boundaries of the three CDRs in the VHH sequence can be defined by IMGT.

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

(i) three complementarity determining regions (CDRs) contained in the VH set forth in SEQ ID NO: 24 or 25, or

(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);

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.

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

(i) three complementarity determining regions (CDRs) contained in the VH set forth in SEQ ID NO: 24 or 25, or

(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);

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.

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 of the present invention comprises or consists of a heavy chain variable region comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3.

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

In some embodiments, VHH CDR2 comprises the amino acid sequence of SEQ ID NO: 12 or 15, or consists of the amino acid sequence, or VHH CDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 12 or 15.

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

In one embodiment, the anti-HTRA1 VHH antibody of the present invention comprises 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:11, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:12, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:13; or

(ii) 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

(iii) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:11 or 14, VHH CDR2 comprises or consists of the amino acid sequence shown in 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.

In one embodiment, the anti-HTRA1 VHH antibody of the invention comprises or consists of a heavy chain variable region, wherein the heavy chain variable region comprises 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:11, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:12, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:13; or

(ii) 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;

(iii) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:11 or 14, VHH CDR2 comprises or consists of the amino acid sequence shown in 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.

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

(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 selected from SEQ ID NO: 24 or 25; or

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

(iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 24 or 25, and preferably, the amino acid changes do not occur in the CDR region.

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.

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 region sequences, in a non-human natural VHH sequence (e.g., a VHH sequence from camelids or alpacas immunization) with residues at corresponding positions of the heavy chain VH of a conventional human antibody. Methods for humanizing VHH 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. 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.

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

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

① Determine the CDR loop structure;

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

③Construct the CDR region of VHH onto the human framework region;

④Use sequence and structural features to determine the amino acid positions in the framework region that maintain the CDR function;

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

⑥Optimize amino acids at risk sites.

IV. Anti-C5 Heavy Chain Antibody or Anti-HTRA1 Heavy Chain Antibody

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.

In some embodiments, the single domain antibody or VHH of the present invention (e.g., camel-derived VHH or humanized form thereof) can be connected to the 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 connected 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.

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.

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.

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.

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, such as a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region, preferably a human IgG1 or human IgG4 Fc region.

In one embodiment, the heavy chain antibody according to the present invention can dimerize with another polypeptide chain (e.g., the same or different another heavy chain antibody) comprising the 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 includes a heavy chain antibody formed by pairing two identical heavy chain antibody chains.

In some embodiments, the anti-C5 antibody of the present invention comprises a heavy chain comprising a heavy chain variable region and an Fc region. In some embodiments, the anti-C5 antibody of the present invention comprises or consists of a heavy chain comprising or consists of a heavy chain variable region and an Fc region of an anti-C5 VHH of the present invention.

In some embodiments, the anti-HTRA1 antibody of the present invention comprises a heavy chain comprising a heavy chain variable region and an Fc region. In some embodiments, the anti-HTRA1 antibody of the present invention comprises or consists of a heavy chain comprising or consists of a heavy chain variable region and an Fc region of an anti-HTRA1 VHH of the present invention.

V. Multispecific Antibodies

In some embodiments, the present invention provides a multispecific antibody, such as a bispecific antibody, which specifically binds to C5, and optionally 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 other antigen binding regions, such as an antigen binding region that specifically binds to HTRA1.

In some embodiments, the present invention provides a multispecific antibody, such as a bispecific antibody, which specifically binds to HTRA1, and optionally other antigens such as C5. In some embodiments, the multispecific antibody comprises an antigen binding region that specifically binds to HTRA1 (such as 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 other antigen binding regions, such as an antigen binding region that specifically binds to C5.

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.

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.

In some embodiments, the antigen binding region that specifically binds C5 is from an anti-C5 antibody, such as an anti-C5 antibody described herein, such as an anti-C5 VHH described herein. In some embodiments, the two different antigen binding regions that specifically bind C5 bind to different C5 epitopes. In some embodiments, the two different antigen binding regions that specifically bind C5 are two different anti-C5 VHH described herein.

In some embodiments, the antigen binding region that specifically binds to HTRA1 is from an anti-HTRA1 antibody, such as an anti-HTRA1 antibody described herein, such as an anti-HTRA1 VHH described herein.

In some embodiments, the bispecific antibody 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 that bind to different C5 epitopes and one antigen binding region that specifically binds to HTRA1.

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 connected in series. In some embodiments, the VHHs of the bispecific antibodies of the present invention are connected by a linker or not. In some embodiments, the VHHs of the bispecific antibodies of the present invention are all connected by a linker.

In some embodiments, in the bispecific antibodies of the present 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.

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

VHH1 that specifically binds to C5 - VHH2 that specifically binds to C5 - VHH that specifically binds to HTRA1,

The VHHs are connected with or without a linker.

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.

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

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

The VHHs are connected via a linker.

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, a VHH1 that specifically binds to C5

(i) contains the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, where

(a) 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 or 7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6 or 30; or

(b) 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, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6 or 30; or

(c) 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:7 or 8, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6;

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

VHH2 that specifically binds to C5

(i) contains the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, where

(a) 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; or

(b) 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, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:3; or

(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.

(ii) comprising a heavy chain variable region comprising, or consisting of, the amino acid sequence of any one of SEQ ID NO: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;

Or vice versa.

In some embodiments, a VHH3 that specifically binds to HTRA1

(i) contains the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, where

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

(b) 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

(c) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11 or 14, VHH CDR2 comprises or consists of the amino acid sequence shown in 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;

(ii) comprises a heavy chain variable region comprising, or consisting of, the amino acid sequence shown in 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.

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

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

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

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.

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.

In some embodiments, the bispecific antibody of the present invention comprises a chain comprising 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.

In some embodiments, the bispecific antibody of the present invention 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.

VI. Polynucleotides, Vectors and Hosts

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, such as 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, such as (HEK 293 or 293F cells or 293FT cells or Expi293 cells). In another embodiment, the host cell is prokaryotic.

In one aspect, the present invention provides a nucleic acid encoding any of the above anti-C5 antibodies or anti-HTRA1 antibodies or bispecific antibodies.

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

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.

In some embodiments, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID 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.

Nucleic acid sequences encoding molecules of the invention can be generated using methods well known in the art, such as by de novo solid phase DNA synthesis, or by PCR amplification.

VII. Vectors and Host Cells

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. The vector includes but is not limited to a virus, a plasmid, a cosmid, a lambda phage or a yeast artificial chromosome (YAC). In a preferred embodiment, the expression vector is pCDNA, such as pCDNA3.1.

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 line (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 cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, 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.

VIII. Production and Purification of the Molecules of the Invention

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

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. Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). Once an expression vector comprising one or more polynucleotides of the present invention for expression has been prepared, 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.

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

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.

IX. Assay

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 a variety of assays known in the art.

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

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

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

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

The effect of the anti-HTRA1 antibody or bispecific antibody of the present invention in blocking HTRA1 enzyme activity (such as 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.

X. Immunoconjugates

In one aspect, the present invention provides an immunoconjugate comprising a molecule of the present 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 present invention, such as a small molecule that enhances the therapeutic effect of the molecule of the present invention).

XI. Pharmaceutical Compositions

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

In one embodiment, the composition further comprises a pharmaceutical excipient, such as a pharmaceutical carrier, a pharmaceutical excipient, including a buffer, known in the art.

As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, isotonic agents and absorption delaying agents that are physiologically compatible. For the use of pharmaceutical excipients and their uses, see also "Handbook of Pharmaceutical Excipients", 8th edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

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.

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

The composition or medicine or preparation 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 composition or medicine or preparation of the present invention is suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion). The preferred form depends on the expected mode of administration and therapeutic use.

XII. Pharmaceutical Combinations and Kits

The present invention also provides a pharmaceutical combination or a pharmaceutical combination product, which comprises the 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.

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

- 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);

- 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).

XIII. Use and Methods

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 or a drug or a preparation comprising the same.

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 a bispecific antibody or an immunoconjugate thereof, etc.), or a composition or a medicament or a preparation comprising the same.

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 an immunoconjugate thereof, etc.), or a composition or medicament or formulation comprising the same.

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 preparation comprising the same.

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.

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 generation of C5a.

In some embodiments, the complement-related disease or disorder may 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.

In some embodiments, the HTRA1-related disease or condition has (e.g., elevated levels, such as nucleic acid or protein levels) HTRA1 in the subject (e.g., compared to healthy subjects). In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels) HTRA1 in a biological sample (e.g., compared to a biological sample of a healthy subject). In some embodiments, the HTRA1-related disease or condition treatment would benefit from inhibition of nucleic acid or protein levels of HTRA1.

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

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

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

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.

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 purposes described herein, e.g., for preventing and/or treating the relevant diseases or disorders mentioned herein.

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.

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 disorders mentioned herein.

In other aspects, the present invention also provides the 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.

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.

XIIII. Diagnosis and testing

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.

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.

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 an 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 against complement C5 (e.g., an antibody against complement C5, such as an anti-C5 antibody or bispecific antibody or an immunoconjugate thereof of the invention, etc.), for example, wherein complement C5 is a biomarker for selecting the subject.

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 an immunoconjugate thereof) under conditions that allow it to bind to HTRA1, and detecting whether a complex is formed 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 an immunoconjugate thereof of the invention, etc.), for example, wherein or HTRA1 is a biomarker for selecting the subject.

In certain embodiments, the method comprises contacting a biological sample with a molecule as described herein (e.g., a bispecific antibody or an immunoconjugate thereof) under conditions that allow it to bind 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 against 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 a bispecific antibody or an immunoconjugate thereof of the invention, etc.), for example, wherein complement C5 and/or HTRA1 is a biomarker for selecting the subject.

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

In certain embodiments, a labeled molecule of the invention (e.g., an anti-C5 antibody or anti-HTRA1 antibody or bispecific antibody or 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 indirectly detected moieties, such as enzymes or ligands, e.g., by enzymatic reactions or molecular interactions.

In some embodiments, the label is a label such as biotin or a His tag.

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

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.

In some embodiments, a method for 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 with 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.

Any or all of the 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

Example 1. Preparation and panning of C5 phage display library

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

1.2 Collect 50 ml of alpaca peripheral blood, separate lymphocytes, add 1 mL of Trizol reagent (Thermo) for every 2.5×10 ⁷ living cells, and extract total RNA by chloroform/isopropanol precipitation method. Take 10 μg RNA as a template and use PrimeScript reverse transcription kit (Takara) for reverse transcription. Use cDNA as a template for the first round of PCR reaction, use forward primer Alp-VhL and reverse primer Alp-2b/2cR to obtain the first round of PCR products. Use the first round of PCR products as templates for the second round of PCR reaction, use forward primer Alp-VhF and reverse primer Alp-JHR-SalI to obtain the second round of PCR products. The pC3-HF vector and the second round of PCR products were double-digested with SacI and SalI (Thermo), respectively, and the digested products were added with T4 ligase (Thermo) for ligation reaction. The ligation products were electrotransformed into TG1 competent cells to construct the VHH antibody immune library.

The above library bacterial solution was inoculated into 100 ml YT-AG medium (Shanghai Bioengineering Co., Ltd.) and cultured to the logarithmic growth phase. M13KO7 helper phage was added for infection. The bacteria were resuspended in 2×YT-AK medium (Shanghai Bioengineering Co., Ltd.) and cultured overnight at 30°C and 200 rpm. The culture supernatant was collected and the recombinant phage was prepared by PEG/NaCl precipitation method.

1.3 The recombinant phage was subjected to 2-3 rounds of panning experiments using the 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, incubated at room temperature for 30 minutes, and then 1x10 ¹² cfu recombinant phage was added and incubated at room temperature for 1 hour. The obtained mixture was added with 1 ml 0.1%-0.05% PBST and washed 10-15 times, and finally 0.5 ml pH2.5 glycine buffer was added to elute the antigen-bound recombinant phage, and the logarithmic growth phase TG1 was infected and coated on the YT-AG plate for overnight culture. At the same time, a small amount of the infected bacterial solution was taken and gradiently diluted and coated on the YT-AG plate to form a single clone on the plate for the Binding ELISA test in 1.4. The next day, the bacterial lawn was scraped off the plate and recombinant phages were prepared according to the recombinant phage preparation method in 1.2 for the next round of panning experiments.

1.4 Binding ELISA to detect binding activity and clone sequencing.

Take the monoclonal plate in 1.3, pick a single clone and inoculate it into a 96-deep-well plate containing YT-A, culture it until OD600 is about 0.5, add IPTG with a final concentration of 1mM at 30℃ to induce expression overnight. Add polymyxin to the culture supernatant and incubate at 37℃ for 30min. After centrifugation, the supernatant is used for binding ELISA detection.

Take C5 antigen (ACRO, Beijing) and dilute it to 0.5 μg/ml with PBS buffer to coat 96-well ELISA plate. Wash the antigen-coated plate 3 times with PBST, add blocking agent to 300 μL/well, and stand at room temperature for 1 hour. Wash 3 times with PBST, add 80 μL blocking agent + 20 μL bacterial expression supernatant, and incubate at room temperature for 1 hour.

Wash 3 times with PBST, add 100 μL/well of Anti-Flag/HRP secondary antibody (Sigma) diluted with blocking agent, and incubate at room temperature for 1 hour. Wash 6 times with PBST, add 100 μL/well of TMB colorimetric solution, and color for 5-15 minutes in the dark. Then add 100 μL/well of stop solution. Read the results with an ELISA reader, measure the OD450nm absorbance, and select bacterial clones with a reading greater than 0.5 and send them to Genewise for sequencing.

Example 2. Panning of HTRA1 phage display library

2.1 Use our own VHH synthetic library and the customized HTRA1 recombinant antigen (UniProtKB/Swiss-Prot: Q92743) of Beijing Sino Company for 2-3 rounds of solid phase panning experiments. The specific experimental method is to add 30pmoL HTRA1 recombinant antigen to each ELISA well and coat it at 4℃ overnight. Wash the well plate 3 times with PBST and add blocking solution to block at room temperature for 1h. Add 5x10 ¹¹ cfu recombinant phage to each ELISA well and incubate at room temperature for 1 hour. After the incubation, wash 10-15 times with 0.1%-0.05% PBST, and each wash time is 3-5 minutes. Finally, elute the antigen-bound recombinant phage with pH2.5 glycine buffer, infect TG1 in the logarithmic growth phase and spread it on YT-AG plate for overnight culture. At the same time, take a small amount of infected bacterial solution, spread it on YT-AG plate after gradient dilution to form a single clone on the plate, which is used for Binding in 2.2 ELISA test. The next day, the bacterial lawn was scraped off the plate and recombinant phages were prepared according to the recombinant phage preparation method in 1.2 for the next round of panning experiments.

2.2 Binding ELISA to detect binding activity and clone sequencing.

Take the monoclonal plate in 2.1, pick a single clone and inoculate it into a 96-deep-well plate containing YT-A, culture it until OD600 is about 0.5, add IPTG at a final concentration of 1mM at 30℃ to induce expression overnight. Add polymyxin to the culture supernatant and incubate at 37℃ for 30min. After centrifugation, the supernatant is used for binding ELISA detection.

Take HTRA1 antigen (customized by Beijing Sino Company) and dilute it to 0.5μg/ml with PBS buffer to coat 96-well ELISA plate, and keep it in a refrigerator at 4℃ overnight. Wash the antigen-coated plate 3 times with PBST, add blocking agent to 300μL/well, and stand at room temperature for 1 hour. Wash 3 times with PBST, add 80μL blocking agent + 20μL expression supernatant of the above TG1 monoclonal clone, and incubate at room temperature for 1 hour.

Wash 3 times with PBST, add 100 μL/well of Anti-Flag/HRP secondary antibody (Sigma) diluted with blocking agent, and incubate at room temperature for 40 minutes. Wash 6 times with PBST, add TMB colorimetric solution to 100 μL/well, and color for 5-15 minutes in the dark. Then add 100 μL/well stop solution. Read the results with an ELISA reader, measure the OD450nm absorbance, and select bacterial clones with a reading greater than 0.5 for sequencing by Genewise.

Example 3. Eukaryotic expression and purification of candidate antibodies

The present invention uses molecular biological technology to amplify the VHH antibody sequence in the anti-C5 or HTRA1 positive phage obtained in Example 1 by PCR and insert it into pCDNA3.1 with a His tag (HHHHHH), transfect HEK-293 (Thermo Fisher Scientific), express and purify it, and finally obtain a VHH-His recombinant antibody with a 6XHis tag at the C-terminus. The VHH antibodies individually verified below are all VHH-His recombinant antibodies.

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.

For 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, please refer to the sequence listing for the specific sequence numbers.

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

The equilibrium dissociation constant (KD) of the antibody of the present invention binding to human C5 or HTRA1 was determined by biofilm thin layer interferometry (BLI). The experimental detection used a ForteBio Octet Red96e instrument, and the affinity determination was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p. 270-8).

Half an hour before the experiment, according to the number of samples, take an appropriate number of SA sensors (18-5019, Satorius) and immerse them in SD buffer (PBS 1×, BSA 0.1%, Tween-20 0.05%).

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 values.

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

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

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

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; the monovalent affinity KD values for human C5 (R885H) were also 1.33E-10M and 1.83E-09M, respectively.

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

Example 5 Anti-C5 VHH antibody blocks hemolysis induced by the classical complement pathway (CH50)

Sensitized sheep red blood cells (immune complexes) can activate the classical complement pathway, resulting in the formation of transmembrane pores on the surface of red blood cells, allowing extracellular water to penetrate, causing red blood cell 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 red blood cells to verify the blocking effect of anti-C5 VHH antibodies on the classical complement pathway.

Weigh 0.825g of anhydrous calcium chloride, 5g of magnesium chloride, hexahydrate, dissolve in 50mL GVB buffer to obtain 1000X calcium and magnesium ion solution, and store at 4℃; add 500μL 1000X calcium and magnesium ion solution to 500mL GVB buffer to obtain gelatin Veronal (GVB) buffer; take 1 human serum complement dry powder (Sigma), add 1mL ultrapure water to dissolve, then add 9mL gelatin Veronal buffer to dilute, and store at 4℃ for use. Use a pipette to draw 10mL of sheep red blood cells (Kejing Bio) into a 50mL centrifuge tube, add 30mL 1XPBS, 500g, 4℃, centrifuge for 10min, and discard the supernatant. Repeat washing until the supernatant is clear. Add 5mL 1XPBS to the centrifuged red blood cells, gently blow and evenly dilute 10,000 times, take 10μL of the diluted red blood cells and mix with 10μL Trypan Blue Stain (0.4%), and count with a cell counting plate. Use 1XPBS to dilute the red blood cells to 1X10E9 cells/mL, and dilute the hemolysin 1000 times with 1XPBS. Mix the two in equal volumes and place them in a 37℃ carbon dioxide shaker at 120r/min for 45min. Then place them on ice and ice bath for 45min. Centrifuge the red blood cells at 500g, 4℃ for 10min, discard the supernatant, add 1XPBS to 40mL, 500g, 4℃, centrifuge for 10min, discard the supernatant, and repeat washing twice. Finally, resuspend the sensitized sheep red blood cells with 5mL of 1XPBS. Dilute the sensitized red blood cells 10,000 times, take 10 μL of the diluted red blood cells and mix with 10 μL Trypan Blue Stain (0.4%), and count them using a cell counting plate. Dilute the red blood cells to 1X10E8 cells/mL with gelatin Veronal buffer and place on ice for later use. Use gelatin Veronal buffer to dilute the antibodies to be tested (test samples: Control: IgG1 antibody; ARC1905 (Beixin Biotechnology); VHH antibody 1 and VHH antibody 293) in a gradient manner, with a starting concentration of 200 nM and a 3-fold gradient dilution. Take human complement serum and dilute it 5 times with gelatin Veronal buffer.

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

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;

Negative control group (data not shown), each well is the same as the positive control group, but no subsequent contact reaction with sheep red blood cells. 37℃ shaker, 120r/min, 45min.

Add sensitized sheep red blood cells at a concentration of 1X10E8 cells/mL, 50μL per well. Shake at 37℃, 120r/min, 30min, pipette evenly and put back, shake at 37℃, 120r/min, 30min. Pipet evenly, 1000g, 15℃, centrifuge for 3min. Aspirate 100μL of supernatant into 96-well plate, and read OD405nm on microplate reader.

The calculated red blood cell hemolysis inhibition rate is as follows:

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%

The blocking results of the two anti-C5 VHH antibodies obtained in the present invention are shown in Figure 1. The candidate molecule 1,29 antibody is able to block the classical complement pathway.

Example 6 Anti-C5 VHH antibody blocks hemolysis induced by the alternative complement pathway (ACH50)

When the complement alternative pathway (AP) is activated, C1, C2, and C4 are not involved, but C3, C5-C9, and factors B, D, P, H, and I are involved. Using EGTA to chelate Ca2+ in the sample can block C1 and block the activation of the traditional complement pathway. Adding rabbit red blood cells that can activate factor B activates the complement alternative pathway, which leads to hemolysis of rabbit red blood cells. Based on the above principle, this experiment reacts the test samples with different concentration gradients with complement and MgEGTA, and then adds rabbit red blood cells to observe the hemolytic reaction to verify the blocking effect of anti-C5 VHH antibodies on the complement alternative pathway.

Take 5mL of rabbit peripheral blood (Suzhou Huqiao Biological) in a 50mL centrifuge tube, add 35mL PBS, 400g, 4℃, centrifuge for 10min, discard the supernatant, and repeat washing until the supernatant is clear. Resuspend the rabbit red blood cells with 5mL PBS, dilute 2000 times, take 10μL of the diluted rabbit red blood cells and mix with 10μL Trypan Blue Stain (0.4%), and count them under an inverted microscope with a hemocytometer. Place the rabbit red blood cells on ice for later use. Take 2 human serum complement dry powders, add 4mL GVB buffer, and shake gently to completely dissolve. Use GVB buffer to dilute each sample to be tested (test samples: Control: IgG1 antibody; ARC1905; VHH antibody 1 and VHH antibody 293) in a gradient manner, with a starting concentration of 6000nM and a 3-fold gradient dilution.

According to the arrangement order of the 96-well plate, add 25μL human serum complement, 75μL sample, 40μL GVB buffer, and 10μL 0.1mol/L MgEGTA to each well in turn.

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

Negative control group (data not shown): Same as the positive control group, but no subsequent contact reaction with rabbit red blood cells. 37℃ constant temperature shaker, 120r/min, 45min. Take rabbit red blood cells and dilute them with GVB buffer to obtain a suspension with a concentration of 1X10E8 cells/mL, add 50μL to each well; aspirate 200μL of rabbit red blood cells into 3 empty wells.

37℃ constant temperature shaker, 120r/min, 30min, take out and blow and aspirate evenly, put back, 37℃ constant temperature shaker, 120r/min, 30min. Take out and blow and aspirate evenly, 1000g, 15℃, centrifuge for 3min. Pipette 50μL of the supernatant with only rabbit red blood cells into the negative control group and mix well. Pipette 100μL of the supernatant into a new 96-well plate and read the OD405nm on the microplate reader.

The calculated red blood cell hemolysis inhibition rate is as follows:

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%

The blocking results of the two anti-C5 VHH antibodies obtained in the present invention are shown in Figure 2. The candidate molecule 1,293 antibody is able to block the complement alternative pathway.

Example 7 Anti-C5 VHH Antibody Blocks the Production of C5a in the Classical Complement Pathway (C5a ELISA)

In the classical and alternative pathways of complement, C5 reacts with C5 convertase to form C5a and C5b. C5b forms the final MAC complex to lyse cells, while C5a can interact with C5a receptors to participate in biological activities. In this study, the Human C5a ELISA Kit II (BD, Cat. No. 557965) was used to detect the content of C5a in the hemolytic system induced by the classical or alternative pathways of complement, and to verify the biological activity of anti-C5 VHH antibodies in blocking the production of C5a.

Take out the test kit from the 4℃ refrigerator and place it at room temperature for half an hour. Take out the sample to be tested (supernatant obtained in Example 5: CH50 supernatant), and dilute the CH50 supernatant 2 times with Standard/Sample Diluent. Add 50μL ELISA Diluent to the plate coated with the antibody. Add 100μL of sample to each well, seal the film, and incubate at room temperature for 2 hours. Dilute 48μL Enzyme Concentrate with 12mL Detection Antibody. Wash 3 times with 300μL Wash Buffer per well, and put the plate on a paper towel for the last time to remove the residual liquid. Add 100μL Working Detector to each well, seal the film, and incubate at room temperature for 1 hour. Wash 3 times with 300μL Wash Buffer per well, and put the plate on a paper towel for the last time to remove the residual liquid. Add 100μL TMB One-Step Substrate Reagent to each well, protect from light, and incubate at room temperature for 30 minutes. Add 50 μL Stop Solution to each well, tap to mix, and protect from light. Use a multifunctional microplate reader to read the fluorescence value, and set it as follows: Optical Configuration is Monochromator, Read Mode is ABS, Read Type is Endpoint, 2 wavelengths (450nm and 620nm), Plate Type is 96Well Standard clrbtm, Shake is Off, and Read Order is Row.

The blocking results of the two anti-C5 VHH antibodies obtained in the present invention are shown in Figure 3. The candidate molecule 1,293 antibody can block the production of C5a in the classical complement pathway.

Example 8 Anti-C5 VHH Antibody Binding Epitope Analysis

The biofilm thin layer interferometry (BLI) technique was used to determine the epitope differences of the antibody of the present invention binding to human C5 (Beijing ACRO Company) and group them. The experimental detection used the ForteBio Octet Red96e instrument. Half an hour before the start of the experiment, according to the number of samples, a suitable number of SA sensors (18-5019, Satorius) were taken and soaked in SD buffer (PBS 1×, BSA 0.1%, Tween-20 0.05%).

The samples to be tested were divided into first Ab (for saturated antigen) and 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 speed is 1000 rpm, and the temperature is 30°C. The second Ab that competes with the first Ab for binding binds to similar antigen epitopes. The second Ab has no competition or partial competition with the first Ab, and the antigen binding epitopes are different.

The binding epitope results of the two anti-C5 VHH antibodies obtained in the present invention are shown in Figure 4, candidate molecule 1,293, and the epitopes of the antibodies are different.

Example 9 Anti-HTRA1 VHH Antibody Blocks H2-Opt Assay

HTRA1 is a serine protease. The peptide Mca-IRRVSYSF(K-Dnp)K (H2-Opt for short) can be cleaved by HTRA1 to produce a fluorescent product. The rate of product generation can be detected by an ELISA instrument to indicate the active HTRA1 content in the system, which is used to verify the activity of anti-HTRA1 VHH antibody in blocking HTRA1 cleavage of H2-Opt.

Weigh 5.85g sodium chloride, 1.25g CHAPS, and 3.0g Tris base, dissolve them in 400mL ultrapure water, adjust to pH 8.0, and dilute to 500mL to obtain Reaction buffer B, filter with a 0.22μm filter, and store in a 4-degree refrigerator; dissolve 5mg H2-optimal substrate peptide (INNOVAGEN) in 300μL ultrapure water to obtain H2-Opt storage solution (10mM), divide it into portions, and store it in a -40-degree refrigerator; dilute the H2-Opt storage solution to 50μM with Reaction buffer B to obtain H2-Opt working solution;

Dilute recombinant human HTRA1 to 2.016 μg/mL (40 nM) with Reaction buffer B to obtain HTRA1 enzyme working solution. Use Reaction buffer B to dilute the test sample in a gradient manner.

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

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;

The negative control group (used for evaluation experiments, data not shown) was 80 μL Reaction buffer B.

Incubate on ice for 30 min. Add 40 μL H2-Opt working solution to each well. 30°C, Ex/Em=320nm/405nm, collect once every minute for 45 min. Export raw data and slope obtained by linear fitting.

The blocking result of an anti-HTRA1 VHH antibody obtained in the present invention is shown in Figure 5. The candidate molecule SA239 antibody can block the activity of HTRA1 enzymatic cleavage of H2-Opt.

Example 10 Anti-HTRA1 VHH antibody blocks Elastase assay

HTRA1 is a serine protease that can cleave some extracellular matrix components and can exhibit elastase activity when the substrate is elastin. Commercial Kit 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 HTRA1 enzymatic cleavage of elastin.

Move the reagent from minus 30 degrees to room temperature. 10mL Component C + 10mL ultrapure water to obtain 1X Assay buffer, dilute Component A with 1X Assay buffer 100X (prepare and use immediately), and obtain Elastase substrate solution. Dilute recombinant human HTRA1 (i.e., the recombinant human HTRA1 antigen of Example 1) with 1X Assay buffer to 20μg/mL to obtain HTRA1 working solution. Use 1X Assay buffer to dilute the test sample in a gradient manner.

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

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

The negative control group (used to evaluate the experiment and calculate the sample reaction rate, data not shown) was 50 μL 1X Assay buffer. Incubate on ice for 30 min. Add 50 μL Elastase substrate solution to each well. Ex/Em = 490nm/520nm, collect every 2min for a total of 30min. Derive the slope obtained by linear fitting.

The sample reaction rate is calculated as follows:

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

The blocking result of an anti-HTRA1 VHH antibody obtained in the present invention is shown in Figure 6. The candidate molecule SA239 antibody can block the activity of HTRA1 enzymatically cleaving elastin.

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

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.

Please refer to the attached table for 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 the sequence numbers.

Humanization of immune library antibody 1 and 293

The antibody 1,293 obtained in Example 5 and Example 6 was humanized by the following steps:

① Determine the CDR loop structure;

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

③Construct the CDR region of VHH onto the human framework region;

④Use sequence and structural features to determine the amino acid positions in the framework region that maintain the CDR function;

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

⑥Optimize amino acids at risk sites.

The amino acid sequences of the three humanized antibodies obtained in the present invention are shown in the attached sequence table.

The specific sequence numbers are shown in Table 3:

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

Protein expression

Expi-293 cells (Thermo Fisher Scientific) were passaged according to the required transfection volume. The cell density was adjusted to 1.5×10 ⁶ cells/ml the day before transfection. The cell density on the day of transfection was about 3×10 ⁶ cells/ml. Opti-MEM medium (Gibco, 11058021) with 1/10 of the final volume was used as the transfection buffer, and the appropriate plasmid was added and mixed. The appropriate polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid (the mass ratio of plasmid to PEI was 1:3), mixed and incubated at room temperature for 10 minutes, and after adding expi293 cells, cultured at 36.5°C and 8% CO ₂ . After 24 hours, 2% of the transfection volume of FEED (100 g/L Phytone Peptone + 100 g/L Difco Select Phytone), a glucose solution with a final concentration of 4 g/L, and VPA (Gibco, catalog number: 11140-050) with a final concentration of 2 mM/L were added, and cultured at 36.5°C, 120 rpm, and 8% CO2. After continuous culture for 6 days, the culture was collected and centrifuged at 4000 rpm for 30 minutes. The cell supernatant was filtered with a 0.45 μM filter membrane and purified by affinity chromatography.

The specific affinity chromatography purification operation steps are as follows: select Amsphere ^™ A3 (JSR Life Sciences, catalog number: 10000327-C05) affinity chromatography column, remove endotoxin from affinity chromatography column and pipeline with 0.1M NaOH for 1 hour before purification, then wash pipeline and column with distilled water. Equilibrate the packing column with 5 column volumes of 1×PBS (Gibco, catalog number: 10010023); pass the collected supernatant through the column, and then wash the packing column with 10 column volumes of 1×PBS to remove non-specific binding proteins; rinse the packing with 5 column volumes of elution buffer (100mM glycine, pH 2.7), collect the eluate, and then adjust the protein pH to 6.0 with 2M Tris.

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;

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

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 by the present invention can completely inhibit the biological activity of C5.

Example 12 Anti-HTRA1 VHH affinity maturation

The SA239 VHH gene was used as a template to introduce random amino acid mutations into the antigen binding region (CDR) for affinity maturation. Degenerate primers containing NNK codons (Suzhou Jinweizhi Company) and framework region specific primers (Suzhou Jinweizhi Company) were designed and synthesized. The antibody mutant gene library was amplified by overlapping extension PCR (OE-PCR), and the PCR fragments and vectors were digested, connected, and transformed into TG1 competent cells in the same manner as in Example 1 to prepare recombinant phage libraries, and three rounds of phage panning and monoclonal ELISA identification were performed. The candidate monoclones were 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 (the 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).

The specific sequence numbers are shown in Table 4:

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

Example 13 Experiment on blocking complement-induced hemolysis by anti-C5 VHH antibody combinations with different epitopes

In the screening of anti-C5 VHH antibodies, 1 and 293 can completely block CH50 (Figure 1), but cannot completely block ACH50 (Figure 2). In this experiment, anti-C5 VHH antibodies with different epitopes were combined to verify their blocking effect on ACH50.

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

Example 14 Experiment of blocking complement-induced hemolysis by dual-epitope anti-C5 antibodies

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

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

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

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

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

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

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

The blocking effect was verified by complement-induced hemolysis experiment.

The experimental detection methods are as in Examples 5 and 6 in the main text (ARC1905 is from BeiXin Biotechnology), and 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).

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

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 2 anti-C5 VHHs and 1 anti-HTRA1 VHH. On the two anti-C5 VHHs, the present invention has constructed 2 identical anti-C5 VHHs (anti-C5 VHH single epitope bivalent form) and 2 different anti-C5 VHHs (anti-C5 VHH dual epitope form).

Two anti-C5 VHH antibodies (different epitopes) and one anti-HTRA1 VHH antibody were constructed into a bispecific antibody, and the molecular structure is shown in Figure 11.

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

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

The binding kinetics of IAR045-001 molecule and antigen were determined by biofilm thin layer interferometry.

The detection method of this experiment is as 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 Corporation. 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), Dog HTRA1 (sequence reference UniProtKB: A0A8C0RQ32, Q30-P485) proteins were expressed in-house. Human HTRA1 (sequence reference UniProtKB: Q92743, Q23-P480) protein was ordered from Sino Company. The affinity of the antibody is shown in Table 5:

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

In the above experiments, the K _D value of the bispecific antibody molecule IAR045-001 for human C5 is less than 2.84E-10M, the K _D value for human C5 (R885H) is 5.59E-10M, the K _D value for monkey C5 is 1.05E-10M, the K D value for mouse C5 is 4.46E-09M, the K _D value for rat C5 is less than 3.44E-10M, the K _D value for rabbit C5 is 2.08E-10M, it does not bind to dog C5, the K _D value for human HTRA1 is 1.58E-09M, the K _D value for monkey HTRA1 is 1.31E-08M, the K _D value for mouse HTRA1 is 7.75E-08M, it does not bind to rat HTRA1, the K _D value for rabbit HTRA1 is 2.61E-09M, and the K _D value for dog HTRA1 is 2.03E-09M.

The blocking effect was verified by complement-induced hemolysis experiment.

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

Example 15 Bispecific antibody molecules block HTRA1 enzyme activity

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.

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 cleaving H2-Opt ( FIG. 14 ) and the activity of HTRA1 cleaving elastin ( FIG. 15 ).

Example 16. In vivo efficacy test of sodium iodate-induced geographic atrophy

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

Experimental animals: hC5 mice; Gender: male; Age: 6-8 weeks; Source: Shanghai Model Organisms Technology Co., Ltd.

In this experiment, sodium iodate was injected into the tail vein to induce damage to the pigment epithelial cells in the mouse retina, thereby causing phenotypes such as thinning of the outer nuclear layer (ONL) and photoreceptor outer segment (POS) of the retina, and establishing a mouse model similar to the human geographic atrophy phenotype. 28 complement C5 humanized mice (hC5 mice) were selected and divided into 5 groups, namely model control group (4 mice), IgG1 group (6 mice), Galegenimab group (6 mice), ARC1905 (Beixin Bio) group (6 mice), and IAR045-001 group (6 mice). Sodium iodate was given on Day 0, and the model control group was not treated. The test article was injected into the vitreous on Day 0 and Day 7, and the samples were collected on Day 10. The ocular tissue was sectioned and H&E stained to analyze the inhibition of the test article on geographic atrophy. The experimental design is shown in Table 6.

Table 6: Experimental design table

H&E staining histopathological examination

Evaluation indicators:

(1) Thickness of the retinal outer nuclear layer

(2) Thickness of the retinal POS layer

(3) Integrity of the retinal pigment epithelium

RPE integrity statistics at 250um from the optic nerve and 40um in length: brown area/total area*100%

As shown in the results of Figure 16, Figure 16A shows a representative H&E staining of eye tissue in each experimental group, in which the arrows 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 mice in the IgG1 group, with thinner ONL and POS layers than normal mice, and discontinuous RPE; the upper right picture shows the retinal tissue of mice in the Galegenimab group, with thicker ONL and POS layers than the IgG1 group, and more complete RPE; the lower left picture shows the retinal tissue of mice in the ARC1905 group, with thicker ONL and POS layers than the IgG1 group, and more complete RPE; the lower middle picture shows the retinal tissue of mice in the IAR045-001 group, with thicker ONL and POS layers than the IgG1 group, and more complete RPE.

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

FIG16C is a statistical diagram of the thickness of the retinal ONL layer of each group of mice, and 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.

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

In summary, the antibody of the present invention can significantly restore the thinning of the ONL layer, the 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.

Sequence Listing

Claims (40)

A VHH antibody that specifically binds to C5, comprising three complementarity determining regions (CDRs) contained in the VHH represented by 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. The VHH antibody that specifically binds to C5 of 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, and 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 shown in SEQ ID NO:1, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:2, and VHH CDR3 comprises or consists of the amino acid sequence shown 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. The VHH antibody that specifically binds to C5 according to claim 1, 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: 23, 17 or 22; or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 23, 17 or 22. A VHH antibody that specifically binds to C5, comprising three complementarity determining regions (CDRs) contained in the VHH represented by 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. The VHH antibody that specifically binds to C5 of 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 shown in SEQ ID NO:4, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:7 or 8, and 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 shown in SEQ ID NO:4, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:5, and VHH CDR3 comprises or consists of the amino acid sequence shown in 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. 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 NO: 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. A heavy chain antibody that specifically binds to C5, comprising the VHH antibody according to any one of claims 1 to 6. 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. A VHH antibody specifically binding to HTRA1, comprising 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. The VHH antibody that specifically binds to HTRA1 of 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 shown in SEQ ID NO:11, VHH CDR2 comprises or consists of the amino acid sequence shown in 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 shown in SEQ ID NO:11 or 14, VHH CDR2 comprises or consists of the amino acid sequence shown in 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. 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 the 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. A heavy chain antibody that specifically binds to HTRA1, comprising the VHH antibody that specifically binds to HTRA1 according to any one of claims 9 to 11. 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. 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. 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. 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. The antibody specifically binding to C5 of claim 16, comprising 1 or 2 VHH antibodies specifically binding to C5 or heavy chain antibodies comprising said VHH antibodies of any one of claims 1 to 3, and 1 or 2 VHH antibodies specifically binding to C5 or heavy chain antibodies comprising said VHH antibodies of any one of claims 4 to 6; preferably, it comprises 1 or 2 VHH antibodies specifically binding to C5 of any one of claims 4 to 6, and 1 VHH antibody specifically binding to C5 of any one of claims 1 to 3, preferably, the 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. 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. 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. 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. 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. A bispecific antibody comprises 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. 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. 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 by a linker. The bispecific antibody of claim 24, wherein the linker comprises (G) _n , wherein n=5-10, such as 5, 6, 7, 8, 9, 10. 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. The bispecific antibody of any one of claims 24 to 26, wherein one antigen-binding region that specifically binds to C5 comprises or consists of the VHH of any one of claims 1 to 3, the other antigen-binding region that specifically binds to C5 comprises or consists of the VHH of any one of claims 4 to 6, or the antigen-binding region that specifically binds to HTRA1 comprises or consists of the VHH of any one of claims 9 to 11. The bispecific antibody of any one of claims 24 to 26, wherein the one antigen-binding region that specifically binds to C5 comprises or consists of the VHH of any one of claims 1 to 3, the other antigen-binding region that specifically binds to C5 comprises or consists of the VHH of any one of claims 4 to 6, and the antigen-binding region that specifically binds to HTRA1 comprises or consists of the VHH of any one of claims 9 to 11. The bispecific antibody of any one of claims 24 to 28, comprising the following chain, wherein the chain comprises, from the N-terminus to the C-terminus: VHH1 that specifically binds to C5 - VHH2 that specifically binds to C5 - VHH3 that specifically binds to HTRA1, The VHHs are connected by a linker; wherein VHH1 comprises or consists of the VHH of any one of claims 4-6, VHH1 comprises or consists of the VHH of any one of claims 1-3, and VHH3 comprises or consists of the VHH of any one of claims 9-11. The bispecific antibody of claim 29, which comprises or consists 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. 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, 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, 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. An expression vector comprising the nucleic acid molecule of claim 31, preferably, the expression vector is pCDNA3.1. 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 CHO cells, 293 cells, such as Expi293 cells. A method for preparing the antibody of any one of claims 1 to 30, the method comprising culturing 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. An immunoconjugate comprising the antibody according to any one of claims 1 to 30, and one or more other active ingredients. A pharmaceutical composition or drug or preparation comprising the antibody according to any one of claims 1 to 30 or the immunoconjugate according to claim 35, and optionally a pharmaceutically acceptable excipient. Drug combination products containing The antibody of any one of claims 1 to 30 or the immunoconjugate of claim 35; and Other Therapeutic Agents. A method for preventing or treating an ocular disease or disorder 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. 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). 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 allowing 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.