Adam9 binding proteins and uses thereof

Abstract

Anti-ADAM9 antibodies or antigen-binding fragments thereof are provided, along with nucleic acid molecules encoding them, methods for their preparation, and relate to the field of disease treatment. The anti-ADAM9 antibodies or antigen-binding fragments thereof exhibit high specificity and high affinity for ADAM9. Further, the use of said antibodies or antigen-binding fragments thereof in the treatment and diagnosis of diseases is discussed.

Description

ADAM9 binding proteins and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311734757.9 filed on December 14, 2023, priority to Chinese patent application No. 202410173238.8 filed on February 6, 2024, and priority to Chinese patent application No. 202411789351.5 filed on December 6, 2024, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention belongs to the field of therapeutic monoclonal antibodies, and more specifically, the present invention relates to an antibody targeting ADAM9; and also relates to the use of the antibody in treating and diagnosing diseases. Technical Background

[0004] A distegrinin and a metalloprotease (ADAM) is a type I transmembrane protein anchored to the cell surface membrane, and over 30 species have been discovered to date. Similar to matrix metalloproteinases (MMPs), ADAMs also contain a prodomain and a zinc-binding metalloprotease domain. ADAMs also contain a disintegrin domain, which is unique among cell surface proteins. Among them, ADAM9 is widely expressed in the human body and regulates multiple biological functions, playing an important role in various diseases, including neurodegenerative diseases, retinal diseases, inflammation, and tumors. ADAM9 participates in various physiological functions primarily through its distegrins domain, which is used for adhesion, and its metalloprotein domain, which is used to shed various cell surface proteins.

[0005] In recent years, increasing evidence has shown that ADAM9 plays an important role in tumors. ADAM9 is involved in the regulation of various tumor processes. In addition to metastasis, ADAM9 plays an important role in tumor proliferation, angiogenesis, and even immune escape. Overexpression of ADAM9 has been found in many cancers and is associated with tumor aggressiveness and poor prognosis. In addition, ADAM9 promotes tumor progression, treatment resistance, and cancer metastasis through proteolytic or non-proteolytic pathways.

[0006] Several studies have shown that overexpression of ADAM9 shortens overall survival. Other studies have shown that ADAM9 plays a key role in several steps of lung cancer metastasis. Shintani et al. first described that overexpression of ADAM9 promotes tumor cell adhesion to vascular endothelial cells, demonstrating the importance of ADAM9 in metastasis. Furthermore, ADAM9 enhances cell migration and stress resistance through a novel mechanism to promote metastasis. Fritzsche et al. demonstrated that overexpression of ADAM9 mRNA and protein is associated with poor recurrence-free survival in prostate cancer. Immunohistochemical analysis demonstrated elevated ADAM9 protein expression in over 60% of recurrent prostate tumors. IL-6 is a major mediator of liver cancer invasion and metastasis, and IL-6 enhances ADAM9 expression in vitro by activating the JNK pathway. Silencing ADAM9 not only reduces primary tumor size but also inhibits the rate of metastasis to the lungs. Conversely, overexpression of ADAM9 accelerates primary tumor growth and promotes lung metastasis. ADAM9 shares the methyltransferase of EGFR, and nuclear receptor-associated SET domain protein 2 is a member of the histone methyltransferase family. It can upregulate the expression of ADAM9 and EGFR and promote the resistance of TNBC cells to EGFR inhibitors. KRAS signaling is necessary to maintain pancreatic cancer tumorigenesis. Yuan et al. found that dysregulated KRAS signaling enhances ADAM9 expression through the NF-kB cascade, and knockout of ADAM9 inhibits the downstream pathways of KRAS and MEK-ERK signaling. In addition, studies have shown that cyclic ADAM9 (circ-ADAM9) is upregulated in pancreatic cancer cells and is associated with poor prognosis. Overexpression of circ-ADAM9 increases ERK signaling, promotes cell proliferation and migration in vitro, and silencing circ-ADAM9 delays the growth of pancreatic tumors in vivo.

[0007] Research to date is sufficient to support the use of ADAM9 as a prognostic marker for cancer. Currently used therapeutic drugs (such as Sorafenib and Regorafenib) enhance cancer treatment by reducing ADAM9 levels, which provides new ideas for ADAM9 targeted therapy. At the 2021 AACR meeting, ImmunoGen introduced the research progress of IMGC936. IMGC936 is the first ADC targeting ADAM9 developed by ImmunoGen and Macrogenics. IMGC936 is an innovative drug composed of a high-affinity humanized monoclonal antibody, a maytansine alkaloid microtubule inhibitor load and a stable tripeptide linker with a DAR of 2. It is currently in the Phase I clinical research stage.

[0008] In terms of safety, no major developmental defects were observed in ADAM9 knockout mice, except for later retinal disorders, suggesting that it may be a relatively safe target in adult patients.

[0009] Nevertheless, the specificity of therapeutic drugs targeting ADAM9 should be considered when treating ADAM9-related diseases, as modulation of the protective activity of MMPs generally causes side effects. Therefore, it is necessary to develop antibodies targeting ADAM9, which will provide patients with more drug options. Summary of the Invention

[0010] In the present application, the inventors have developed high-affinity humanized antibodies with excellent properties, which can specifically recognize / bind to ADAM9 and can be used to prevent and / or treat cancers that express ADAM9, especially overexpress ADAM9.

[0011] Antibodies of the present invention

[0012] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to ADAM9, wherein the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):

[0013] (a) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 54 or 52; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 55 or 53;

[0014] (b) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 3 or 1; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 4 or 2;

[0015] (c) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 9 or 7; and / or CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 10 or 8;

[0016] (d) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 50 or 48; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO: 51 or 49; or

[0017] (e) CDR-H1, CDR-H2 and CDR-H3 contained in the following heavy chain variable region (VH), and / or CDR-L1, CDR-L2 and CDR-L3 contained in the following light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation compared to the heavy chain variable region and / or light chain variable region according to any one of (a) to (d), and the mutation is a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids).

[0018] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):

[0019] (i) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 54; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 55;

[0020] (ii) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 52; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 53;

[0021] (iii) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 3; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 4;

[0022] (iv) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 1; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 2;

[0023] (v) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 9; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 10;

[0024] (vi) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 7; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 8;

[0025] (vii) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 50; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 51;

[0026] (viii) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 48; and / or CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 49; or

[0027] (ix) CDR-H1, CDR-H2 and CDR-H3 contained in the following heavy chain variable region (VH), and / or CDR-L1, CDR-L2 and CDR-L3 contained in the following light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation compared to the heavy chain variable region and / or light chain variable region described in any one of (i) or (iv), and the mutation is a substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids).

[0028] In certain embodiments, the substitutions are conservative substitutions.

[0029] In certain embodiments, the CDRs are defined according to the IMGT, Kabat, Chothia, or AbM numbering systems.

[0030] In certain embodiments, the ADAM9 comprises human ADAM9 and / or monkey ADAM9. In certain embodiments, the monkey is a rhesus monkey (Macaca mulatta).

[0031] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0032] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 81 or a variant thereof; CDR-H2 of SEQ ID NO: 82 or a variant thereof; CDR-H3 of SEQ ID NO: 83 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 84 or a variant thereof; CDR-L2 of SEQ ID NO: 85 or a variant thereof; and CDR-L3 of SEQ ID NO: 76 or a variant thereof;

[0033] (1b-i) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 21 or a variant thereof; CDR-H2 of SEQ ID NO: 22 or a variant thereof; and CDR-H3 of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 24 or a variant thereof; CDR-L2 of SEQ ID NO: 25 or a variant thereof; and CDR-L3 of SEQ ID NO: 16 or a variant thereof;

[0034] (1b-ii) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 21 or a variant thereof; CDR-H2 of SEQ ID NO: 22 or a variant thereof; and CDR-H3 of SEQ ID NO: 94 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 24 or a variant thereof; CDR-L2 of SEQ ID NO: 25 or a variant thereof; and CDR-L3 of SEQ ID NO: 16 or a variant thereof;

[0035] (1c-i) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 35 or a variant thereof; CDR-H2 of SEQ ID NO: 36 or a variant thereof; and CDR-H3 of SEQ ID NO: 37 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 38 or a variant thereof; CDR-L2 of SEQ ID NO: 39 or a variant thereof; and CDR-L3 of SEQ ID NO: 30 or a variant thereof;

[0036] (1c-ii) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 103 or a variant thereof; CDR-H2 of SEQ ID NO: 104 or a variant thereof; CDR-H3 of SEQ ID NO: 37 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 105 or a variant thereof; CDR-L2 of SEQ ID NO: 39 or a variant thereof; CDR-L3 of SEQ ID NO: 30 or a variant thereof; or

[0037] (1d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 65 or a variant thereof; CDR-H2 of SEQ ID NO: 66 or a variant thereof; CDR-H3 of SEQ ID NO: 67 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 68 or a variant thereof; CDR-L2 of SEQ ID NO: 69 or a variant thereof; and CDR-L3 of SEQ ID NO: 70 or a variant thereof;

[0038] The variant described in any one of (1a), (1b-i), (1b-ii), (1c-i), (1c-ii), and (1d) has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0039] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0040] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 71 or a variant thereof; CDR-H2 of SEQ ID NO: 72 or a variant thereof; CDR-H3 of SEQ ID NO: 73 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 74 or a variant thereof; CDR-L2 of SEQ ID NO: 75 or a variant thereof; and CDR-L3 of SEQ ID NO: 76 or a variant thereof;

[0041] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof; CDR-H2 of SEQ ID NO: 12 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 16 or a variant thereof;

[0042] (2c-i) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof; CDR-H2 of SEQ ID NO: 26 or a variant thereof; and CDR-H3 of SEQ ID NO: 27 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 28 or a variant thereof; CDR-L2 of SEQ ID NO: 29 or a variant thereof; and CDR-L3 of SEQ ID NO: 30 or a variant thereof;

[0043] (2c-ii) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 95 or a variant thereof; CDR-H2 of SEQ ID NO: 96 or a variant thereof; CDR-H3 of SEQ ID NO: 27 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 97 or a variant thereof; CDR-L2 of SEQ ID NO: 98 or a variant thereof; CDR-L3 of SEQ ID NO: 30 or a variant thereof; or

[0044] (2d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 56 or a variant thereof; CDR-H2 of SEQ ID NO: 57 or a variant thereof; CDR-H3 of SEQ ID NO: 58 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 59 or a variant thereof; CDR-L2 of SEQ ID NO: 60 or a variant thereof; and CDR-L3 of SEQ ID NO: 61 or a variant thereof;

[0045] The variant described in any one of (2a), (2b), (2c-i), (2c-ii), and (2d) has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0046] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0047] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 79 or a variant thereof; CDR-H2 of SEQ ID NO: 80 or a variant thereof; and CDR-H3 of SEQ ID NO: 73 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 74 or a variant thereof; CDR-L2 of SEQ ID NO: 75 or a variant thereof; and CDR-L3 of SEQ ID NO: 76 or a variant thereof;

[0048] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 19 or a variant thereof; CDR-H2 of SEQ ID NO: 20 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 16 or a variant thereof;

[0049] (3c-i) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 33 or a variant thereof; CDR-H2 of SEQ ID NO: 34 or a variant thereof; and CDR-H3 of SEQ ID NO: 27 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 28 or a variant thereof; CDR-L2 of SEQ ID NO: 29 or a variant thereof; and CDR-L3 of SEQ ID NO: 30 or a variant thereof;

[0050] (3c-ii) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 101 or a variant thereof; CDR-H2 of SEQ ID NO: 102 or a variant thereof; CDR-H3 of SEQ ID NO: 27 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 97 or a variant thereof; CDR-L2 of SEQ ID NO: 98 or a variant thereof; CDR-L3 of SEQ ID NO: 30 or a variant thereof; or

[0051] (3d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 62 or a variant thereof; CDR-H2 of SEQ ID NO: 63 or a variant thereof; and CDR-H3 of SEQ ID NO: 64 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 59 or a variant thereof; CDR-L2 of SEQ ID NO: 60 or a variant thereof; and CDR-L3 of SEQ ID NO: 61 or a variant thereof;

[0052] The variant described in any one of (3a), (3b), (3c-i), (3c-ii), and (3d) has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0053] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0054] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 77 or a variant thereof; CDR-H2 of SEQ ID NO: 78 or a variant thereof; CDR-H3 of SEQ ID NO: 73 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 74 or a variant thereof; CDR-L2 of SEQ ID NO: 75 or a variant thereof; and CDR-L3 of SEQ ID NO: 76 or a variant thereof;

[0055] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 17 or a variant thereof; CDR-H2 of SEQ ID NO: 18 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 16 or a variant thereof;

[0056] (4c-i) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 31 or a variant thereof; CDR-H2 of SEQ ID NO: 32 or a variant thereof; and CDR-H3 of SEQ ID NO: 27 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 28 or a variant thereof; CDR-L2 of SEQ ID NO: 29 or a variant thereof; and CDR-L3 of SEQ ID NO: 30 or a variant thereof;

[0057] (4c-ii) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 99 or a variant thereof; CDR-H2 of SEQ ID NO: 100 or a variant thereof; CDR-H3 of SEQ ID NO: 27 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 97 or a variant thereof; CDR-L2 of SEQ ID NO: 98 or a variant thereof; CDR-L3 of SEQ ID NO: 30 or a variant thereof; or

[0058] (4d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 62 or a variant thereof; CDR-H2 of SEQ ID NO: 63 or a variant thereof; CDR-H3 of SEQ ID NO: 64 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 59 or a variant thereof; CDR-L2 of SEQ ID NO: 60 or a variant thereof; and CDR-L3 of SEQ ID NO: 61 or a variant thereof;

[0059] The variant described in any one of (4a), (4b), (4c-i), (4c-ii), and (4d) has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0060] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0061] (ai) a VH comprising the sequence shown in SEQ ID NO: 54 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 55 or a variant thereof;

[0062] (a-ii) a VH comprising the sequence shown in SEQ ID NO: 52 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 53 or a variant thereof;

[0063] (bi) a VH comprising the sequence shown in SEQ ID NO: 3 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 4 or a variant thereof;

[0064] (b-ii) a VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof;

[0065] (ci) a VH comprising the sequence shown in SEQ ID NO: 9 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 10 or a variant thereof;

[0066] (c-ii) a VH comprising the sequence shown in SEQ ID NO: 7 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 8 or a variant thereof;

[0067] (di) a VH comprising the sequence shown in SEQ ID NO: 50 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 51 or a variant thereof; or

[0068] (d-ii) a VH comprising the sequence shown in SEQ ID NO: 48 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 49 or a variant thereof;

[0069] wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0070] In certain embodiments of the antibodies or antigen-binding fragments disclosed herein, the heavy chain constant domain may comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid. In some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid.

[0071] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0072] In certain embodiments, provided herein are compositions comprising the antibodies or antigen-binding fragments disclosed herein, wherein each antibody or antigen-binding fragment may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, the N-terminal amino acid cyclized to pyroglutamic acid, or the N-terminal amino acid cyclized to pyroglutamate.

[0073] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to an antigen and may include post-translational modifications thereof (e.g., cleavage of a C-terminal lysine in a heavy chain, conversion of an N-terminal glutamine or glutamic acid in a heavy or light chain to pyroglutamic acid or pyroglutamate), which may occur upon recombinant expression in a host cell (e.g., a CHO cell) or during purification / storage.

[0074] In certain embodiments, the N-terminal glutamine of the VH comprising the sequence as shown in SEQ ID NO: 54, 52, 3, 1, 9, 7, 50 or 48 or a variant thereof undergoes cyclization to form pyroglutamate or pyroglutamate. In certain embodiments, the N-terminal glutamine of the VH comprising the sequence as shown in SEQ ID NO: 54, 3, 9 or 50 or a variant thereof undergoes cyclization to form pyroglutamate or pyroglutamate.

[0075] In certain embodiments, the antibody or antigen-binding fragment thereof having the features of any one of (1a), (2a), (3a), (4a), (ai), or (a-ii) of the above embodiments further has a feature selected from the following:

[0076] (1) at an EC of less than about 100 ng / mL, for example, less than about 80 ng / mL, 50 ng / mL, 20 ng / mL, 15 ng / mL, 14 ng / mL, 13 ng / mL, 12 ng / mL, 11 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL or less. 50 Binds to ADAM9 (eg, human or monkey ADAM9); preferably, the EC 50 Measured by ELISA;

[0077] (2) binds to ADAM9 (e.g., human or monkey ADAM9) with a KD of less than about 100 nM, such as less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 5 nM, or less; preferably, the KD is determined by biofilm interferometry (BLI) (e.g., ForteBio ) measured;

[0078] (3) having CDC activity, such as inducing killing of cells expressing ADAM9 (e.g., tumor cells) through CDC;

[0079] (4) No ADCC activity;

[0080] (5) inducing ADAM9 internalization, e.g., as measured by flow cytometry;

[0081] (6) does not bind to ADAM12 and ADAM15 proteins;

[0082] (7) inhibiting cell (such as tumor cell) proliferation; and / or

[0083] (8) Inhibit tumor growth.

[0084] In certain embodiments, the antibody or antigen-binding fragment thereof having the features of any one of (1b-i), (1b-ii), (2b), (3b), (4b), (bi), or (b-ii) in the above embodiments further has a feature selected from the following:

[0085] (1) at an EC of less than about 100 ng / mL, for example, less than about 80 ng / mL, 50 ng / mL, 20 ng / mL, 15 ng / mL, 14 ng / mL, 13 ng / mL, 12 ng / mL, 11 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL or less. 50 Binds to ADAM9 (eg, human or monkey ADAM9); preferably, the EC 50 Measured by ELISA;

[0086] (2) binds to ADAM9 (e.g., human or monkey ADAM9) with a KD of less than about 100 nM, such as less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 5 nM, or less; preferably, the KD is determined by biofilm interferometry (BLI) (e.g., ForteBio ) measured;

[0087] (3) having CDC activity, such as inducing killing of cells expressing ADAM9 (e.g., tumor cells) through CDC;

[0088] (4) No ADCC activity;

[0089] (5) inducing ADAM9 internalization, e.g., as measured by flow cytometry;

[0090] (6) does not bind to ADAM12 and ADAM15 proteins;

[0091] (7) inhibiting cell (such as tumor cell) proliferation; and / or

[0092] (8) Inhibit tumor growth.

[0093] In certain embodiments, the antibody or antigen-binding fragment thereof having the features of any one of (1c-i), (1c-ii), (2c-i), (2c-ii), (3c-i), (3c-ii), (4c-i), (4c-ii), (ci) or (c-ii) in the above embodiments further has a feature selected from the following:

[0094] (1) at an EC of less than about 100 ng / mL, for example, less than about 80 ng / mL, 50 ng / mL, 20 ng / mL, 15 ng / mL, 14 ng / mL, 13 ng / mL, 12 ng / mL, 11 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL or less. 50Binds to ADAM9 (eg, human or monkey ADAM9); preferably, the EC 50 Measured by ELISA;

[0095] (2) binds to ADAM9 (e.g., human or monkey ADAM9) with a KD of less than about 100 nM, such as less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 5 nM, or less; preferably, the KD is determined by biofilm interferometry (BLI) (e.g., ForteBio ) measured;

[0096] (3) having CDC activity, such as inducing killing of cells expressing ADAM9 (e.g., tumor cells) through CDC;

[0097] (4) No ADCC activity;

[0098] (5) inducing ADAM9 internalization, e.g., as measured by flow cytometry;

[0099] (6) does not bind to ADAM12 and ADAM15 proteins;

[0100] (7) inhibiting cell (such as tumor cell) proliferation; and / or

[0101] (8) Inhibit tumor growth.

[0102] In certain embodiments, the antibody or antigen-binding fragment thereof having the features of any one of (1d), (2d), (3b), (3d), (4d), (di), or (d-ii) in the above embodiments further has a feature selected from the following:

[0103] (1) at an EC of less than about 100 ng / mL, for example, less than about 80 ng / mL, 50 ng / mL, 20 ng / mL, 15 ng / mL, 14 ng / mL, 13 ng / mL, 12 ng / mL, 11 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL or less. 50 Binds to ADAM9 (eg, human or monkey ADAM9); preferably, the EC 50 Measured by ELISA;

[0104] (2) binds to ADAM9 (e.g., human or monkey ADAM9) with a KD of less than about 100 nM, such as less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 5 nM, or less; preferably, the KD is determined by biofilm interferometry (BLI) (e.g., ForteBio ) measured;

[0105] (3) having CDC activity, such as inducing killing of cells expressing ADAM9 (e.g., tumor cells) through CDC;

[0106] (4) No ADCC activity;

[0107] (5) inducing ADAM9 internalization, e.g., as measured by flow cytometry;

[0108] (6) does not bind to ADAM12 and ADAM15 proteins;

[0109] (7) inhibiting cell (such as tumor cell) proliferation; and / or

[0110] (8) Inhibit tumor growth.

[0111] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments may comprise a constant region from or derived from a human immunoglobulin.

[0112] In certain embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises from or is derived from the heavy chain constant region of human immunoglobulin (such as IgG1, IgG2, IgG3 or IgG4).In certain embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises wild-type Fc district, or comprises mutated or chemically modified Fc district, which has the effector function (such as reduced ADCC activity) of change compared with wild-type Fc district.In certain exemplary embodiments, the antibody or its antigen-binding fragment of the present invention comprises the variant of human IgG1 heavy chain constant region, and the variant has the following displacement compared with the wild-type sequence from which it is derived: Leu234Ala, Leu235Ala and Gly237Ala (according to the position of EU numbering system).In such embodiments, the antibody or its antigen-binding fragment of the present invention has reduced ADCC activity. In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a sequence as set forth in SEQ ID NO: 5 or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared thereto. In certain embodiments, the heavy chain constant region (CH) as set forth in SEQ ID NO: 5 or a variant thereof lacks a C-terminal lysine.

[0113] In certain embodiments, the light chain of the antibody or its antigen-binding fragment comprises a light chain constant region from or derived from a human immunoglobulin (e.g., κ or λ). In certain embodiments, the light chain of the antibody or its antigen-binding fragment comprises a sequence as shown in SEQ ID NO:6 or a variant thereof, the variant having a conservative substitution of up to 20 amino acids (e.g., a conservative substitution of up to 15, up to 10, or up to 5 amino acids; e.g., a conservative substitution of 1, 2, 3, 4, or 5 amino acids) compared thereto.

[0114] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0115] (1) a heavy chain comprising the VH region represented by SEQ ID NO: 54 and the heavy chain constant region (CH) represented by SEQ ID NO: 5, and a light chain comprising the VL region represented by SEQ ID NO: 55 and the light chain constant region (CL) represented by SEQ ID NO: 6;

[0116] (2) a heavy chain comprising the VH represented by SEQ ID NO: 52 and the heavy chain constant region (CH) represented by SEQ ID NO: 5, and a light chain comprising the VL represented by SEQ ID NO: 53 and the light chain constant region (CL) represented by SEQ ID NO: 6;

[0117] (3) a heavy chain comprising the VH region of SEQ ID NO: 3 and the heavy chain constant region (CH) of SEQ ID NO: 5, and a light chain comprising the VL region of SEQ ID NO: 4 and the light chain constant region (CL) of SEQ ID NO: 6;

[0118] (4) a heavy chain comprising the VH region of SEQ ID NO: 1 and the heavy chain constant region (CH) of SEQ ID NO: 5, and a light chain comprising the VL region of SEQ ID NO: 2 and the light chain constant region (CL) of SEQ ID NO: 6;

[0119] (5) a heavy chain comprising the VH region of SEQ ID NO: 9 and the heavy chain constant region (CH) of SEQ ID NO: 5, and a light chain comprising the VL region of SEQ ID NO: 10 and the light chain constant region (CL) of SEQ ID NO: 6;

[0120] (6) a heavy chain comprising the VH region of SEQ ID NO: 7 and the heavy chain constant region (CH) of SEQ ID NO: 5, and a light chain comprising the VL region of SEQ ID NO: 8 and the light chain constant region (CL) of SEQ ID NO: 6;

[0121] (7) a heavy chain comprising the VH represented by SEQ ID NO: 50 and the heavy chain constant region (CH) represented by SEQ ID NO: 5, and a light chain comprising the VL represented by SEQ ID NO: 51 and the light chain constant region (CL) represented by SEQ ID NO: 6; or

[0122] (8) A heavy chain comprising the VH represented by SEQ ID NO: 48 and the heavy chain constant region (CH) represented by SEQ ID NO: 5, and a light chain comprising the VL represented by SEQ ID NO: 49 and the light chain constant region (CL) represented by SEQ ID NO: 6.

[0123] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0124] (1) a heavy chain having the sequence shown in SEQ ID NO: 88 and a light chain having the sequence shown in SEQ ID NO: 89;

[0125] (2) a heavy chain having the sequence shown in SEQ ID NO: 40 and a light chain having the sequence shown in SEQ ID NO: 41; or,

[0126] (3) a heavy chain having the sequence shown in SEQ ID NO: 42 and a light chain having the sequence shown in SEQ ID NO: 43; or,

[0127] (4) A heavy chain having the sequence shown in SEQ ID NO: 86 and a light chain having the sequence shown in SEQ ID NO: 87.

[0128] In certain embodiments, the N-terminal glutamine of the heavy chain having the sequence shown as SEQ ID NO: 88, 40, 42 or 86 or a variant thereof undergoes cyclization to form pyroglutamate or pyroglutamate salt.

[0129] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments is a murine antibody, a chimeric antibody, or a humanized antibody.

[0130] In certain embodiments, the variable region of the antibody or antigen-binding fragment thereof described in any of the above embodiments is of human origin.

[0131] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments is selected from ScFv, Fab, Fab', F(ab')2, Fab'-SH, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody and multispecific antibody.

[0132] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments is labeled. In certain embodiments, the antibody or antigen-binding fragment thereof is labeled with a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

[0133] The present invention also provides methods for treating tumors using the antibodies or antigen-binding fragments provided herein or pharmaceutical compositions thereof.

[0134] Derivatized antibodies

[0135] The antibody of the present invention or its antigen-binding fragment can be derivatized, for example, connected to another molecule (such as another polypeptide or protein). Generally, the derivatization (for example, labeling) of the antibody or its antigen-binding fragment will not adversely affect its binding to ADAM9 (particularly human ADAM9). Therefore, the antibody of the present invention or its antigen-binding fragment is also intended to include such derivatized forms. For example, the antibody of the present invention or its antigen-binding fragment can be functionally connected (by chemical coupling, gene fusion, non-covalent connection or other means) to one or more other molecular groups, such as another antibody (for example, forming a bispecific antibody), detection reagent, pharmaceutical agent, and / or a protein or polypeptide (for example, avidin or polyhistidine tag) that can mediate the binding of the antibody or antigen-binding fragment to another molecule.

[0136] As one of the derivatives of the antibody, the present invention provides a conjugate comprising the antibody or antigen-binding fragment thereof of the present invention and a conjugated moiety.

[0137] In certain embodiments, the coupling moiety is selected from a detectable label. The detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In certain embodiments, the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme. In certain embodiments, the detectable label as described above can be linked to the antibody or antigen-binding fragment thereof of the present invention by linkers of varying lengths to reduce potential steric hindrance.

[0138] In certain embodiments, the conjugated moiety is selected from a therapeutic agent. In certain embodiments, the therapeutic agent is preferably an anti-tumor agent, such as a cytotoxic agent, a cytokine, a toxin or a radionuclide.

[0139] In certain embodiments, the conjugated moiety is selected from substances that can improve the biological properties of the antibody (eg, increase serum half-life), for example, a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a sugar group.

[0140] As one of the antibody derivatives, the present invention provides a multispecific antibody comprising the antibody of the present invention or an antigen-binding fragment thereof.

[0141] In certain embodiments, the multispecific antibody comprises an antibody of the present invention or an antigen-binding fragment thereof as a first antigen-binding domain and further comprises at least one second antigen-binding domain directed against another target.

[0142] In certain embodiments, each antigen-binding domain of the multispecific antibody retains its respective original binding specificity.

[0143] In certain embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0144] As one of the derivatives of antibodies, the present invention provides a chimeric antigen receptor, which includes an antibody of the present invention or its antigen-binding fragment. In certain embodiments, the chimeric antigen receptor includes an antibody of the present invention or its antigen-binding fragment (e.g., ScFv) as an extracellular antigen binding domain that specifically binds to ADAM9, as well as a transmembrane domain and one or more intracellular T cell signaling domains. The present invention also provides host cells (e.g., immune cells, such as T lymphocytes, NK cells, DC cells, macrophages) containing or expressing the chimeric antigen receptor.

[0145] Antibody preparation

[0146] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0147] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture fluid. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0148] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the present invention, or a heavy chain variable region and / or a light chain variable region thereof. According to codon degeneracy in the art, in certain embodiments, the nucleotide sequence can be replaced according to codon degeneracy. In certain embodiments, the nucleotide sequence is codon optimized.

[0149] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence set forth in SEQ ID NO:92, (ii) a sequence substantially identical to SEQ ID NO:92 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:92), or (iii) a degenerate sequence of (i) or (ii) above; and / or the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence set forth in SEQ ID NO:93, (v) a sequence substantially identical to SEQ ID NO:93 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:93), or (vi) a degenerate sequence of (iv) or (v) above.

[0150] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence set forth in SEQ ID NO:44, (ii) a sequence substantially identical to SEQ ID NO:44 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:44), or (iii) a degenerate sequence of (i) or (ii) above; and / or the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence set forth in SEQ ID NO:45, (v) a sequence substantially identical to SEQ ID NO:45 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:45), or (vi) a degenerate sequence of (iv) or (v) above.

[0151] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence set forth in SEQ ID NO:46, (ii) a sequence substantially identical to SEQ ID NO:46 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:46), or (iii) a degenerate sequence of (i) or (ii) above; and / or the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence set forth in SEQ ID NO:47, (v) a sequence substantially identical to SEQ ID NO:47 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:47), or (vi) a degenerate sequence of (iv) or (v) above.

[0152] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence set forth in SEQ ID NO:90, (ii) a sequence substantially identical to SEQ ID NO:90 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:90), or (iii) a degenerate sequence of (i) or (ii) above; and / or the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence set forth in SEQ ID NO:91, (v) a sequence substantially identical to SEQ ID NO:91 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:91), or (vi) a degenerate sequence of (iv) or (v) above.

[0153] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the present invention. In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a lentivirus, or the like. In certain embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof of the present invention in a subject (e.g., a mammal, such as a human).

[0154] In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding the light chain or light chain variable region thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0155] In certain embodiments, the antibodies or their antigen-binding fragments of the present invention can be used to construct a chimeric antigen receptor (CAR), which comprises an extracellular antigen binding domain (e.g., ScFv) specifically binding to ADAM9, a transmembrane domain, and one or more intracellular T cell signaling domains. In such embodiments, the detached nucleic acid molecules of the present invention may include a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further includes a nucleotide sequence encoding an antibody of the present invention or its antigen-binding fragment (e.g., ScFv). In certain embodiments, the detached nucleic acid molecules of the present invention encode a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention.

[0156] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention can be used to construct chimeric antigen receptor-modified immune cells, wherein the chimeric antigen receptor-modified immune cells comprise a chimeric antigen receptor (CAR) and an immune cell (e.g., T lymphocytes, NK cells, DC cells, macrophages).

[0157] The present invention further provides a host cell comprising an isolated nucleic acid molecule of the present invention or a vector of the present invention. The host cell can be a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44, CHO-EBNA).

[0158] In certain embodiments, the host cell of the present invention may be a chimeric antigen receptor T cell (CAR-T). In such embodiments, the isolated nucleic acid molecule contained in the host cell may include a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further includes a nucleotide sequence encoding an antibody of the present invention or its antigen-binding fragment (e.g., ScFv). In certain embodiments, the isolated nucleic acid molecule contained in the host cell encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention.

[0159] In another aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell of the present invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0160] Therapeutic applications

[0161] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or chimeric antigen receptor or host cell expressing the chimeric antigen receptor of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0162] In certain embodiments, the pharmaceutical compositions of the present invention comprise the antibodies or antigen-binding fragments thereof of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0163] In certain embodiments, the pharmaceutical composition of the present invention comprises a vector or host cell of the present invention, and a pharmaceutically acceptable carrier and / or excipient. In such embodiments, the isolated nucleic acid molecule contained in the vector comprises a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further comprises a nucleotide sequence encoding an antibody of the present invention or its antigen-binding fragment (e.g., ScFv); the host cell comprises an isolated nucleic acid molecule or vector as described above. In certain embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention. In certain embodiments, the host cell is an immune cell, such as a T cell. In certain embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0164] In certain embodiments, the pharmaceutical composition may also include another pharmaceutically active agent. In certain embodiments, the other pharmaceutically active agent is a medicine with anti-tumor activity. In certain embodiments, the other pharmaceutically active agent is selected from ADAM9 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutics or any combination thereof. In certain embodiments, the antibody of the present invention or its antigen-binding fragment and the other pharmaceutically active agent are provided as independent components or as mixed components. Therefore, the antibody of the present invention or its antigen-binding fragment and the other pharmaceutically active agent can be used simultaneously, separately or successively.

[0165] In certain embodiments, the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or chimeric antigen receptor or host cell expressing the chimeric antigen receptor in the pharmaceutical composition of the present invention is sufficient (e.g., in a subject):

[0166] (a) Inhibit cell (such as tumor cell) proliferation;

[0167] (b) inhibiting tumor growth;

[0168] (c) inducing and / or increasing antibody-dependent cellular cytotoxicity;

[0169] (d) inhibiting ADAM9-mediated signal transduction;

[0170] (e) preventing and / or treating ADAM9-mediated diseases / disorders; or

[0171] (f) Any combination of (a)-(e).

[0172] In certain embodiments, the disease / disorder mediated by ADAM9 is a tumor, such as a tumor expressing ADAM9. In certain embodiments, the tumor is selected from glioma, cervical cancer, oral squamous cell carcinoma, liver cancer, melanoma, prostate cancer, pancreatic ductal adenocarcinoma, gastric cancer, lung cancer such as non-small cell lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer such as triple-negative breast cancer, or any combination thereof.

[0173] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention in the preparation of a medicament, wherein the medicament is used for: use in a medicament for inhibiting cell proliferation, or preventing and / or treating and / or adjuvant treating tumors, neurodegenerative diseases, retinal diseases or inflammation.

[0174] In certain embodiments, the medicament is used to inhibit the proliferation of cells expressing ADAM9 (eg, tumor cells).

[0175] In another aspect, the present invention provides a method for inhibiting cell proliferation, comprising contacting the cell with an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor, or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention. In certain embodiments, the cell is a cell expressing ADAM9, such as a tumor cell.

[0176] In another aspect, the present invention provides a method for preventing and / or treating and / or adjuvant treating tumors, neurodegenerative diseases, retinal diseases or inflammation in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention.

[0177] In certain embodiments, the method further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof. In certain embodiments, the second therapy can be applied simultaneously, separately, or sequentially with the above method.

[0178] In any of the above embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention can be any tumor type. In certain embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention is an ADAM9 positive tumor. In certain embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention is selected from glioma, cervical cancer, oral squamous cell carcinoma, liver cancer, melanoma, prostate cancer, pancreatic ductal adenocarcinoma, gastric cancer, lung cancer such as non-small cell lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer such as triple-negative breast cancer or any combination thereof.

[0179] The antibodies or antigen-binding fragments thereof of the present invention, and the pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended route of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of production and storage. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: incorporating the required dose of the antibody of the present invention into an appropriate solvent, and optionally, other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for ease of storage and use. Such sterile lyophilized powders can be dispersed in a suitable vehicle, such as sterile pyrogen-free water, before use.

[0180] Furthermore, the antibodies or antigen-binding fragments thereof of the present invention can be presented in a pharmaceutical composition in a unit dosage form for ease of administration.

[0181] Antibody of the present invention or its Fab, pharmaceutical composition can be used by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intravesical, local (such as, powder, ointment or drops), or nasal route. But, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that route of administration and / or mode will change according to intended purpose. In a preferred embodiment, antibody of the present invention or its Fab, pharmaceutical composition is given by intravenous infusion or injection.

[0182] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding fragment thereof of the present invention. A "prophylactically effective amount" is an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other concurrently administered therapies.

[0183] In the present invention, the dosage regimen can be adjusted to obtain the best desired response (e.g., therapeutic or preventive response). For example, the dosage can be a single dose, multiple doses can be administered over a period of time, or the dosage can be proportionally reduced or increased according to the urgency of the treatment situation.

[0184] In the present invention, the subject may be a mammal, such as a human.

[0185] Detection Application

[0186] The antibodies or antigen-binding fragments thereof of the present invention can specifically bind to ADAM9 and thus can be used to detect the presence or level of ADAM9 in a sample.

[0187] Therefore, in another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof of the present invention. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention carries a detectable label. In a preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the present invention. Preferably, the second antibody further comprises a detectable label.

[0188] In the present invention, the detectable label can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Particularly preferably, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In certain embodiments, the detectable labels described above can be attached to the antibodies of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0189] In another aspect, the invention provides methods for detecting the presence or level of ADAM9 in a sample, comprising the step of using an antibody of the present invention or its Fab. In a preferred embodiment, the antibody of the present invention or its Fab also carries a detectable label. In another preferred embodiment, the method further comprises detecting the antibody of the present invention or its Fab using a reagent with a detectable label. The method may be used for diagnostic purposes, or non-diagnostic purposes (e.g., the sample is a cell sample, rather than a sample from a patient).

[0190] In certain embodiments, the method comprises contacting the sample with the antibody or antigen-binding fragment thereof of the present invention under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and ADAM9, and detecting formation of the complex.

[0191] In view of the low expression or non-expression of ADAM9 in normal tissues, the expression or high expression in some cancers, tumors can be diagnosed by detecting the presence or level of ADAM9 in a sample. Therefore, in certain embodiments, the method is used to diagnose tumors, such as ADAM9 positive tumors, such as gliomas, cervical cancer, oral squamous cell carcinoma, liver cancer, melanoma, prostate cancer, pancreatic ductal adenocarcinoma, gastric cancer, lung cancer such as non-small cell lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer such as triple negative breast cancer or its combination in any.

[0192] In certain embodiments, the method comprises detecting the expression level of ADAM9 in a test sample from a subject, and comparing the expression level with a reference value (eg, a healthy control), wherein an increase in the expression level compared to the reference value is indicative of a tumor.

[0193] In another aspect, provided is a use of the antibody or antigen-binding fragment thereof of the present invention in preparing a kit for detecting the presence or level of ADAM9 in a sample and / or diagnosing a tumor.

[0194] In another aspect, the present invention provides a diagnostic or therapeutic kit comprising an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, or multispecific antibody as described herein, and instructions for use. The kit may also include a drug delivery device for topical administration. The drug delivery device may include a prefilled syringe or a needle-free device. Beneficial effects

[0195] The antibodies of the present invention have high binding affinity for ADAM9 and extremely strong specificity, and are devoid of ADCC activity, effectively avoiding side effects caused by ADCC function. The antibodies of the present invention can inhibit tumor cell proliferation and therefore have the potential to be used for the prevention and / or treatment of tumors. Furthermore, the antibodies of the present invention are humanized antibodies that can be safely administered to human subjects without triggering an immunogenic response. Therefore, the antibodies of the present invention have significant clinical value.

[0196] Abbreviations CDR Complementarity determining region of immunoglobulin variable region FR Antibody framework region: amino acid residues in antibody variable region other than CDR residues VH Antibody heavy chain variable region VL Antibody light chain variable region IgG Immunoglobulin G IMGT Based on the international Immunogenetics information system initiated by Lefranc et al. The immunoglobulin (CDR) numbering system is described in Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). Chothia The immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the location of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). The definition of AbM CDR is derived from the research of Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272). mAb monoclonal antibody EC 50The concentration that produces 50% efficacy or binding is IC 50 Concentration that produces 50% inhibition ELISA Enzyme-linked immunosorbent assay PCR Polymerase chain reaction HRP Horseradish peroxidase K D Equilibrium dissociation constant Ka Association rate constant Kd Dissociation rate constant ADCC Antibody-dependent cell-mediated cytotoxicity CDC Complement-dependent cytotoxicity FACS Flow cytometry CDR-H1 Complementarity determining region 1 of the immunoglobulin heavy chain variable region CDR-H2 Complementarity determining region 2 of the immunoglobulin heavy chain variable region CDR-H3 Complementarity determining region 3 of the immunoglobulin heavy chain variable region CDR-L1 Complementarity determining region 1 of the immunoglobulin light chain variable region CDR-L2 Complementarity determining region 2 of the immunoglobulin light chain variable region CDR-L3 Complementarity determining region 3 of the immunoglobulin light chain variable region

[0197] definition

[0198] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, immunology, and other laboratory procedures used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0199] As used herein, the term "antibody" is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule can be composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a single domain, CL. The constant domain is not directly involved in antibody-antigen binding but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly variable regions, known as complementarity-determining regions (CDRs), interspersed with more conserved regions known as framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding site. The distribution of amino acids among regions or domains can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0200] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0201] The term "antibody" also includes embodiments in which the heavy chain constant region comprises a C-terminal lysine, or lacks a C-terminal lysine or a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized to a pyroglutamate. Thus, in a composition comprising the antibodies disclosed herein, each antibody therein may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid, or have the N-terminal amino acid cyclized to a pyroglutamate.

[0202] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0203] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be identified according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably identified by the IMGT, Kabat, Chothia or AbM numbering systems.

[0204] The following general rules (published at www.bioinf.org.uk: Professor Andrew CR Martin's research group) can be used to define CDRs in antibody sequences, which include amino acids that specifically interact with amino acids that make up the epitope to which the antibody binds. In rare cases, these generally constant features do not appear; however, Cys residues are the most conserved feature.

[0205] V HThe entire amino acid sequence of a V is generally numbered according to Kabat, and the three CDRs within the variable region may be defined according to any of the above numbering systems. H The amino acid positions in the sequence may be numbered sequentially starting from amino acid position 1 to the end of the sequence, or according to Kabat numbering. H and V L The amino acid positions in are defined according to sequential numbering.

[0206] Amino acid positions in the heavy chain constant region can be numbered sequentially starting from amino acid position 1 and continuing to the end of the sequence, or numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence numbered according to Eu begins at position 118 and ends at position 447. Unless otherwise indicated, amino acid positions in the heavy and light chains described herein are defined according to sequential numbering.

[0207] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0208] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0209] As used herein, the term "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody and binds to the antigen bound by the intact antibody. For example, an antigen-binding fragment can be a polypeptide that is a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology from Domantis), domain antibodies (technology from Ablynx), and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0210] As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length heavy chains" or "heavy chains" and two "full-length light chains" or "light chains." A "full-length heavy chain" or "heavy chain" refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; and, when the full-length antibody is of the IgE isotype, optionally further comprising a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" or "light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between the CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibodies of the present invention can be derived from a single species, such as humans; they can also be chimeric antibodies or humanized antibodies. The full-length antibodies of the present invention comprise two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to the same antigen.

[0211] As used herein, the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and the Fd fragment of the heavy chain (consisting of the VH and CH1 domains); the term "Fab'-SH" refers to a Fab fragment containing a free sulfhydryl group.

[0212] As used herein, the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. An Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.

[0213] As used herein, the term "Fc fragment" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0214] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv.

[0215] As used herein, the term "diabody" means an antibody whose VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0216] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen. In this article, those skilled in the art can use known conventional techniques to obtain an antigen-binding fragment of an antibody (e.g., an antibody provided by the present invention) from a given antibody (e.g., an antibody provided by the present invention), and screen the antigen-binding fragment of the antibody for specificity in the same manner as for the complete antibody.

[0217] As used herein, the term "multispecific antibody" refers to an antibody with multiple different antigen-binding specificities, including, for example, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies. A "bispecific antibody" refers to an antibody with two different antigen-binding specificities, which is a conjugate formed by a first antibody (or a fragment thereof) and a second antibody (or a fragment thereof) or an antibody analog through a coupling arm, and the coupling method includes but is not limited to chemical reaction, gene fusion, and enzymatic reaction. "Multispecific antibodies" include, for example, trispecific antibodies and tetraspecific antibodies. Trispecific antibodies are antibodies with three different antigen-binding specificities, and tetraspecific antibodies are antibodies with four different antigen-binding specificities.

[0218] As used herein, the terms "monoclonal antibody," "single antibody," and "mAb" have the same meaning and are used interchangeably to refer to an antibody or an antibody fragment from a population of highly homologous antibody molecules, that is, a population of identical antibody molecules except for possible spontaneous natural mutations. A monoclonal antibody has high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies, and these different antibodies typically recognize different epitopes on an antigen. In addition, the modifier "monoclonal" only indicates the characteristic of the antibody as being obtained from a population of highly homologous antibodies, and is not to be understood as requiring the antibody to be prepared by any particular method.

[0219] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen. For example, the term "chimeric antibody" may include an antibody in which the heavy and light chain variable regions are derived from a first antibody (e.g., human), while the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., mouse).

[0220] As used herein, the term "humanized antibody" refers to an antibody that can be prepared by replacing a portion of a human antibody with a portion of a non-human antibody prepared by immunizing a mammal other than a human. Typically, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). For example, a humanized antibody can be prepared by transplanting CDR sequences derived from the germline of other mammalian species onto human framework sequences.

[0221] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived.

[0222] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured in terms of the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC). 50 )express.

[0223] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rates of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant, KD (see Davies et al., Annual Rev Biochem, 1990;59:439-473). KD, kon, and kdis values ​​can be measured using any valid method. In certain embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., the ForteBio Octet method). Surface plasmon resonance techniques (e.g., Biacore) or Kinexa can also be used to measure the dissociation constant.

[0224] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0225] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and it includes an origin of replication or an autonomous replication sequence. The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide, which comprises an expression regulatory sequence operably linked to the nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by host cells or in vitro expression systems. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0226] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, NSO cells, Vero cells, Hela cells, COS cells, CHO cells (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44 or CHO-EBNA cells), ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.

[0227] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0228] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0229] The twenty conventional amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0230] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), etc.

[0231] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or entirely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared to the expected survival (if not receiving treatment), extending the survival period.

[0232] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) suffers from a tumor, or is at risk of suffering from the above-mentioned disease.

[0233] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0234] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region), and varies with the antibody isotype. Examples of antibody effector functions include, but are not limited to, Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, half-life / clearance rate of antibodies and antigen-antibody complexes, and the like. Methods for altering the effector functions of an antibody are known in the art, for example, by introducing mutations in the Fc region.

[0235] As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which Ig binds to Fc receptors (FcRs) present on cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, or macrophages), enabling these cytotoxic effector cells to specifically bind to antigen-attached target cells and then kill the target cells by secreting cytotoxins.

[0236] As used herein, the term "complement-dependent cytotoxicity (CDC)" refers to the cytotoxic effect in which complement participates, i.e., specific antibodies bind to corresponding antigens on the cell membrane surface to form a complex that activates the classical complement pathway. The membrane attack complex formed exerts a lytic effect on target cells.

[0237] As used herein, "combination" includes therapies that can be administered separately, such as those formulated separately for separate administration (e.g., they can be provided in a kit), and therapies that can be administered together in a single formulation (i.e., a "co-formulation"). In certain embodiments, the anti-ADAM9 antibodies or antigen-binding fragments thereof of the present invention can be administered sequentially. In other embodiments, the anti-ADAM9 antibodies or antigen-binding fragments thereof can be administered simultaneously. The anti-antibody or antigen-binding fragment thereof of the present invention can be used in combination with at least one other (active) agent in any manner.

[0238] In such combination therapies, various active agents often have different complementary mechanisms of action, and combination therapies may result in synergistic effects. Combination therapies include therapeutic agents that affect immune responses (e.g., enhance or activate responses) and therapeutic agents that affect (e.g., inhibit or kill) tumor / cancer cells. Combination therapies can reduce the likelihood of drug-resistant cancer cells occurring. Combination therapies can allow one or more of the reagents in the reagent to be dosed to reduce or eliminate adverse effects associated with one or more of the reagents. Such combination therapies can have a synergistic therapeutic or preventive effect on potential diseases, disorders, or conditions.

[0239] In this article, ADAM9 positivity was obtained by immunohistochemistry and staining intensity evaluation by professional clinical pathologists.

[0240] The terms "cancer" and "tumor" are used interchangeably to refer to a broad category of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors, or cells that invade neighboring tissues and may spread to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0241] Below in conjunction with embodiment, embodiment of the present invention is described in detail, but those skilled in the art will understand that the following embodiment is only used to illustrate the present invention, and is not intended to limit the scope of the invention. According to the following detailed description of the accompanying drawings and preferred embodiments, various objects and advantages of the present invention will become practicable to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0242] Figure 1: Affinity test results of anti-human ADAM9 mouse antibody and EBC-1 cells.

[0243] Figure 2: Affinity test results of anti-human ADAM9 chimeric and humanized antibodies to EBC-1 cells.

[0244] Figure 3: Affinity test results of anti-human ADAM9 chimeric and humanized antibodies to HCC1806 cells.

[0245] Figure 4: Endocytosis activity detection results of anti-human ADAM9 chimeric and humanized antibodies.

[0246] Figure 5: Specificity detection results of anti-human ADAM9 humanized antibodies.

[0247] Sequence information

[0248] The information of the sequences involved in the present invention is described in the following table:

[0249] Table 1: Sequence description DETAILED DESCRIPTION

[0250] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0251] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0252] Example 1: Preparation of ADAM9 antigen and control antibody protein

[0253] 1.1 Preparation of ADAM9 antigen

[0254] The extracellular sequences of human ADAM9 (UniProt: Q13443) and monkey ADAM9 (UniProt: A0A2K5X4X8) were synthesized at GenScript and constructed into the pTT5 vector. Six histidine residues were introduced at the C-terminus. The recombinant plasmids were extracted and transiently transfected into HEK293 cells for transient expression after sequencing. After 6 days, the cell supernatants were collected and purified to obtain human ADAM9 protein and monkey ADAM9 protein, respectively.

[0255] 1.2 ADAM9 control antibody expression

[0256] The ADAM9 control antibody is derived from hMAB-A (2I.2) in patent CN201980049672. After codon optimization by GenScript, its heavy and light chain constant regions are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively, in the patent application. The antibody heavy and light chain nucleotide sequences were synthesized and cloned into the pTT5 vector. The pTT5 plasmids corresponding to the antibody heavy and light chain nucleotide sequences were then simultaneously transfected into CHOS cells. The cell supernatant was collected by centrifugation and purified using Protein A (MabSelect SuRe, GE) to obtain the control antibody protein.

[0257] 1.3 Preparation of ADAM9-overexpressing cell lines

[0258] Based on the full-length proteins of human ADAM9 (NP_003807.1), monkey ADAM9 (XP_045253681.1), rat ADAM9 (NP_001014772.2), and mouse ADAM9 (NP_001257925.1), Nanjing GenScript Biotechnology Co., Ltd. was commissioned to perform codon optimization and gene synthesis, and constructed into the lentiviral vector pLVX-IRES-puro. The virus was packaged and used to infect BaF3 cells. After pressure screening and flow cytometry verification, overexpression cell lines stably expressing human, monkey, rat, and mouse ADAM9 were obtained.

[0259] Example 2: Preparation of anti-human ADAM9 monoclonal antibodies

[0260] 2.1 Mouse immunization

[0261] Wild-type mice were immunized with the human ADAM9 protein prepared in 1.1. Specifically, Freund's complete adjuvant was used for the first immunization, and then Freund's incomplete adjuvant was used for booster immunization every week. During the immunization period, the serum titer of anti-ADAM9 antibodies was monitored every two weeks by protein ELISA and cell flow cytometry. Specifically, ELISA: human ADAM9-his was diluted to 1 μg / ml with CBS coating solution, 100 μl / well, and incubated at 4°C overnight; washed once with 300 μl PBST, added 100 μl PBS+2% BSA, and blocked at 37°C for 2 hours; discarded the blocking solution, and the serum was diluted 3-fold starting from 100 times with PBS+2% BSA, and incubated at 37°C for 2 hours; washed 3 times with 300 μl PBST; HRP-Goat-Anti-mouse IgG (1:10000) was diluted with PBS (2% BSA), 100 μl was added to the well plate, and incubated at 37°C for 1 hour; washed 5 times with 300 uL PBST; added 80 μl TMB (Huzhou Yingchuang Biotechnology Co., Ltd.), and added 40 μl 2N Stop with H2SO4 and read on a microplate reader at 450 nm. Flow cytometry: HCT116 cells were harvested and washed three times with PBS. Mouse serum was serially diluted with PBS + 1% BSA solution, starting at 50-fold and then three-fold, for a total of 11 concentration points. The volume per well was 50 μL. Blank cell wells and negative mouse serum control wells were also set up. NCI-H226 cells (1×10 5 Cells were then washed three times with PBS (100 μL / well) and resuspended in 100 μL of APC anti-mouse IgG Fc antibody (1 μL / well, 100 μL) diluted in PBS + 1% BSA solution. The cells were then incubated at 4°C for 0.5 h. The cells were then washed three times with PBS and resuspended in 300 μL of PBS for analysis. The optimal mouse hybridoma was selected for hybridoma fusion based on protein binding and cell titer.

[0262] 2.2 Hybridoma fusion screening

[0263] 7 days after hybridoma fusion, supernatant screening was performed using ELISA. Specifically, the coating solution diluted monkey ADAM9 protein to 1 μg / ml, 100 μl / well, and coated overnight at 4°C. Wash once with 300 μL PBST, add 100 μl PBS + 2% BSA to each well, incubate at 37°C for 1 hour, directly take 20 μl of hybridoma supernatant and add it to the ELISA plate, incubate at 37°C for 2 hours. Discard the solution, wash the ELISA plate 3 times with PBST, 320 μl per well. Control the dryness of the ELISA plate, add 100 μL (1:10000) diluted HRP-Goat anti-mouse IgG (Thermo Fisher) to each well, and incubate at 37°C for 1 hour. The solution was discarded, and the plate was washed five times with PBST. The plate was drained and 80 μL of TMB (Huzhou Yingchuang Biotechnology Co., Ltd.) was added to each well in the dark for color development. The reaction was then terminated with 40 μL of 2N H₂SO₄ and the absorbance was read at 450 nm using a microplate reader. Clones with a signal value five times greater than the negative control were further validated using human ADAM9 protein ELISA and flow cytometry using the overexpressing cells prepared in 1.3. Positive clones were subcloned, and single clones were screened using the above method. The optimal clone was selected for expansion and culture.

[0264] Example 3: Evaluation of anti-human ADAM9 mouse antibodies

[0265] 3.1 Evaluation of Binding Activity of Anti-Human ADAM9 Mouse Antibodies

[0266] All preferred monoclones were amplified and cultured in serum-free culture. 5-10 ml of culture supernatant was affinity purified using Protein-A beads. The eluted product was neutralized with Tris solution. The antibody protein concentration was quantified using Nanodrop and then used for candidate evaluation.

[0267] StemProTM Accutase (Gibco) solution was used to digest adherent lung squamous cell carcinoma cells EBC-1 (Nanjing Kebai), count and take appropriate amount of cells, wash twice with 1xPBS, resuspend in 1% BSA solution, and then transfer the cells to 96-well conical bottom plates, 50μL per well; dilute the candidate antibody with 1% BSA, starting at 10μg / mL, 3-fold serial dilution, then take 50μL of the diluted antibody to the conical bottom plate containing cells, incubate at 4 degrees for 45min; wash the cells twice with PBS, then add 50μL of diluted secondary antibody to each well, mix well, and incubate at 4 degrees for 30min; wash the cells twice with PBS, then resuspend the cells in 400μL PBS and detect on flow cytometry. Data processing: Export the Median PE value and then import it into GraphPad Prism 6 software to calculate EC 50,The results are shown in Figure 1 and Table 2. ,The affinity of Ab01 to Ab04 antibodies is high, all at the 10E-10 level.

[0268] Table 2: Affinity determination of anti-human ADAM9 mouse antibody and EBC-1 cells

[0269] 3.2 Sequencing of anti-human ADAM9 mouse antibodies and construction of chimeric antibodies

[0270] After the hybridoma cells reached approximately 8,000 cells, they were lysed and first-strand cDNA was synthesized using a cDNA reverse transcription kit (Thermo Fisher). The VH and VK genes were amplified from the cDNA using primers by PCR and sequenced. The resulting anti-human ADAM9 antibody variable region sequences are shown in Table 3.

[0271] Table 3. Anti-human ADAM9 mouse antibody variable region and CDR amino acid sequences

[0272] Example 4: Evaluation of humanization of anti-human ADAM9 antibodies

[0273] 4.1 Humanization and expression of anti-human ADAM9 antibodies

[0274] Murine antibodies Ab01, Ab02, Ab03, and Ab04 were humanized using a CDR-grafted antibody humanization method. Briefly, humanization involves the following steps: The amino acid sequences of the murine monoclonal antibodies are compared with those of human germline antibodies to identify sequences with high homology and superior physicochemical properties, which serve as human germline framework sequences; HLA-DR affinity is analyzed and examined to select human germline framework sequences with low affinity; and the six CDRs of the murine antibodies are then transplanted onto the selected heavy and light chain framework sequences.

[0275] Computer simulations and molecular docking were then used to analyze the variable region and its surrounding framework amino acid sequences, examining their spatial binding patterns. By calculating electrostatic forces, van der Waals forces, hydrophilicity, and entropy, the key amino acids in the mouse antibody's amino acid sequence that interact with the ADAM9 protein and maintain the spatial framework were identified. These mouse amino acids were then retained in the transplanted antibody. Specifically, a series of back mutations were performed on the amino acid residues in the FR region of the humanized template to ensure that the humanized antibody retained the antigen-binding ability of the mouse antibody as much as possible.

[0276] Nanjing GenScript Biotechnology Co., Ltd. was commissioned to perform codon optimization for the humanized antibody. The cDNA was synthesized and ligated into the expression plasmid pTT5. The heavy and light chain expression plasmids for the humanized antibody were simultaneously transfected into CHO-S cells. After 7 days of expression, the supernatant was collected by centrifugation. The recombinant antibody in the supernatant was purified using Protein A (MabSelect SuRe, GE) to obtain a chimeric, humanized anti-human ADAM9 antibody. The variable region and CDR sequences of the humanized antibody are shown in Table 4.

[0277] Table 4. Anti-human ADAM9 humanized antibody variable region and CDR amino acid sequences

[0278] 4.2 Protein affinity detection of humanized anti-human ADAM9 antibody

[0279] The dynamic affinity of chimeric antibodies Ab01-CH, Ab02-CH and humanized antibodies Ab01-HZ43, Ab02-HZ42, Ab03-HZ43, and Ab04-HZ81 to human and monkey ADAM9-His was detected using ForteBio (Pall life sciences).

[0280] The specific method is as follows: the test antibody is diluted to 5 μg / ml with PBST (0.02% Tween 20), and the human and monkey ADAM9-His proteins are diluted in a gradient dilution to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, and 0 nM. The test antibody is then captured with Protein A Senso (Pall life sciences) in PBST (0.02% Tween-20) solution for 60 s, then bound to the human or monkey ADAM9-His protein for 60 s, and then dissociated for 180 s. The results are measured and opened in Data Analysis 11.0 software. The software is opened in 1:1 mode and global fitting mode. The results are analyzed to obtain the association rate, dissociation rate and affinity constant.

[0281] The results are shown in Table 5. The dissociation rate constants (kdis) of Ab04-HZ81, Ab01-HZ43, and Ab02-HZ42 for human ADAM9-His were lower than those of the positive control hMAB-A (2I.2), demonstrating higher dynamic affinity. The dissociation constant and dissociation rate constant of Ab04-HZ81 for monkey ADAM9-His were comparable to those of the positive control hMAB-A (2I.2), demonstrating higher dynamic affinity.

[0282] Table 5 Dynamic affinity test of anti-human ADAM9 humanized antibody and human monkey ADAM9-His

[0283] 4.3 Cellular affinity detection of humanized anti-human ADAM9 antibody

[0284] StemProTM Accutase (Gibco) solution was used to digest adherent lung squamous cell carcinoma EBC-1 (Nanjing Kebai) and human breast cancer cell HCC1806 (Nanjing Kebai). The cells were counted and an appropriate amount of cells were taken, washed twice with 1xPBS, resuspended in 1% BSA solution, and then the cells were transferred to a 96-well conical bottom plate, 50 μL per well; the candidate antibody was diluted with 1% BSA, starting at 10 μg / mL, and diluted 3-fold, and then 50 μL of the diluted antibody was added to the conical bottom plate containing the cells and incubated at 4 degrees for 45 minutes; the cells were washed twice with PBS, and then 50 μL of the diluted secondary antibody was added to each well, mixed, and incubated at 4 degrees for 30 minutes; the cells were washed twice with PBS, and then the cells were resuspended in 400 μL PBS and detected by flow cytometry. Data processing: Median PE values ​​were exported and then imported into GraphPad Prism 6 software to calculate EC 50 ,The results are shown in Figure 2, Figure 3 and Table 6. ,The affinities of Ab01-CH, Ab01-HZ43, Ab02-CH, Ab02-HZ42, Ab03-HZ43 and Ab04-HZ81 antibodies are all high.

[0285] Table 6 Cell affinity detection of anti-human ADAM9 humanized antibodies

[0286] 4.4 Endocytic activity detection of humanized anti-human ADAM9 antibody

[0287] The endocytic activity of anti-human ADAM9 humanized antibodies in human lung squamous cell carcinoma cells EBC-1 (Nanjing Kebai) was detected using a flow cytometer (Thermo, model Attune NxT). The specific steps are as follows: 10,000 EBC-1 cells were plated per well overnight, the antibody to be tested was diluted with culture medium, starting at 0.3 μg / ml, 2-fold serial dilution, pHrodo was diluted with culture medium to 12 μg / ml, the serially diluted antibody and pHrodo were mixed 1:1, incubated at room temperature in the dark for 30 minutes, 50 μL of fresh complete culture medium and 50 μL of labeled antibody were added to the cells, incubated at 37°C, 5% CO2 for 24 hours, and the cells were washed with PBS and detected by flow cytometry. Data processing: The fluorescence signal value was exported and then imported into GraphPad Prism 6 software to calculate EC 50The results are shown in Figure 4 and Table 7. The chimeric antibody Ab01-CH and the corresponding humanized antibody Ab01-HZ43 have the strongest endocytic activity, and the endocytic activity of Ab02-HZ42, Ab03-HZ43, Ab04-HZ81 and the control antibody hMAB-A (2I.2) are similar.

[0288] Table 7 Endocytosis activity detection of anti-human ADAM9 humanized antibodies

[0289] 4.5 Hydrophobicity detection of anti-human ADAM9 humanized antibody

[0290] Hydrophobicity was determined using hydrophobic interaction chromatography (HIC) using a TOSOH TSKgel Butyl-NPR 100*4.6mm, 2.5μm column; mobile phase A: 25mmol / L sodium hydrogen phosphate, pH 7.0, 25% isopropanol; mobile phase B: 1.5mol / L ammonium sulfate. The sample was diluted to 1mg / ml with 0.75mol / L ammonium sulfate, and 40μg was injected. Gradient elution was used, with hydrophilic samples eluting first and hydrophobic samples eluting later. The hydrophilicity of the samples was determined based on their retention times. The results are shown in Table 8: Ab01-HZ43, Ab02-HZ42, Ab03-HZ43, and Ab04-HZ81 exhibited high hydrophilicity, all superior to the control antibody hMAB-A (2I.2).

[0291] Table 8 Hydrophilicity detection of anti-human ADAM9 humanized antibodies

[0292] 4.6 Specificity detection of humanized anti-human ADAM9 antibodies

[0293] To test the specificity of humanized anti-human ADAM9 antibodies, human ADAM9, ADAM12 (Sino Biological), and ADAM15 (Sino Biological) proteins were diluted to 1 μg / ml in CBS coating buffer, and 100 μL / well was coated onto a 96-well microtiter plate and incubated at 4°C overnight. The plate was washed once with PBS, drained, and then 100 μL of 2% BSA was added to each well. The plate was incubated at 37°C for 2 hours, and the blocking buffer was removed. The test antibody was diluted to 10 μg / ml in 2% BSA, and then 100 μL was added to each well of the microtiter plate and incubated at 37°C for 2 hours. The plate was washed three times with PBST (containing 0.2% Tween-20), drained, and then 100 μL of HRP Goat Anti-Human IgG (H+L) (Jackson) was added to each well and incubated at 37°C for 1 hour. The plate was washed five times with PBST (0.2% Tween-20). After the plate was drained, 100 μL of TMB (Huzhou Yingchuang) chromogenic substrate was added to each well. After reacting at room temperature for 3-5 min, 50 μL of 2N H2SO4 was added to each well to terminate the color reaction. The absorbance at OD450 nm was read with a microplate reader.

[0294] The results are shown in Figure 5 , and all the ADAM9 antibodies tested only bound to ADAM9 protein, and did not bind to ADAM12 and ADAM15 proteins.

[0295] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to ADAM9, wherein: The antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): (a) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 54 or 52; and / or, CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO: 55 or 53; (b) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 3 or 1; and / or, CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO: 4 or 2; (c) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 9 or 7; and / or, CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO: 10 or 8; (d) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 50 or 48; and / or, CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO: 51 or 49; or (e) CDR-H1, CDR-H2 and CDR-H3 contained in the following heavy chain variable region (VH), and / or CDR-L1, CDR-L2 and CDR-L3 contained in the following light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation compared to the heavy chain variable region and / or light chain variable region of any one of (a) to (d), and the mutation is a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitution is a conservative substitution; Preferably, the CDRs are defined according to the IMGT, Kabat, Chothia or AbM numbering systems.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 81 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 82 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 83 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 84 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 85 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 76 or a variant thereof; (1b-i) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 21 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 22 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 23 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 24 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 25 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 16 or a variant thereof; (1b-ii) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 21 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 22 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 94 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 24 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 25 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 16 or a variant thereof; (1c-i) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 35 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 36 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 37 or a variant thereof; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 38 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 39 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 30 or a variant thereof; (1c-ii) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 103 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 104 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 37 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 105 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 39 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 30 or a variant thereof; or (1d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 65 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 66 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 67 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 68 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 69 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 70 or a variant thereof; or, (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 71 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 72 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 73 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 74 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 75 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 76 or a variant thereof; (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 11 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 12 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 14 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 15 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 16 or a variant thereof; (2c-i) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 11 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 26 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 27 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 28 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 29 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 30 or a variant thereof; (2c-ii) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 95 or a variant thereof; CDR-H2 of SEQ ID NO: 96 or a variant thereof; CDR-H3 of SEQ ID NO: 27 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 97 or a variant thereof; CDR-L2 of SEQ ID NO: 98 or a variant thereof; CDR-L3 of SEQ ID NO: 30 or a variant thereof; or (2d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 56 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 57 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 58 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 59 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 60 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 61 or a variant thereof; or, (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 79 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 80 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 73 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 74 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 75 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 76 or a variant thereof; (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 19 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 20 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 14 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 15 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 16 or a variant thereof; (3c-i) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 33 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 34 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 27 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 28 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 29 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 30 or a variant thereof; (3c-ii) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 101 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 102 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 27 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 97 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 98 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 30 or a variant thereof; or (3d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 62 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 63 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 64 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 59 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 60 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 61 or a variant thereof; or, (4) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system: (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 77 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 78 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 73 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 74 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 75 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 76 or a variant thereof; (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 17 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 18 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 14 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 15 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 16 or a variant thereof; (4c-i) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 31 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 32 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 27 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 28 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 29 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 30 or a variant thereof; (4c-ii) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 99 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 100 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 27 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 97 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 98 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 30 or a variant thereof; or (4d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 62 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 63 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 64 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 59 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 60 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 61 or a variant thereof; Among them, the variant described in any one of (1a), (1b-i), (1b-ii), (1c-i), (1c-ii), (1d), (2a), (2b), (2c-i), (2c-ii), (2d), (3a), (3b), (3c-i), (3c-ii), (3d), (4a), (4b), (4c-i), (4c-ii), and (4d) has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The antibody or antigen-binding fragment thereof comprises: (ai) a VH comprising the sequence shown in SEQ ID NO: 54 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 55 or a variant thereof; (a-ii) a VH comprising the sequence shown in SEQ ID NO: 52 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 53 or a variant thereof; (bi) a VH comprising the sequence shown in SEQ ID NO: 3 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 4 or a variant thereof; (b-ii) a VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; (ci) a VH comprising the sequence shown in SEQ ID NO: 9 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 10 or a variant thereof; (c-ii) a VH comprising the sequence shown in SEQ ID NO: 7 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 8 or a variant thereof; (di) a VH comprising the sequence shown in SEQ ID NO: 50 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 51 or a variant thereof; or (d-ii) a VH comprising the sequence shown in SEQ ID NO: 48 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 49 or a variant thereof; Wherein, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antibody or antigen-binding fragment thereof is a mouse antibody, a chimeric antibody, or a humanized antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The antibody or antigen-binding fragment thereof further comprises a constant region from or derived from a human immunoglobulin; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region from or derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4); preferably, the antibody or antigen-binding fragment thereof comprises a wild-type Fc region, or comprises a mutated or chemically modified Fc region having altered effector function compared to a wild-type Fc region; Preferably, the antibody or antigen-binding fragment thereof comprises a variant of the human IgG1 heavy chain constant region having the following substitutions compared to the wild-type sequence from which it is derived: Leu234Ala, Leu235Ala and Gly237Ala (positions according to the EU numbering system); Preferably, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region from or derived from a human immunoglobulin (eg, κ or λ); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 5 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 5 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4 or 5 conservative substitutions of amino acids); Preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 6 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 6 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4 or 5 conservative substitutions of amino acids); More preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO:5 and a light chain constant region (CL) as shown in SEQ ID NO:

6.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein The heavy chain constant region (CH) as shown in SEQ ID NO: 5 or a variant thereof lacks the C-terminal lysine.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein The antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising the VH set forth in SEQ ID NO:54 and the heavy chain constant region (CH) set forth in SEQ ID NO:5, and a light chain comprising the VL set forth in SEQ ID NO:55 and the light chain constant region (CL) set forth in SEQ ID NO:6; (2) a heavy chain comprising the VH set forth in SEQ ID NO:52 and the heavy chain constant region (CH) set forth in SEQ ID NO:5, and a light chain comprising the VL set forth in SEQ ID NO:53 and the light chain constant region (CL) set forth in SEQ ID NO:6; (3) a heavy chain comprising the VH shown in SEQ ID NO:3 and the heavy chain constant region (CH) shown in SEQ ID NO:5, and a light chain comprising the VL shown in SEQ ID NO:4 and the light chain constant region (CL) shown in SEQ ID NO:6; (4) a heavy chain comprising the VH shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 5, and a light chain comprising the VL shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 6; (5) a heavy chain comprising the VH set forth in SEQ ID NO:9 and the heavy chain constant region (CH) set forth in SEQ ID NO:5, and a light chain comprising the VL set forth in SEQ ID NO:10 and the light chain constant region (CL) set forth in SEQ ID NO:6; (6) a heavy chain comprising the VH set forth in SEQ ID NO:7 and the heavy chain constant region (CH) set forth in SEQ ID NO:5, and a light chain comprising the VL set forth in SEQ ID NO:8 and the light chain constant region (CL) set forth in SEQ ID NO:6; (7) a heavy chain comprising the VH set forth in SEQ ID NO:50 and the heavy chain constant region (CH) set forth in SEQ ID NO:5, and a light chain comprising the VL set forth in SEQ ID NO:51 and the light chain constant region (CL) set forth in SEQ ID NO:6; or (8) A heavy chain comprising the VH shown in SEQ ID NO:48 and the heavy chain constant region (CH) shown in SEQ ID NO:5, and a light chain comprising the VL shown in SEQ ID NO:49 and the light chain constant region (CL) shown in SEQ ID NO:

6.

8. The antibody or antigen-binding fragment thereof according to claim 3 or 7, wherein The N-terminal glutamine of the VH comprising a sequence as shown in SEQ ID NO:54, SEQ ID NO:52, SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:7, SEQ ID NO:50 or SEQ ID NO:48 or a variant thereof undergoes cyclization to form pyroglutamate or pyroglutamate salt.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein The antibody or antigen-binding fragment thereof comprises: (1) a heavy chain having the sequence shown in SEQ ID NO:88 and a light chain having the sequence shown in SEQ ID NO:89; (2) a heavy chain having the sequence shown in SEQ ID NO:40 and a light chain having the sequence shown in SEQ ID NO:41; (3) a heavy chain having the sequence shown in SEQ ID NO:42 and a light chain having the sequence shown in SEQ ID NO:43; or, (4) A heavy chain having the sequence shown in SEQ ID NO:86 and a light chain having the sequence shown in SEQ ID NO:

87.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein The N-terminal glutamine of the heavy chain having a sequence as shown in SEQ ID NO: 88, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 86 or a variant thereof undergoes cyclization to form pyroglutamate or pyroglutamate salt.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein: The antibody or antigen-binding fragment thereof is selected from ScFv, Fab, Fab', Fab'-SH, F(ab')2, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody and multispecific antibody.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein The antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof is detectably labeled, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance) or biotin.

13. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof has one or more characteristics selected from the following: (1) less than about 100 ng / mL, such as less than about 80 ng / mL, 50 ng / mL, 20 ng / mL, 15 ng / mL, 14 ng / mL, 13 ng / mL, 12 ng / mL, 11 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL or less EC 50 Binds to ADAM9 (eg, human or monkey ADAM9); preferably, the EC 50 Measured by ELISA; (2) binds to ADAM9 (e.g., human or monkey ADAM9) with a KD of less than about 100 nM, such as less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 5 nM or less; preferably, the KD is determined by biofilm interferometry (BLI) (e.g., ForteBio ) measured; (3) having CDC activity, such as inducing killing of cells expressing ADAM9 (such as tumor cells) through CDC; (4) No ADCC activity; (5) inducing ADAM9 internalization, e.g., as measured by flow cytometry; (6) does not bind to ADAM12 and ADAM15 proteins; (7) inhibiting cell (such as tumor cell) proliferation; and / or (8) Inhibit tumor growth.

14. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof, the heavy chain and / or light chain thereof, or the heavy chain variable region and / or light chain variable region thereof according to any one of claims 1 to 13.

15. The isolated nucleic acid molecule of claim 14, comprising a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein: (a) the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence of SEQ ID NO:92, (ii) a sequence substantially identical to SEQ ID NO:92 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:92), or (iii) a degenerate sequence of (i) or (ii) above; and / or the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of SEQ ID NO:93, (v) a sequence substantially identical to SEQ ID NO:93 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:93), or (vi) a degenerate sequence of (iv) or (v) above; or (b) the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence of SEQ ID NO:44, (ii) a sequence substantially identical to SEQ ID NO:44 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:44), or (iii) a degenerate sequence of (i) or (ii) above; and / or the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of SEQ ID NO:45, (v) a sequence substantially identical to SEQ ID NO:45 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:45), or (vi) a degenerate sequence of (iv) or (v) above; or (c) the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence of SEQ ID NO:46, (ii) a sequence substantially identical to SEQ ID NO:46 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:46), or (iii) a degenerate sequence of (i) or (ii) above; and / or, the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence of SEQ ID NO:47, (v) a sequence substantially identical to SEQ ID NO:47 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:47), or (vi) a degenerate sequence of (iv) or (v) above; or (d) the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:90, (ii) a sequence substantially identical to SEQ ID NO:90 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:90), or (iii) a degenerate sequence of the above (i) or (ii); and / or the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence shown in SEQ ID NO:91, (v) a sequence substantially identical to SEQ ID NO:91 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:91), or (vi) a degenerate sequence of the above (iv) or (v).

16. A vector comprising the nucleic acid molecule according to any one of claims 14 to 15; preferably, the vector is a cloning vector or an expression vector.

17. A host cell comprising the nucleic acid molecule according to any one of claims 14 to 15 or the vector according to claim 16.

18. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, comprising culturing the host cell according to claim 17 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

19. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 and a conjugated moiety connected thereto; Preferably, the coupling moiety is selected from a detectable label (such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme) or a therapeutic agent (such as a cytotoxic agent, a cytokine, a toxin or a radionuclide).

20. A multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13; Preferably, the multispecific antibody comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 as a first antigen-binding domain, and further comprises at least one second antigen-binding domain for other targets; Preferably, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

21. A chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof (eg, ScFv) of any one of claims 1-13, a transmembrane domain, and one or more intracellular T cell signaling domains.

22. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the isolated nucleic acid molecule according to claim 14 or 15, or the vector according to claim 16, or the host cell according to claim 17, or the conjugate according to claim 19, or the multispecific antibody according to claim 20, or the chimeric antigen receptor according to claim 21 or a host cell expressing the chimeric antigen receptor, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug having anti-tumor activity; Preferably, the additional pharmaceutically active agent is selected from: ADAM9 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic drugs or any combination thereof; Preferably, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as mixed components.

23. A diagnostic or therapeutic kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the isolated nucleic acid molecule according to claim 14 or 15, or the vector according to claim 16, or the host cell according to claim 17, or the conjugate according to claim 19, or the multispecific antibody according to claim 20, or the chimeric antigen receptor according to claim 21 or a host cell expressing the chimeric antigen receptor, or the pharmaceutical composition according to claim 22, and optionally instructions for use and / or a drug delivery device.

24. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the isolated nucleic acid molecule according to claim 14 or 15, or the vector according to claim 16, or the host cell according to claim 17, or the conjugate according to claim 19, or the multispecific antibody according to claim 20, or the chimeric antigen receptor according to claim 21 or the host cell expressing the chimeric antigen receptor, or the pharmaceutical composition according to claim 22 in the preparation of a medicament for inhibiting cell proliferation (e.g., cells expressing ADAM9, such as tumor cells) or preventing and / or treating and / or adjuvanting tumors, neurodegenerative diseases, retinal diseases or inflammation; Preferably, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or pharmaceutical composition is administered in combination with another pharmaceutically active agent, such as simultaneously, separately or sequentially; Preferably, the additional pharmaceutically active agent is a drug having anti-tumor activity; Preferably, the additional pharmaceutically active agent is selected from: ADAM9 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic drugs or any combination thereof.

25. The use of claim 24, wherein the tumor is an ADAM9-positive tumor; Preferably, the tumor is selected from glioma, cervical cancer, oral squamous cell carcinoma, liver cancer, melanoma, prostate cancer, pancreatic ductal adenocarcinoma, gastric cancer, lung cancer such as non-small cell lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer such as triple-negative breast cancer or any combination thereof.

26. A method for inhibiting cell proliferation, comprising contacting the cell with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the isolated nucleic acid molecule according to claim 14 or 15, or the vector according to claim 16, or the host cell according to claim 17, or the conjugate according to claim 19, or the multispecific antibody according to claim 20, or the chimeric antigen receptor or the host cell expressing the chimeric antigen receptor according to claim 21, or the pharmaceutical composition according to claim 22; Preferably, the cell is a cell expressing ADAM9, such as a tumor cell; Preferably, the tumor cells overexpress ADAM9.

27. A method for preventing and / or treating and / or adjuvanting tumors, neurodegenerative diseases, retinal diseases or inflammation in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the isolated nucleic acid molecule according to claim 14 or 15, or the vector according to claim 16, or the host cell according to claim 17, or the conjugate according to claim 19, or the multispecific antibody according to claim 20, or the chimeric antigen receptor according to claim 21 or the host cell expressing the chimeric antigen receptor, or the pharmaceutical composition according to claim 22.

28. The method of claim 27, further comprising administering to the subject a second therapy selected from surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy, and any combination thereof; Optionally, the second therapy may be applied simultaneously, separately or sequentially with the method of claim 27.

29. The method of claim 27 or 28, wherein The tumor is an ADAM9-positive tumor; Preferably, the tumor is selected from glioma, cervical cancer, oral squamous cell carcinoma, liver cancer, melanoma, prostate cancer, pancreatic ductal adenocarcinoma, gastric cancer, lung cancer such as non-small cell lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer such as triple-negative breast cancer or any combination thereof.

30. A method for detecting the presence or level of ADAM9 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1 to 13 under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and ADAM9, and detecting formation of the complex; Preferably, the method is used to diagnose a tumor, such as an ADAM9-positive tumor, such as a glioma, cervical cancer, oral squamous cell carcinoma, liver cancer, melanoma, prostate cancer, pancreatic ductal adenocarcinoma, gastric cancer, lung cancer such as non-small cell lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer such as triple-negative breast cancer, or any combination thereof; Preferably, the method comprises detecting the expression level of ADAM9 in a test sample from a subject, and comparing the expression level with a reference value, wherein an increase in the expression level compared to the reference value is indicative of a tumor.

31. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or the isolated nucleic acid molecule according to claim 14 or 15, or the vector according to claim 16, or the host cell according to claim 17, or the conjugate according to claim 19, or the multispecific antibody according to claim 20 in the preparation of a detection kit for detecting the presence or level of ADAM9 in a sample and / or diagnosing a tumor; Preferably, the tumor is an ADAM9-positive tumor; Preferably, the tumor is selected from glioma, cervical cancer, oral squamous cell carcinoma, liver cancer, melanoma, prostate cancer, pancreatic ductal adenocarcinoma, gastric cancer, lung cancer such as non-small cell lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, breast cancer such as triple-negative breast cancer or any combination thereof.

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