Antibodies to adam9 and uses thereof

Abstract

The invention relates to an anti-ADAM9 antibody or antigen binding fragment, and a preparation method and medical application thereof. Specifically, the invention provides a pharmaceutical composition containing the humanized anti-ADAM9 antibody and a pharmaceutical composition containing the humanized anti-ADAM9 antibody or an antigen binding fragment thereof, and application of the pharmaceutical composition as an anti-cancer drug. The anti-ADAM9 antibody or the antigen binding fragment of the anti-ADAM9 antibody has a good endocytosis effect, and is suitable for being coupled with a drug to construct an ADC (Analog to Digital Converter); the affinity and endocytosis of the ADAM9 antibody are superior to those of an ADAM9 antibody of IMGC936 which is clinically used at present; while high specificity and killing activity are maintained, the immunogenicity is lower, and the stability is higher. The anti-ADAM9 antibody or antigen binding fragment disclosed by the invention has better potential as an anti-cancer drug, and ensures medication safety.

Description

Technical Field

[0001] The present disclosure belongs to the fields of genetic engineering and immunology, and relates to antibodies against ADAM9 and their uses. Specifically, it relates to an anti-ADAM9 antibody or its antigen-binding fragment that specifically binds to the human ADAM9 receptor, a humanized anti-ADAM9 antibody, a pharmaceutical composition comprising the humanized anti-ADAM9 antibody and its antigen-binding fragment, and its uses as a cell growth inhibitor and an anti-cancer drug, and for detecting or diagnosing tumors. Background Art

[0002] The a disintegrin and metalloproteinase (ADAM) protein family consists of 33 members that regulate a series of intracellular physiological processes, including cell fusion, cell adhesion, and migration (The ADAM Metalloproteinases. Molecular Aspects of Medicine, 2008, 29, 258 - 289.). ADAMs are a class of proteases with sheddase function that can regulate corresponding cell physiological processes by modulating the shedding of extracellular cytokines, growth factors, and receptors. Among them, approximately half of the ADAMs mainly associated with the cell membrane have proteolytic ability and have been found to regulate membrane-active cytokines and growth factors, their receptors, and cell adhesion molecules, which are significant for many aspects of tumorigenesis (The ADAM: Signalling Scissors in the Tumour Microenvironment. Nature Reviews Cancer, 2008, 8, 929 - 941.). In addition, there are many examples of the expression or upregulation of various ADAMs in tumor tissues and cell lines (Emerging Roles of ADAM and ADAMTS Metalloproteinases in Cancer. Biochimie, 2008, 90, 369 - 379.).

[0003] As a member of the ADAM family, ADAM9 is widely involved in the occurrence, development, invasion, metastasis and prognosis of various malignant tumors such as liver cancer, breast cancer, lung cancer, gastric cancer, kidney cancer, prostate cancer, etc. (ADAM9 Silencing Inhibits Breast TumorCell Invasion in Vitro. Biochimie, 2013, 95, 1371-1378.; Increased Expression of aDisintegrin and Metalloprotease-9in Hepatocellular Carcinoma: Implications forTumor Progression and Prognosis. Japanese Journal of Clinical Oncology, 2010, 40, 645-651.). Based on its potential to shed some membrane-bound ligands that can stimulate migration, adhesion and proliferation, the prognostic and diagnostic value of ADAM9 for different solid tumors has been determined. Currently, the identified substrates of ADAM9 include amyloid precursor protein (APP), heparin-binding epidermal growth factor (HB-EGF), collagen XVII, tumor necrosis factor-p75, fibronectin, fibroblast growth factor receptor 2IIIB, insulin β-chain and gelatin, and these substrates are involved in the pathological environment of various diseases including cancer. Overexpression of ADAM9 in solid tumors is associated with invasive phenotype tumors and poor clinical prognosis. For example, ADAM9 mRNA expression is related to the tumor grade and histological type of glioma. In patients with low-grade neoplasia, there is a significant correlation between high expression of ADAM9 and poor clinical outcomes. Studies have shown that ADAM9 expression can be used as a prognostic indicator for patients with low-grade neoplasia and is also a potential therapeutic target. Kim et al. found that ADAM9 plays an important role in the proliferation and invasion of gastric cancer, and ADAM9 may be an effective therapeutic target for advanced gastric cancer (The Effect ofDisintegrin-Metalloproteinase ADAM9 in Gastric Cancer Progression. MolecularCancer Therapeutics, 2014, 13, 3074-3085.). Mechanistically, ADAM9 may drive tumor progression through non-proteolytic or proteolytic mechanisms. The proteolytic mechanism may involve the shedding or processing of cell surface proteins, such as CDCP1 or MHC class I polypeptide-related sequence A (MICA), which directly drive tumor growth.

[0004] Based on this, there is an urgent need to develop a new anti-ADAM9 antibody with better binding efficiency for anti-tumor target therapy. SUMMARY OF THE INVENTION

[0005] In order to solve the problems existing in the prior art, the purpose of the present disclosure is to provide an anti-ADAM9 antibody or antigen-binding fragment thereof, a preparation method thereof, and a pharmaceutical use thereof.

[0006] In order to achieve the above purpose, the present disclosure adopts the following specific solutions:

[0007] According to some embodiments of the present disclosure, there is provided an anti-ADAM9 antibody or an antigen-binding fragment thereof, which comprises an antibody light chain variable region and an antibody heavy chain variable region, wherein,

[0008] The light chain variable region comprises: LCDR1 having an amino acid sequence shown in any one of SEQ ID NOs: 7-10 or any variant thereof, LCDR2 having an amino acid sequence shown in any one of SEQ ID NOs: 11-12 or any variant thereof, and LCDR3 having an amino acid sequence shown in any one of SEQ ID NOs: 13-16 or any variant thereof;

[0009] The heavy chain variable region comprises: HCDR1 having an amino acid sequence shown in any one of SEQ ID NOs: 17-19 or any variant thereof, HCDR2 having an amino acid sequence shown in any one of SEQ ID NOs: 20-22 or any variant thereof, and HCDR3 having an amino acid sequence shown in any one of SEQ ID NOs: 23-24 or any variant thereof.

[0010] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or antigen-binding fragment thereof of the present disclosure comprises at least 1 LCDR selected from the following sequences: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15,

[0011] The heavy chain variable region of the antibody comprises at least 1 HCDR selected from the following sequences: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

[0012] In some embodiments, the light chain variable region of an anti-ADAM9 antibody or an antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13.

[0013] In some embodiments, the light chain variable region of an anti-ADAM9 antibody or an antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14.

[0014] In some embodiments, the light chain variable region of an anti-ADAM9 antibody or an antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:14.

[0015] In some embodiments, the light chain variable region of an anti-ADAM9 antibody or an antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15.

[0016] In some embodiments, the heavy chain variable region of an anti-ADAM9 antibody or an antigen-binding fragment thereof according to the present disclosure comprises: HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23.

[0017] In some embodiments, the heavy chain variable region of an anti-ADAM9 antibody or an antigen-binding fragment thereof according to the present disclosure comprises: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22.

[0018] In some embodiments, the heavy chain variable region of an anti-ADAM9 antibody or an antigen-binding fragment thereof according to the present disclosure comprises: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24.

[0019] In some embodiments, the heavy chain variable region of the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises: HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23.

[0020] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; and the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises: HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23.

[0021] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; and the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22.

[0022] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; and the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24.

[0023] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23.

[0024] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; and the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises: HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23.

[0025] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; and the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22.

[0026] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; and the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24.

[0027] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; and the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23. In some embodiments, the light chain variable region of the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; and the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises: HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23.

[0028] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; and the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ IDNO:21, and HCDR3 shown in SEQ ID NO:22.

[0029] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; and the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises: HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ IDNO:21, and HCDR3 shown in SEQ ID NO:24.

[0030] In some embodiments, LCDR1 as shown in SEQ ID NO:8, LCDR2 as shown in SEQ ID NO:12, and LCDR3 as shown in SEQ ID NO:14 according to the present disclosure; the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:19, LCDR2 as shown in SEQ ID NO:22, and HCDR3 as shown in SEQ ID NO:23; the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:19, LCDR2 as shown in SEQ ID NO:22, and HCDR3 as shown in SEQ ID NO:23. In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure comprises: LCDR1 as shown in SEQ ID NO:10, LCDR2 as shown in SEQ ID NO:11, and LCDR3 as shown in SEQ ID NO:15; and the heavy chain variable region of the antibody or its antigen-binding fragment comprises: HCDR1 as shown in SEQ ID NO:17, HCDR2 as shown in SEQ ID NO:20, and HCDR3 as shown in SEQ ID NO:23.

[0031] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure comprises: LCDR1 as shown in SEQ ID NO:10, LCDR2 as shown in SEQ ID NO:11, and LCDR3 as shown in SEQ ID NO:15; and the heavy chain variable region of the antibody or its antigen-binding fragment comprises: HCDR1 as shown in SEQ ID NO:87, HCDR2 as shown in SEQ ID NO:21, and HCDR3 as shown in SEQ ID NO:22.

[0032] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure comprises: LCDR1 as shown in SEQ ID NO:10, LCDR2 as shown in SEQ ID NO:11, and LCDR3 as shown in SEQ ID NO:15; and the heavy chain variable region of the antibody or its antigen-binding fragment comprises: HCDR1 as shown in SEQ ID NO:16, HCDR2 as shown in SEQ ID NO:21, and HCDR3 as shown in SEQ ID NO:24.

[0033] In some embodiments, the light chain variable region of the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure comprises: LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15; and the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23.

[0034] In some embodiments, the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure is selected from: murine antibodies or their antigen-binding fragments, chimeric antibodies or their antigen-binding fragments, human antibodies or their antigen-binding fragments, and humanized antibodies or their antigen-binding fragments.

[0035] In some embodiments, for the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a light chain constant region or a variant thereof derived from human κ chain, λ chain;

[0036] Preferably, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a light chain constant region derived from human κ chain;

[0037] More preferably, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a light chain constant region as shown in SEQ ID NO:5.

[0038] In some embodiments, for the anti-ADAM9 antibody or its antigen-binding fragment according to the present disclosure, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a heavy chain constant region or a variant thereof derived from human IgG1, IgG2, IgG3 or IgG4;

[0039] Preferably, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a heavy chain constant region derived from human IgG1, IgG2 or IgG4;

[0040] More preferably, the anti-ADAM9 antibody or its antigen-binding fragment further comprises a heavy chain constant region as shown in SEQ ID NO:6.

[0041] In some embodiments, an anti-ADAM9 antibody or an antigen-binding fragment thereof according to the present disclosure, wherein the anti-ADAM9 antibody or the antigen-binding fragment thereof comprises a light chain variable region selected from the sequences shown below, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO:25, SEQID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32 or SEQ ID NO:34.

[0042] In some embodiments, an anti-ADAM9 antibody or an antigen-binding fragment thereof according to the present disclosure, wherein the anti-ADAM9 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region selected from the sequences shown below, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO:26, SEQID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:36.

[0043] In preferred embodiments, an anti-ADAM9 antibody or an antigen-binding fragment thereof according to the present disclosure, wherein the anti-ADAM9 antibody or the antigen-binding fragment thereof comprises:

[0044] a light chain variable region shown in SEQ ID NO: 25 and a heavy chain variable region shown in SEQ ID NO: 26;

[0045] a light chain variable region shown in SEQ ID NO: 27 and a heavy chain variable region shown in SEQ ID NO: 28;

[0046] a light chain variable region shown in SEQ ID NO: 27 and a heavy chain variable region shown in SEQ ID NO: 26;

[0047] a light chain variable region shown in SEQ ID NO: 29 and a heavy chain variable region shown in SEQ ID NO: 26;

[0048] a light chain variable region shown in SEQ ID NO: 30 and a heavy chain variable region shown in SEQ ID NO: 31;

[0049] a light chain variable region shown in SEQ ID NO: 32 and a heavy chain variable region shown in SEQ ID NO: 33;

[0050] a light chain variable region shown in SEQ ID NO: 32 and a heavy chain variable region shown in SEQ ID NO: 31;

[0051] The light chain variable region shown in SEQ ID NO: 34 and the heavy chain variable region shown in SEQ ID NO: 35; or

[0052] The light chain variable region shown in SEQ ID NO: 34 and the heavy chain variable region shown in SEQ ID NO: 36.

[0053] In some embodiments, the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure, wherein the anti-ADAM9 antibody or antigen-binding fragment thereof comprises a light chain selected from the following sequences, or a light chain having at least 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46.

[0054] In some embodiments, the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure, wherein the anti-ADAM9 antibody or antigen-binding fragment thereof comprises a heavy chain selected from the following sequences, or a heavy chain having at least 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 or SEQ ID NO: 48.

[0055] In a specific embodiment, the anti-ADAM9 antibody or antigen-binding fragment thereof according to the present disclosure, wherein the anti-ADAM9 antibody comprises:

[0056] (1) The light chain shown in SEQ ID NO: 37 and the heavy chain shown in SEQ ID NO: 38;

[0057] (2) The light chain shown in SEQ ID NO: 39 and the heavy chain shown in SEQ ID NO: 40;

[0058] (3) The light chain shown in SEQ ID NO: 39 and the heavy chain shown in SEQ ID NO: 38;

[0059] (4) The light chain shown in SEQ ID NO: 41 and the heavy chain shown in SEQ ID NO: 38;

[0060] (5) The light chain shown in SEQ ID NO: 42 and the heavy chain shown in SEQ ID NO: 43;

[0061] (6) The light chain shown in SEQ ID NO: 44 and the heavy chain shown in SEQ ID NO: 45;

[0062] (7) The light chain shown in SEQ ID NO: 44 and the heavy chain shown in SEQ ID NO: 43;

[0063] (8) The light chain shown in SEQ ID NO: 46 and the heavy chain shown in SEQ ID NO: 47; or

[0064] (9) The light chain shown in SEQ ID NO: 46 and the heavy chain shown in SEQ ID NO: 48.

[0065] The present disclosure includes variants of the ADAM9 antigen-binding fragment, including functionally equivalent polypeptides that do not significantly affect the properties of such molecules and variants with enhanced or reduced activity. Modification of polypeptides is routine in the art. Examples of modified polypeptides include polypeptides that have conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that do not significantly or detrimentally alter functional activity, or the use of chemical analogs. Amino acid residues that can be conservatively substituted for one another include, but are not limited to: glycine / alanine; serine / threonine; valine / isoleucine / leucine; asparagine / glutamine; aspartic acid / glutamic acid; lysine / arginine; and phenylalanine / tyrosine. These polypeptides also include glycosylated and non-glycosylated polypeptides and polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. Preferably, the amino acid substitutions should be conservative, i.e., the substituted amino acid would have chemical properties similar to the original amino acid. Such conservative substitutions are known in the art. Amino acid modifications can range from altering or modifying one or more amino acids to complete redesign of regions such as variable domains. Changes in variable domains can alter binding affinity and / or specificity. Other methods of modification include using conjugation techniques known in the art, including but not limited to enzymatic means, oxidative substitution, and chelation. Modifications can be used, for example, to attach tags for immunoassays, such as attaching a radioactive moiety for radioimmunoassay. Modified polypeptides are prepared using methods established in the art and can be screened using standard assays known in the art.

[0066] According to some embodiments of the present disclosure, the present disclosure includes fusion proteins that comprise one or more of the light chain variable region (VL) and / or heavy chain variable region (VH) in the ADAM9 antigen-binding fragment of the present invention. In one embodiment, a fusion polypeptide is provided that comprises a light chain, a heavy chain, or both a light chain and a heavy chain. In another embodiment, the fusion polypeptide comprises a heterologous immunoglobulin constant region. In another embodiment, the fusion polypeptide comprises the light chain variable domain and the heavy chain variable domain of an antibody.

[0067] According to some embodiments of the present disclosure, the present disclosure further includes an ADAM9 antibody or antigen-binding fragment (e.g., antibody, diabody, trivalent binding molecule, etc.) conjugated to a diagnostic or therapeutic moiety. For diagnostic purposes, the ADAM9 antibody or antigen-binding fragment of the present disclosure can be conjugated to a detectable substance. Such ADAM9 antigen-binding fragments can be used to monitor and / or predict the development or progression of a disease as part of a clinical testing procedure, such as determining the efficacy of a particular treatment. Examples of detectable substances include various enzymes (e.g., horseradish peroxidase, β-galactosidase, etc.), cofactors (e.g., streptavidin / biotin), fluorescent substances (e.g., 7-hydroxycoumarin, fluorescein, or phycoerythrin), luminescent substances (e.g., luminol), bioluminescent substances (e.g., luciferase or aequorin), radioactive substances (e.g., carbon-14, manganese-54, strontium-85, or zinc-65), positron-emitting metals, and non-radioactive paramagnetic metal ions. The detectable substance can be directly conjugated or linked to the ADAM9 antibody or antigen-binding fragment using techniques known in the art, or indirectly conjugated or linked to the ADAM9 antibody or antigen-binding fragment through an intermediate (e.g., a linker).

[0068] According to some embodiments of the present disclosure, for therapeutic purposes, the ADAM antibody or antigen-binding fragment of the present invention can be conjugated to a therapeutic moiety such as a cytotoxin (e.g., a cell growth inhibitor or a cytocidal agent), a therapeutic agent, or a radioactive metal ion (e.g., an α-emitter). Cytotoxins or cytotoxic agents include any reagent that is harmful to cells, such as Pseudomonas exotoxin, diphtheria toxin, botulinum toxins A to F, ricin, abrin, saporin, and cytotoxic fragments of these reagents. Therapeutic agents include any reagent having a prophylactic or therapeutic treatment effect for treating a disorder. Such therapeutic agents can be chemotherapeutic agents, protein or polypeptide therapeutic agents, and include therapeutic agents having the desired biological activity and / or altering a given biological response. Examples of therapeutic agents include alkylating agents, angiogenesis inhibitors, antimitotic agents, hormonal therapeutic agents, and antibodies for treating cell proliferative disorders. The therapeutic moiety can be directly conjugated or linked to the ADAM9 antibody or antigen-binding fragment using techniques known in the art, or indirectly conjugated or linked to the ADAM9 antibody or antigen-binding fragment through an intermediate (e.g., a linker).

[0069] According to some embodiments of the present disclosure, a polynucleotide is provided that encodes the anti-ADAM9 antibody or antigen-binding fragment of the present disclosure.

[0070] According to some embodiments of the present disclosure, an expression vector is provided that contains the polynucleotide of the present disclosure.

[0071] According to some embodiments of the present disclosure, a host cell is provided, which imports or contains the polynucleotide or expression vector of the present disclosure. In a specific embodiment, the host cell is a bacterium, preferably Escherichia coli.

[0072] In another specific embodiment, the host cell is a yeast, preferably Pichia pastoris.

[0073] In another specific embodiment, the host cell is a mammalian cell, preferably a CHO cell or a HEK293 cell.

[0074] According to some embodiments of the present disclosure, a method for producing an anti-ADAM9 antibody is provided, comprising the steps of culturing the host cell of the present disclosure, isolating the antibody from the culture, and purifying the antibody.

[0075] According to some embodiments of the present disclosure, a pharmaceutical composition is provided, which contains the anti-ADAM9 antibody or its antigen-binding fragment of the present disclosure and a pharmaceutically acceptable excipient, diluent or carrier.

[0076] According to some embodiments of the present disclosure, a detection or diagnostic kit is provided, which contains the anti-ADAM9 antibody or its antigen-binding fragment of the present disclosure and an excipient, diluent or carrier for detection or diagnosis.

[0077] According to some embodiments of the present disclosure, a detection or diagnostic kit is provided, which contains the anti-ADAM9 antibody or its antigen-binding fragment of the present disclosure, and optionally, further comprises one or more reagents for detecting the binding of the anti-ADAM9 antibody or its antigen-binding fragment to ADAM9 or its epitope.

[0078] According to some embodiments of the present disclosure, the use of the anti-ADAM9 antibody or its antigen-binding fragment of the present disclosure, or the above-mentioned pharmaceutical composition in the preparation of a drug is provided, wherein the drug is used for treating or preventing ADAM9-mediated diseases or disorders.

[0079] According to some embodiments of the present disclosure, the use of the anti-ADAM9 antibody or its antigen-binding fragment of the present disclosure, or the above-mentioned pharmaceutical composition in the preparation of a kit is provided, wherein the kit is used for detecting or diagnosing ADAM9-mediated diseases or disorders.

[0080] In some embodiments, according to the above-mentioned use of the present disclosure, the disease or disorder is cancer.

[0081] In a preferred embodiment, the disease or disorder is a cancer expressing ADAM9.

[0082] In a further preferred embodiment, the cancer is selected from bladder cancer, breast cancer (especially triple-negative breast cancer), cervical cancer, colorectal cancer (especially adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma, melanoma or squamous cell carcinoma), brain cancer, esophageal cancer, gastric cancer, head and neck cancer, liver cancer, non-small cell lung cancer (especially squamous cell carcinoma, adenocarcinoma or large cell undifferentiated carcinoma), myeloma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, thyroid cancer, testicular cancer, endometrial cancer, gallbladder cancer.

[0083] In a specific embodiment, the disease or disorder is selected from: gastric cancer, non-small cell lung cancer, prostate cancer, brain cancer, pancreatic cancer, liver cancer, colorectal cancer, triple-negative breast cancer.

[0084] According to some embodiments of the present disclosure, a method for treating or preventing an ADAM9-mediated disease is provided, comprising the step of: administering to a subject a therapeutically effective amount or a prophylactically effective amount of an anti-ADAM9 antibody or an antigen-binding fragment thereof of the present disclosure.

[0085] According to some embodiments of the present disclosure, a method for treating or preventing an ADAM9-mediated disease is provided, comprising the step of: administering to a subject a therapeutically effective amount or a prophylactically effective amount of a pharmaceutical composition of the present disclosure.

[0086] In some embodiments, the subject is suspected of having, has, or is susceptible to an ADAM9-mediated disease, and the ADAM9-mediated disease is selected from bladder cancer, breast cancer (especially triple-negative breast cancer), cervical cancer, colorectal cancer (especially adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma, melanoma or squamous cell carcinoma), esophageal cancer, brain cancer, gastric cancer, head and neck cancer, liver cancer, non-small cell lung cancer (especially squamous cell carcinoma, adenocarcinoma or large cell undifferentiated carcinoma), myeloma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, thyroid cancer, testicular cancer, endometrial cancer, gallbladder cancer.

[0087] The anti-ADAM9 antibody or antigen-binding fragment of the present disclosure can specifically bind to an ADAM9 antigen (or its epitope) and cells expressing ADAM9, and has good species specificity, CDC activity, and ADCC activity, and is a potential tumor treatment antibody.

[0088] In addition, the anti-ADAM9 antibody or its antigen-binding fragment of the present disclosure has good endocytosis and is suitable for conjugation with drugs to construct ADCs. The affinity and endocytosis of the anti-ADAM9 antibody or antigen-binding fragment of the present disclosure are superior to those of the ADAM9 antibody of IMGC936 currently in clinical trials. While maintaining high specificity and killing activity, it has lower immunogenicity and higher stability. The anti-ADAM9 antibody or antigen-binding fragment of the present disclosure has better potential as an anti-cancer drug, ensuring the safety of drug use. Detailed Description of the Invention

[0089] I. Terms

[0090] To make the present disclosure easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure pertains.

[0091] The three-letter and single-letter codes for amino acids used in the present disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0092] As used in the present disclosure, the term "antibody" refers to an immunoglobulin, which is a four-peptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same class of Ig can be further divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain according to the difference in the constant region. Each of the five classes of Ig can have κ chain or λ chain.

[0093] In the present disclosure, the variable region of the antibody light chain of the present disclosure may further include a light chain constant region, and the light chain constant region includes human or murine κ, λ chain or variants thereof.

[0094] In the present disclosure, the variable region of the antibody heavy chain of the present disclosure may further include a heavy chain constant region, and the heavy chain constant region includes human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0095] The sequences of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly and are the variable regions (V regions); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant regions (C regions). The variable region includes three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each variable region of the light chain (VL) and the variable region of the heavy chain (VH) consists of three CDR regions and four FR regions, and the order arranged sequentially from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment described in the present disclosure conform to the known Kabat numbering rules and the Kabat or ABM definition rules (http: / / bioinf.org.uk / abs / ) in terms of quantity and position.

[0096] The term "ADAM9" includes any variant or isotype of ADAM9 naturally expressed by cells. The antibodies of the present disclosure can cross-react with ADAM9 obtained from non-human species. As an alternative, the antibody can also be specific for human ADAM9 and may not exhibit cross-reactivity with other species. ADAM9 or any of its variants or isotypes can be isolated from cells or tissues that naturally express them, or can be produced by recombinant techniques using techniques common in the art and those described herein. Preferably, the anti-ADAM9 antibody targets human ADAM9 with a normal glycosylation pattern.

[0097] The term "recombinant human antibody" includes human antibodies prepared, expressed, created, or isolated by recombinant methods, and the techniques and methods involved are well-known in the art, such as:

[0098] 1. Antibodies isolated from transgenic, transchromosomal animals (such as mice) of human immunoglobulin genes or hybridomas prepared therefrom;

[0099] 2. Antibodies isolated from host cells transformed to express antibodies, such as transfectomas;

[0100] 3. Antibodies isolated from recombinant combinatorial human antibody libraries; and

[0101] 4. Antibodies prepared, expressed, created, or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.

[0102] Such recombinant human antibodies contain variable and constant regions that utilize specific germline immunoglobulin sequences encoded by germline genes of the human species, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.

[0103] The term "murine antibody" in the present disclosure refers to a monoclonal antibody against human ADAM9 prepared according to the knowledge and skills in the art. When preparing, the test subject is injected with the ADAM9 antigen, and then the hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated. In a preferred embodiment of the present disclosure, the murine ADAM9 antibody or its antigen-binding fragment may further comprise a light chain constant region of murine κ, λ chain or its variant, or further comprise a heavy chain constant region of murine IgG1, IgG2, IgG3 or IgG4 or its variant.

[0104] The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutations). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted onto a human framework sequence (i.e., "humanized antibody").

[0105] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting the CDR sequences of a mouse into the framework of the variable region of a human antibody. Humanized antibodies can overcome the drawback of chimeric antibodies that induce a strong immune response due to carrying a large amount of mouse protein components. To avoid a decrease in activity while reducing immunogenicity, minimal back mutations can be made to the variable region of the human antibody to maintain activity.

[0106] The term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen-binding region or variable region (such as one or more CDRs) of the parental antibody. Antibody fragments retain at least some of the binding specificities of the parental antibody. Generally, when expressed on a molar basis, antibody fragments retain at least 10% of the parental binding activity. Preferably, antibody fragments retain at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody for the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab’, F(ab’)2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23: 1126-1136.

[0107] The "Fab fragment" consists of a light chain and the CH1 and variable regions of a heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0108] The "Fc" region contains two heavy chain fragments that include the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0109] The "Fab' fragment" contains a light chain and a portion of a heavy chain that includes the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0110] The "F(ab')2 fragment" contains two light chains and two heavy chains that include portions of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0111] The "Fv region" contains the variable regions from both the heavy and light chains but lacks the constant regions.

[0112] The term "multispecific antibody" is used in its broadest sense and encompasses antibodies with multiepitope specificity. These multispecific antibodies include, but are not limited to: antibodies containing the heavy chain variable region VH and the light chain variable region VL, where the VH-VL unit has multiepitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or different epitopes of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments covalently or non-covalently linked together, etc.

[0113] The term "single-chain antibody" is a single-chain recombinant protein formed by linking the heavy chain variable region VH and the light chain variable region VL of an antibody with a linker peptide, and it is the smallest antibody fragment with a complete antigen-binding site.

[0114] The term "domain antibody fragment" is an immunoglobulin fragment with immunological function that contains only the heavy chain variable region or the light chain variable region chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment can target the same or different antigens.

[0115] The term "bind to ADAM9" in the present disclosure means being able to interact with human ADAM9.

[0116] The term "antigen-binding site" of the present disclosure refers to the three-dimensional spatial site recognized by the antibodies or antigen-binding fragments of the present disclosure.

[0117] The term "epitope" refers to the site on an antigen that specifically binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids or by non-adjacent amino acids juxtaposed through the tertiary folding of a protein. Epitopes formed by adjacent amino acids generally remain after exposure to a denaturing solvent, while epitopes formed by tertiary folding generally are lost after treatment with a denaturing solvent. An epitope typically includes at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0118] The terms "specifically bind" and "selectively bind" as used in the present disclosure mean that an antibody binds to an epitope on a predetermined antigen. Generally, when using human ADAM9 as an analyte and an antibody as a ligand, as measured by surface plasmon resonance (SPR) technology in an instrument, the antibody binds to the predetermined antigen with an equilibrium dissociation constant (KD) of approximately less than 10-7 M or even smaller, and its affinity for binding to the predetermined antigen is at least twice its affinity for binding to a non-specific antigen (such as BSA, etc.) other than the predetermined antigen or a closely related antigen. The term "antibody that recognizes an antigen" can be used interchangeably herein with the term "antibody that specifically binds".

[0119] The term "cross-reactivity" refers to the ability of the antibodies of the present disclosure to bind to ADAM9 from different species. For example, an antibody of the present disclosure that binds human ADAM9 can also bind ADAM9 of another species. Cross-reactivity is measured by detecting specific reactivity with a purified antigen, or binding or functional interaction with cells physiologically expressing ADAM9 in a binding assay (such as SPR and ELISA). Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

[0120] The term "reverse mutation" refers to a mutant that has undergone a first mutation and then, through a second mutation, completely or partially reverts to its original genotype and phenotype.

[0121] The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blockade. Inhibition / blockade of a ligand preferably reduces or alters the normal level or type of activity that occurs upon ligand binding in the absence of inhibition or blockade. Inhibition and blockade are also intended to include any measurable decrease in ligand binding affinity upon contact with an anti-ADAM9 antibody compared to a ligand that has not been contacted with the anti-ADAM9 antibody.

[0122] The term "inhibit growth" (e.g., as it pertains to cells) is intended to include any measurable decrease in cell growth.

[0123] Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as in the Antibody Engineering Laboratory Manual from Cold Spring Harbor, Chapters 5-8 and 15. For example, mice can be immunized with human ADAM9 or a fragment thereof, and the resulting antibodies can be renatured, purified, and their amino acid sequences determined using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments of the invention are engineered to have one or more human FR regions in the non-human CDR regions. Human FR germline sequences can be obtained from the website of ImMunoGeneTics (IMGT) at http: / / imgt.cines.fr, or from the Journal of Immunology (2001, ISBN 012441351).

[0124] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred prior art, mammalian expression systems result in glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. The positive clones are expanded in serum-free medium in a bioreactor to produce the antibody. The culture broth secreting the antibody can be purified and collected using conventional techniques. The antibody can be concentrated by filtration using conventional methods. Soluble aggregates and multimers can also be removed using conventional methods, such as size exclusion chromatography and ion exchange chromatography. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0125] The antibodies of the present disclosure refer to monoclonal antibodies. The monoclonal antibodies (mAbs) described in the present disclosure refer to antibodies obtained from a single clone of cells, and the cell lines are not limited to eukaryotic, prokaryotic, or phage clone cell lines. Monoclonal antibodies or antigen-binding fragments can be recombinantly produced using techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other prior art techniques.

[0126] "Administering", "giving", and "treating", when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, mean the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering", "giving", and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treating a cell includes contacting the cell with a reagent and contacting the reagent with a fluid that contacts the cell. "Administering", "giving", and "treating" also mean treating a cell in vitro and ex vivo by a reagent, diagnostic, binding composition, or by another cell. "Treating", when applied to humans, veterinary medicine, or research subjects, means therapeutic treatment, prophylactic or preventive measures, research, and diagnostic applications.

[0127] "Treatment" means administering to a patient a therapeutic agent, either internally or externally, such as any one of the antibodies of the present disclosure, the patient having one or more symptoms of a disease, and the therapeutic agent being known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered to the treated patient or population in an amount effective to relieve one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of the therapeutic agent effective to relieve any particular disease symptom (also referred to as the "therapeutically effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, as well as the ability of the drug to produce the desired effect in the patient. Whether the disease symptoms have been alleviated can be evaluated by any clinical test method commonly used by a doctor or other professional healthcare provider to evaluate the severity or progression of the symptom. Although the embodiments of the present disclosure (such as treatment methods or articles) may be ineffective in relieving the target disease symptoms of each patient, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test method known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0128] The term "consisting essentially of" or variations thereof used throughout the specification and claims means including all of the recited elements or groups of elements, and optionally including other elements of a similar or different nature, which do not significantly alter the basic or novel properties of the specified dosing regimen, method, or composition.

[0129] The term "naturally occurring" as applied to an object in the present disclosure refers to the fact that the object can be found in nature. For example, a polypeptide sequence or polynucleotide sequence that exists in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified in the laboratory by humans is naturally occurring.

[0130] "Effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or disorders of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the method and route of administration and dosage, and the severity of side effects. The effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0131] "Exogenous" refers to a substance that is produced outside of a biological, cellular, or human body, depending on the context.

[0132] "Endogenous" refers to a substance that is produced within a cell, organism, or human body, depending on the context.

[0133] "Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparison is made when the maximum percentage of homology is obtained by aligning the two sequences.

[0134] As used herein, the expressions "cell", "cell line", and "cell culture" are used interchangeably, and all such names include their progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or inadvertent mutations, all progeny may not be precisely identical in DNA content. This includes mutant progeny having the same function or biological activity as screened in the originally transformed cell. In cases where different names are meant, it is clear from the context.

[0135] "Optionally" or "optionally" means that the subsequently described event or circumstance can but does not have to occur, and this description includes the instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1 - 3 variable regions of an antibody heavy chain" means that the variable regions of the antibody heavy chain of a specific sequence can but do not have to be present.

[0136] "Pharmaceutical composition" means a composition containing one or more antibodies or antigen - binding fragments thereof described herein, as well as other components such as a physiological / pharmaceutically acceptable carrier and excipient. The purpose of the pharmaceutical composition is to facilitate administration to an organism, enhance the absorption of the active ingredient, and thereby exert its biological activity.

[0137] The following embodiments are used to further describe the present disclosure, but these embodiments do not limit the scope of the present disclosure. For the experimental methods without specific conditions in the embodiments of the present disclosure, they are generally carried out according to conventional conditions, such as the antibody technology experimental manual of Cold Spring Harbor, the molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturers. The reagents without specific sources are conventional reagents purchased from the market.

[0138] Embodiment

[0139] A further understanding of the present disclosure can be obtained by referring to some specific embodiments given herein. These embodiments are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure in any way. Obviously, various modifications and changes can be made to the present disclosure without departing from the essence of the present disclosure. Therefore, these modifications and changes are also within the scope claimed in this application.

[0140] Embodiment 1: Antigen, Immunization Protocol and Antibody Preparation

[0141] 1. Antigen

[0142] The protein antigen used for immunization is the amino acids at positions 206-297 of human ADAM9 recombinant protein (NCBI Reference Sequence: NP_003807.1) (SEQ ID NO: 1), that is, the extracellular domain of ADAM9 (ADAM9-ECD, SEQ ID NO: 2), with a 6-His tag at the C-terminus (SEQ ID NO: 3), that is, human ADAM9-ECD-His protein (SEQ ID NO: 4), which is provided by Sanyou Biopharmaceuticals (Shanghai) Co., Ltd.

[0143] 2. Immunization Protocol

[0144] The above-mentioned human ADAM9-ECD-His protein was used for animal immunization. A total of 10 Balb / C mice, female, 10 weeks old, were selected, and all the mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. For the first immunization, the antigen was emulsified with Freund's complete adjuvant, and the antigen amount was 100 μg / mouse. For subsequent immunizations, the adjuvant was Freund's incomplete adjuvant, and the antigen amount was 50 μg / mouse. The injection method was intraperitoneal plus subcutaneous multi-point injection. For cellular immunity, the cell amount for the first immunization was 1×10 7 cells / mouse, and the cell amount for subsequent immunizations was 5×10 6 cells / mouse. The injection method was intraperitoneal injection. The number of immunizations was 4 times, and the immunization time was set to be immunized every other week, that is, immunize again one week after immunization. After the last immunization, a booster immunization was carried out 21 days later.

[0145] 3. Serum Titer Detection

[0146] 3.1. Sample Preparation

[0147] The sera of immunized mice were serially diluted with 5% PBST at gradients of 1500, 4500, 13500, and 40500 - fold dilutions.

[0148] 3.2. Detection of serum titer by ELISA

[0149] 1) Coating: 2 μg / mL antigen, 30 μL per well, overnight at 4°C, wash the plate 3 times with PBST.

[0150] 2) Blocking: Block with 5% PBST at room temperature for 2 h, wash the plate 3 times with PBST.

[0151] 3) Primary antibody: Add the serially diluted sera mentioned above, sera from non - immunized mice as negative control, 30 μL per well, incubate at room temperature for 1 h, wash the plate 6 times with PBST.

[0152] 4) Secondary antibody: Add the serum samples diluted with 1:5000 - diluted goat anti - mouse - IgG - HRP (Rockland, cat#609 - 103 - 123) diluted with 5% PBST; add 1:5000 - diluted goat anti - human IgG - HRP (Shanghai Enzyme - linked Biotechnology Co., Ltd., ml087062) to the positive control group, diluted with 5% PBST. 30 μL per well, incubate at room temperature for 50 min, wash the plate 6 times with PBST.

[0153] 5) Termination: Add 30 μL / well of TMB (Suzhou Yakoo Chemical Reagent Co., Ltd., cat#S0025) for color development at room temperature for 5 - 10 min, then add 30 μL / well of 2M termination solution (Suzhou Yakoo Chemical Reagent Co., Ltd.) to terminate the reaction, read the data at OD450 with an ELISA reader.

[0154] Example 2: Preparation of hybridoma cells and acquisition of murine antibodies

[0155] As described above, the sera of the immunized Balb / C mice in Example 1 were detected for titer using human ADAM9 - ECD - His protein to evaluate the serum titer and the ability to bind cell - surface antigens. Based on the control titer detection results (greater than 100,000 - fold dilution), cell fusion was initiated. Immunized mice with strong serum titers were selected for a final immunization and then sacrificed. Spleen cells were fused with SP2 / 0 myeloma cells and plated to obtain hybridomas. Target hybridomas were screened by indirect ELISA and established as monoclonal cell lines by limiting dilution. The obtained positive antibody clones were further screened using indirect ELISA to select hybridomas that bind to the recombinant protein. Log - phase hybridoma cells were collected, and RNA was extracted with Trizol (Invitrogen, 15596 - 018) and reverse - transcribed (PrimeScript TMReverse Transcriptase, Takara #2680A). The cDNA obtained by reverse transcription was amplified by PCR using the mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sequenced, and finally the sequences of murine antibodies X1 and X2 were obtained. The CDR sequences of the variable regions of the heavy and light chains of the murine antibodies are shown in Table 1.

[0156] Table 1. CDR Region Sequences of Murine Antibodies

[0157]

[0158] Example 3: Humanization Experiment of Mouse Antibodies

[0159] The humanization of murine anti-human ADAM9 monoclonal antibodies was carried out as described in many published methods in the art. Briefly, the human constant domains were used to replace the constant domains of the parental (murine antibodies), and the human antibody sequences were selected according to the homology between murine antibodies and human antibodies. In this example, murine antibodies X1 and X2 were humanized.

[0160] Specifically, the human germline sequences were used as the acceptor framework for humanizing murine antibodies X1 and X2. To find the closest germline sequences, the most similar expressed light chain and the most similar heavy chain were identified in the germline sequence database by NCBI IgBLAST (ncbi.nlm.nih.gov / igblast / ). In this search, the CDR sequences of X1 and X2 were masked. The selection criteria for the most suitable expressed sequences included checking the sequence identity of classical residues and interface residues, as well as checking the similarity of CDR loop lengths.

[0161] Based on the obtained typical structures of murine antibody VH / VL CDRs, the variable region sequences of the heavy and light chains were compared with the human antibody germline database to obtain human germline templates with high homology.

[0162] The CDR regions of the murine antibodies were transplanted onto the selected corresponding humanized templates. Then, based on the three-dimensional structure of the murine antibodies, back mutations were made to the buried residues, the residues that directly interact with the CDR regions, and the residues that have an important impact on the conformations of VL and VH, and the chemically unstable amino acid residues in the CDR regions were optimized. After expression testing and comparison of the number of back mutations, antibodies composed of the sequences of the humanized heavy chain variable region HCVR and the light chain variable region LCVR were selected. The CDR sequences of the variable regions of the heavy and light chains are shown in Table 2 below. The specific CDR combinations and sequence information of the humanized antibodies are shown in Table 3 below, and the sequence information of the variable regions of the heavy and light chains of the humanized antibodies is shown in Table 4 below.

[0163] Table 2. CDR Sequences of the Variable Regions of Humanized Antibody Heavy and Light Chains

[0164]

[0165]

[0166] Table 3. Sequences of CDR regions of humanized antibodies

[0167]

[0168] Table 4. Variable region sequences of heavy and light chains of humanized antibodies

[0169]

[0170]

[0171]

[0172] Note: The underlines indicate the CDR sequences of the monoclonal antibody.

[0173] The designed variable region sequences of the heavy and light chains are ligated with the constant region sequences of the IgG1 heavy chain and light chain. Exemplarily, the constant region of the antibody light chain is selected from the constant region of the human κ chain shown in SEQ ID NO:5, and the constant region of the heavy chain is selected from the native constant region of human IgG1 shown in the sequence as SEQ ID NO:6, and the obtained heavy and light chain sequences are shown in Table 5:

[0174] Table 5. Heavy and light chain sequences of humanized antibodies, and heavy and light chain constant region sequences

[0175]

[0176]

[0177]

[0178]

[0179] Note: The underlines indicate the CDR sequences of the monoclonal antibody, and the double underlines indicate the constant region sequences of the monoclonal antibody.

[0180] Example 4: Expression and purification of humanized antibodies

[0181] cDNA fragments were synthesized based on the amino acid sequences of the light and heavy chains of the above-mentioned humanized antibodies and inserted into the pcDNA3.1 expression vector (Life Technologies Cat.No.V790-20). The expression vector and the transfection reagent PEI (Polysciences, Inc. Cat.No.23966) were transfected into HEK293 cells (Life Technologies Cat.No.11625019) at a ratio of 1:2 and incubated in a CO 2 incubator for 4 - 5 days. The cell culture medium was collected, centrifuged and filtered, and then loaded onto an antibody purification affinity column. After washing the column with phosphate buffer, eluting with glycine hydrochloride buffer (pH 2.7 0.1M Gly-HCl), neutralizing with 1M Tris-HCl pH 9.0, and dialyzing with phosphate buffer, the humanized antibody protein of the present disclosure was obtained, and its molecular weight and purity were detected by SDS-PAGE.

[0182] SDS-PAGE experimental method:

[0183] 1. Preparation of purified sample solution: For the non-reduced sample, 1 μg of the sample with the concentration determined by A280 was added with 4× loading buffer, iodoacetamide (final concentration 40 mM), and heated in a dry bath at 75 °C for 10 min. For the reduced sample, 2 μg of the sample was added with 4× loading buffer and DTT (final concentration 5 mM), and heated in a dry bath at 100 °C for 10 min;

[0184] 2. Electrophoresis: 140 V, 75 min, 12% SDS-PAGE separating gel;

[0185] 3. Stain the gel with Coomassie Brilliant Blue, decolorize it, and then scan it with an EPSON V550 color scanner;

[0186] 4. Use Image J to calculate the purity of the reduced band according to the peak area normalization method, or the purity of the sum of the reduced heavy chain and light chain;

[0187] The results of purity and molecular weight are shown in Table 6 below:

[0188] Table 6. Molecular weight and purity of humanized antibody

[0189] Antibody Molecular weight (kDa) Purity (%) H01-1 146.92 ＞95 H01-2 146.78 ＞95 H01-3 146.86 ＞95 H02-1 147.10 ＞95 H02-2 146.80 ＞95 H03-1 147.12 ＞95 H03-2 147.00 ＞95 H04-1 146.26 ＞95 H04-2 146.42 ＞95

[0190] Example 5: In vitro binding affinity of humanized antibody

[0191] The affinity of the humanized antibody was detected according to the method of the ELISA binding experiment described in Example 1. At the same time, the antibody of IMMGC936 from IMMUNOMEDICS was used as a positive control. The detection results are shown in Table 7 below:

[0192] Table 7. Affinity of Humanized Antibodies for Human ADAM9 Antigen (EC 50 )

[0193]

[0194]

[0195] The results showed that the humanized antibodies of the present disclosure all had high affinity for human ADAM9 antigen.

[0196] Example 6: Affinity Kinetic Constants of Humanized Antibodies

[0197] Using GATOR (ProbeLife), probe: ProA (LN 2205052 T5), select the Kientics experimental mode to detect the affinity kinetic constants of humanized antibodies for human ADAM9 antigen.

[0198] Using the Fortebio BLItz instrument to detect the affinity kinetic constants of the Fab 20 antibody for the N protein antigen of SARS-CoV-2.

[0199] 1. Prepare a kinetic buffer (without IgG and protease) with a final concentration of 0.1% BSA + 0.05% Tween 20 in 1×PBS, that is, 1×Q buffer.

[0200] 2. Dilute the ADAM9 antibody to 5 μg / mL with 1×Q buffer.

[0201] 3. Dilute human ADAM9 antigen with 1×Q buffer at a 2-fold gradient ratio to 300 nM - 4.69 nM respectively.

[0202] 4. Turn on the GATOR instrument and related software, and select the Kientics experimental mode.

[0203] 5. Set the analysis program according to Table 8 below.

[0204] Table 8. GATOR Running Method

[0205]

[0206] By fitting the binding and dissociation curves of the humanized ADAM9 antibody and antigen in the fitting mode, the KD values of the humanized ADAM9 antibody and antigen are shown in Table 9 below.

[0207] Table 9. Affinity Kinetic Detection Results

[0208]

[0209]

[0210] Note: KD: Dissociation equilibrium constant; Ka: Association rate constant; Kd: Dissociation rate constant; R 2 : Linear fitting constant; Rmax: Highest response value of curve fitting

[0211] As can be seen from Table 9, the obtained humanized antibodies all have good affinity for ADAM9.

[0212] Example 7: In vitro FACS binding experiment of humanized antibody

[0213] 1. Experimental method

[0214] Culture ADAM9-overexpressing cells (CHO-S cells overexpressing human or monkey ADAM-9, and human colon cancer cells HT-29 expressing ADAM9). After digestion with trypsin, centrifuge to collect the cells, adjust the cell density with FACS buffer (1×PBS containing 2% FBS), and then seed them in a 96-well U-bottom plate, 1×10 5 to 2×10 5 cells per well. Centrifuge at 1200 g for 5 min, discard the supernatant, add 100 μL of antibody solution serially diluted with FACS buffer, and incubate at 4°C for 1 hour. Centrifuge at 1200 g for 5 min, discard the supernatant, wash the cells twice with PBS, then add the working solution of fluorescently labeled secondary antibody prepared with FACS buffer, PE anti-human IgG Fc Antibody ((abcam 98596 1:300) or FITC anti-mouse IgG Antibody (Biolegend, Cat#406001), resuspend the cells at 100 μL per well, and incubate at 4°C for 1 h. Centrifuge at 1200 g for 5 min, discard the supernatant. After washing the cells twice with PBS, resuspend them in PBS, detect the fluorescence signal using a flow cytometer DxFlex, and perform curve analysis to obtain the EC 50 concentration of the antibody binding to the cells.

[0215] Determine the affinity (EC 50 ) of each humanized antibody for ADAM9 antigen-expressing cells. The results are shown in Table 10:

[0216] Table 10. Affinity (EC 50 value) of humanized antibodies for human ADAM9-overexpressing cells

[0217]

[0218] 2. Experimental results

[0219] As can be seen from the experimental results in Table 10, the humanized antibodies have high affinity for stable cell lines overexpressing human or monkey ADAM9 antigen and can bind to human colon cancer cells HT-29.

[0220] Example 8: Endocytosis of Humanized Antibody

[0221] To detect whether the humanized antibody can be co - endocytosed into cells together with human ADAM9 after binding to ADAM9, in this example, human colon cancer cells HT - 29 (CBP60204, purchased from Nanjing Kebai Biotechnology Co., Ltd.) were selected for evaluation.

[0222] 1. Experimental Method

[0223] (1) Place HT29 - pLVX - human - ADAM9 cells at 3000 cells / well, add 50 μL of complete medium of McCoys 5A (Modified) Medium (Hyclone), and incubate at 37 °C for 12 h.

[0224] (2) Dilute Fab - ZAP (Atsbio, IT - 51 - 100) to 4 nM (final concentration) with DMEM medium (Hyclone).

[0225] (3) Dilute the antibody at 1 nM (final concentration) to a series of concentrations (0.01, 0.1, and 1 nM) with Fab - ZAP diluent, add 50 μL of the dilution, and incubate at 37 °C for 72 h.

[0226] (4) Detect cell viability using the cell viability assay kit MTS (Promega Cat.no.G3581, 100 mL, 0.1 mg / mL), incubate at 37 °C for 2 h, and read the absorbance at OD492.

[0227] Use the antibody of IMMUNOMEDICS' IMGC936 as a positive control, and the EC of the endocytosis of the humanized antibody disclosed in this application 50 The results are shown in Table 11 below:

[0228] Table 11. Endocytosis Results of Humanized Antibody

[0229] Antibody <![CDATA[EC 50 Value (nM)]]> IMGC936 0.1153 H01-1 0.06896 H01-2 0.07847 H01-3 0.07017 H02-1 0.0782 H02-2 0.07148 H03-1 0.08748 H03-2 0.1081 H04-1 0.0920 H04-2 0.1078

[0230] 2. Experimental Results

[0231] From the results in Table 11, it can be seen that the humanized antibody of the present invention has good endocytosis in HT29 cells.

[0232] The above-described embodiments merely represent several implementation manners of the present disclosure. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present disclosure, several modifications and improvements can still be made, and these all fall within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure patent shall be subject to the appended claims.

Claims

1. An anti-ADAM9 antibody or an antigen-binding fragment thereof, which comprises a heavy-chain variable region and a light-chain variable region, wherein, the light-chain variable region comprises: LCDR1 having an amino acid sequence shown in any one of SEQ ID NOs: 7-10 or any variant thereof, LCDR2 having an amino acid sequence shown in any one of SEQ ID NOs: 11-12 or any variant thereof, and LCDR3 having an amino acid sequence shown in any one of SEQ ID NOs: 13-16 or any variant thereof; the heavy-chain variable region comprises: HCDR1 having an amino acid sequence shown in any one of SEQ ID NOs: 17-19 or any variant thereof, HCDR2 having an amino acid sequence shown in any one of SEQ ID NOs: 20-22 or any variant thereof, and HCDR3 having an amino acid sequence shown in any one of SEQ ID NOs: 23-24 or any variant thereof.

2. The anti-ADAM9 antibody or an antigen-binding fragment thereof according to claim 1, wherein, the light-chain variable region comprises: (1) LCDR1 shown in SEQ ID NO: 7, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 13, or, (2) LCDR1 shown in SEQ ID NO: 8, LCDR2 shown in SEQ ID NO: 12, and LCDR3 shown in SEQ ID NO: 14, or, (3) LCDR1 shown in SEQ ID NO: 9, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 16; or, (4) LCDR1 shown in SEQ ID NO: 10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 15; the heavy-chain variable region comprises: (1) HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 20, and HCDR3 shown in SEQ ID NO: 23, or, (2) HCDR1 shown in SEQ ID NO: 18, HCDR2 shown in SEQ ID NO: 21, and HCDR3 shown in SEQ ID NO: 22, or, (3) HCDR1 shown in SEQ ID NO: 18, LCDR2 shown in SEQ ID NO: 21, and HCDR3 shown in SEQ ID NO: 24; (4) HCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 22, and HCDR3 shown in SEQ ID NO:

23.

3. The anti-ADAM9 antibody or an antigen-binding fragment thereof according to claim 1 or 2, wherein: (1) The light chain variable region comprises LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23; or, (2) The light chain variable region comprises LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22; or, (3) The light chain variable region comprises LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24; or, (4) The light chain variable region comprises LCDR1 shown in SEQ ID NO:7, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:13; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23; or, (5) The light chain variable region comprises LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23; or, (6) The light chain variable region comprises LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22; or, (7) The light chain variable region comprises LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24; or, (8) The light chain variable region comprises LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:12, and LCDR3 shown in SEQ ID NO:14; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23; or, (9) The light chain variable region comprises LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23; or, (10) The light chain variable region comprises LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22; or, (11) The light chain variable region comprises LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24; or, (12) The light chain variable region comprises LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:16; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23; or, (13) The light chain variable region comprises LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:17, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:23; or, (14) The light chain variable region comprises LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22; or, (15) The light chain variable region comprises LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:24; or (16) The light chain variable region comprises LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:15; the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:

23.

4. The anti-ADAM9 antibody or antigen-binding fragment thereof according to any one of claims 1-3, which is selected from a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a human antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

5. The anti-ADAM9 antibody or antigen-binding fragment thereof according to claim 4, wherein the anti-ADAM9 antibody or antigen-binding fragment thereof further comprises a light chain constant region or variant thereof derived from a human κ chain or λ chain; Preferably, the anti-ADAM9 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human κ chain; More preferably, the anti-ADAM9 antibody or antigen-binding fragment thereof further comprises a light chain constant region shown in SEQ ID NO:

5.

6. The anti-ADAM9 antibody or antigen-binding fragment thereof according to claim 4, wherein the anti-ADAM9 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region or variant thereof derived from human IgG1, IgG2, IgG3, or IgG4; Preferably, the anti-ADAM9 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2, or IgG4; Optionally, the anti-ADAM9 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO:

6.

7. The anti-ADAM9 antibody or antigen-binding fragment thereof according to claim 4, wherein the anti-ADAM9 antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the sequences shown below, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32 or SEQ ID NO: 34; and / or a heavy chain variable region selected from the sequences shown below, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 or SEQ ID NO:

36.

8. The anti-ADAM9 antibody or antigen-binding fragment thereof according to claim 7, wherein the anti-ADAM9 antibody or antigen-binding fragment thereof comprises: a light chain variable region shown in SEQ ID NO: 25 and a heavy chain variable region shown in SEQ ID NO: 26; a light chain variable region shown in SEQ ID NO: 27 and a heavy chain variable region shown in SEQ ID NO: 28; a light chain variable region shown in SEQ ID NO: 27 and a heavy chain variable region shown in SEQ ID NO: 26; a light chain variable region shown in SEQ ID NO: 29 and a heavy chain variable region shown in SEQ ID NO: 26; a light chain variable region shown in SEQ ID NO: 30 and a heavy chain variable region shown in SEQ ID NO: 31; a light chain variable region shown in SEQ ID NO: 32 and a heavy chain variable region shown in SEQ ID NO: 33; a light chain variable region shown in SEQ ID NO: 32 and a heavy chain variable region shown in SEQ ID NO: 31; a light chain variable region shown in SEQ ID NO: 34 and a heavy chain variable region shown in SEQ ID NO: 35; or a light chain variable region shown in SEQ ID NO: 34 and a heavy chain variable region shown in SEQ ID NO:

36.

9. The anti-ADAM9 antibody or antigen-binding fragment thereof according to claim 7, wherein the anti-ADAM9 antibody or antigen-binding fragment thereof contains a light chain selected from the sequences shown below, or a light chain having at least 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46; and / or A heavy chain selected from the heavy chains shown in the following sequences, or a heavy chain having at least 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47 or SEQ ID NO:

48.

10. The anti-ADAM9 antibody or antigen-binding fragment thereof according to claim 9, wherein the anti-ADAM9 antibody comprises: (1) a light chain shown in SEQ ID NO: 37 and a heavy chain shown in SEQ ID NO: 38; (2) a light chain shown in SEQ ID NO: 39 and a heavy chain shown in SEQ ID NO: 40; (3) a light chain shown in SEQ ID NO: 39 and a heavy chain shown in SEQ ID NO: 38; (4) a light chain shown in SEQ ID NO: 41 and a heavy chain shown in SEQ ID NO: 38; (5) a light chain shown in SEQ ID NO: 42 and a heavy chain shown in SEQ ID NO: 43; (6) a light chain shown in SEQ ID NO: 44 and a heavy chain shown in SEQ ID NO: 45; (7) a light chain shown in SEQ ID NO: 44 and a heavy chain shown in SEQ ID NO: 43; (8) a light chain shown in SEQ ID NO: 46 and a heavy chain shown in SEQ ID NO: 47; or (9) a light chain shown in SEQ ID NO: 46 and a heavy chain shown in SEQ ID NO:

48.

11. A polynucleotide encoding the anti-ADAM9 antibody or antigen-binding fragment thereof according to any one of claims 1-10.

12. An expression vector containing the polynucleotide according to claim 11.

13. A host cell into which or containing the polynucleotide according to claim 11 or the expression vector according to claim 12.

14. The host cell according to claim 13, wherein the host cell is selected from bacteria, yeast or mammalian cells; wherein, the bacteria are preferably Escherichia coli; the yeast is preferably Pichia pastoris; the mammalian cells are preferably CHO cells or HEK293 cells.

15. A method for producing an anti-ADAM9 antibody, comprising the steps of: 1) culturing the host cell according to any one of claims 13-14; 2) isolating the antibody from the culture; and 3) purifying the antibody.

16. A pharmaceutical composition containing the anti-ADAM9 antibody or antigen-binding fragment thereof according to any one of claims 1-10, and a pharmaceutically acceptable excipient, diluent or carrier.

17. A detection or diagnostic kit containing the anti-ADAM9 antibody or antigen-binding fragment thereof according to any one of claims 1-10, and optionally further comprising one or more reagents for detecting the binding of the anti-ADAM9 antibody or antigen-binding fragment thereof to ADAM9 or its epitope.

18. Use of the anti-ADAM9 antibody or antigen-binding fragment thereof according to any one of claims 1-10 or the pharmaceutical composition according to claim 16 in the preparation of a drug for treating or preventing an ADAM9-mediated disease or disorder.

19. Use of the anti-ADAM9 antibody or antigen-binding fragment thereof according to any one of claims 1-10 or the pharmaceutical composition according to claim 16 in the preparation of a kit, wherein, the kit is used for detecting, diagnosing, and prognosticating an ADAM9-mediated disease or disorder.

20. The use according to claim 18 or 19, wherein: the disease or disorder is cancer; preferably, the disease or disorder is cancer expressing ADAM9; more preferably, the cancer is selected from bladder cancer, breast cancer (especially triple-negative breast cancer), cervical cancer, colorectal cancer (especially adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma, melanoma or squamous cell carcinoma), brain cancer, esophageal cancer, gastric cancer, head and neck cancer, liver cancer, non-small cell lung cancer (especially squamous cell carcinoma, adenocarcinoma or large cell undifferentiated carcinoma), myeloma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, thyroid cancer, testicular cancer, endometrial cancer, gallbladder cancer; more preferably, the cancer is selected from: gastric cancer, non-small cell lung cancer, prostate cancer, brain cancer, pancreatic cancer, liver cancer, colorectal cancer, triple-negative breast cancer.

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