Antibody against disintegrin and metalloproteinase 9 of human and use thereof

Abstract

Provided are an antibody against a disintegrin and metalloproteinase 9 (ADAM9) of human and the use thereof. The provided antibody can specifically bind to human ADAM9, is rapidly internalized into tumor cells, can effectively block the metalloproteinase activity of ADAM9, and can be conjugated with a small molecule cytotoxic compound to prepare a targeted-killing antibody-drug conjugate, which has great application potential in tumor-targeted therapy, the development of targeted-killing ADCs, combination therapies of targeted therapy and immunotherapy, the development of bispecific or multispecific antibodies, etc.

Description

Antibodies against human disintegrin and metalloproteinase 9 and uses thereof

[0001] Cross-reference to Related Applications

[0002] This patent application claims the priority benefit of Chinese patent application No. CN202411380427.9, filed on September 30, 2024, the entire contents of which are incorporated herein for all purposes. TECHNICAL FIELD

[0003] The present application belongs to the field of antibody drugs, and specifically, the present application relates to antibodies against human ADAM9 and their use for the preparation of a medicament and antibody drug conjugates targeting human ADAM9. BACKGROUND

[0004] A distegrinin and a metalloprotease 9 (ADAM9) is a membrane-anchored protein belonging to the ADAM family of metalloproteases. ADAM9 is widely expressed in the human body, regulates a variety of biological functions, and also plays an important role in various diseases including neurodegenerative diseases, retinal diseases, inflammation and tumors.

[0005] Overexpression of ADAM9 has been found in a variety of cancers. For example, immunohistochemical analysis using commercial antibodies showed that ADAM9 was expressed on most tumors, with some expressing at higher levels, such as lung cancer, triple-negative breast cancer, colorectal cancer, cholangiocarcinoma, esophageal cancer, glioblastoma, glioma, pancreatic cancer, etc., with significantly higher expression than normal tissue (GEPIA2 Database). In addition, ADAM9 was found to be associated with tumor invasiveness and poor prognosis. ADAM9 promotes tumor progression, treatment resistance and tumor metastasis through proteolytic or non-proteolytic pathways.

[0006] Studies have shown that ADAM9 is also widely expressed in a variety of normal human tissues and exists in multiple structural forms, so developing anti-ADAM9 antibodies with tumor differential selection ability is of great significance for improving anti-tumor effects and reducing the impact on normal tissues.

[0007] In addition, based on the important role of ADAM9 in tumor occurrence and progression, ADAM9 has also become one of the hot target for developing antibody-drug conjugate (ADC). Currently, there are ADCs targeting ADAM9 in clinical research stage, among which IMGC936 is an ADC drug targeting ADAM9 developed by MacroGenics in cooperation with ImmunoGen (now acquired by AbbVie). IMGC936 is composed of three parts: a high-affinity humanized monoclonal antibody, a maytansinoid microtubule inhibitor load and a stable tripeptide linker, with a DAR of 2. The I / II phase clinical trial of IMGC936 for advanced malignant solid tumors (NCT04622774) has been completed, and the development has been terminated due to the failure to achieve the expected safety and effectiveness targets. Therefore, there is still a need to develop more effective and safer ADCs targeting ADAM9. SUMMARY

[0008] The technical problem to be solved by the present application is to provide a novel anti-ADAM9 antibody, which should be able to specifically bind to ADAM9 and have higher tumor selectivity, i.e. have stronger binding ability to ADAM9 expressed in tumor cells and weaker binding ability to ADAM9 expressed in normal tissues, ensuring that the drug prepared based on the antibody has better use safety; further, the novel antibody should also have stronger internalization into ADAM9-expressing tumor cells, thereby being suitable for preparing an antibody-drug conjugate, providing an antibody-drug conjugate with better targeting and killing effect on ADAM9-expressing tumor cells.

[0009] To solve the above technical problems, the inventors of the present application provide a series of new antibodies capable of specifically binding to human ADAM9 through hybridoma screening, biological activity characterization of hybridoma antibodies, antibody humanization modification, etc. The new antibodies can be efficiently internalized into tumor cells via binding to ADAM9, and compared with the humanized monoclonal antibody Mab-A (WO2020 / 005945A1, molecular code hMAB-A(2I.2)) in the first global antibody drug conjugate targeting ADAM9, IMGC936, the new antibodies show better anti-tumor effect in vivo after being conjugated with the same toxin compound to prepare ADC. Moreover, the affinity of the antibodies to human ADAM9 is moderate, and the binding of the antibodies to human ADAM9 in normal tissues is significantly lower than that of Mab-A, indicating that the antibodies have better safety potential. In addition, it is found that the anti-ADAM9 antibodies provided in the present application can effectively block the metalloprotease activity of ADAM9, and the activity is stronger than that of Mab-A. Further, the inventors of the present application conjugate the series of new antibodies with small-molecule cytotoxic compounds of specific structure to obtain a group of new antibody drug conjugates, and select an antibody drug conjugate with strong targeting and killing effect.

[0010] Therefore, an object of the present application is to provide an antibody or fragment thereof that specifically binds to ADAM9, particularly ADAM9 expressed on tumor cells. Another object of the present application is to provide an antibody drug conjugate targeting ADAM9 or a salt thereof prepared using the antibody or fragment thereof.

[0011] In the context of the present application, halogen refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

[0012] In the context of the present application, “linker” is used interchangeably with “linker compound” and “linker compound”.

[0013] In the context of the present application, the term “drug-containing linker” refers to a compound obtained by directly or indirectly covalently bonding a drug (e.g., a small-molecule drug, such as a camptothecin compound) to a linker.

[0014] The present application provides the following technical solutions.

[0015] First aspect

[0016] The present application provides an antibody or antigen-binding fragment thereof against disintegrin and metalloproteinase 9 (ADAM9), which can specifically bind to ADAM9, particularly human ADAM9.

[0017] In the context of the present application, the term "ADAM9" encompasses any form of ADAM9, such as active and inactive forms or membrane-bound and soluble forms, unless otherwise specified; and encompasses any structural region of ADAM9, such as the extracellular domain (ECD) and the structural domains comprised therein.

[0018] In the context of the present application, the term "antigen-binding fragment" encompasses various functional fragments of the antibody specifically binding to ADAM9, which retain the binding capacity of the antibody to the antigen and the corresponding biological activity. It is well known in the art that the binding capacity of an antibody to an antigen and the corresponding biological activity can be achieved by fragments of the intact antibody, which can be obtained using routine techniques known to the person skilled in the art and are screened for functionality in the same way as for the intact antibody. For example, antigen-binding fragments of the antibody can be produced by recombinant DNA techniques or by enzymatic or chemical fragmentation of the intact antibody.

[0019] In particular, the anti-ADAM9 antibody or antigen-binding fragment thereof provided by the present application comprises the complementarity determining regions (CDRs) of the heavy chain, i.e. heavy chain CDR1 (H-CDR1), heavy chain CDR2 (H-CDR2), heavy chain CDR3 (H-CDR3), and the complementarity determining regions (CDRs) of the light chain, i.e. light chain CDR1 (L-CDR1), light chain CDR2 (L-CDR2), light chain CDR3 (L-CDR3). According to a particular embodiment of the present application, the anti-ADAM9 antibody or antigen-binding fragment thereof comprises heavy chain CDRs derived from the amino acid sequence shown in any one of SEQ ID NO. 5, SEQ ID NO. 7, and SEQ ID NO. 9 to SEQ ID NO. 17; and / or, the anti-ADAM9 antibody or antigen-binding fragment thereof comprises light chain CDRs derived from the amino acid sequence shown in any one of SEQ ID NO. 6, SEQ ID NO. 8, and SEQ ID NO. 18 to SEQ ID NO. 35.

[0020] The amino acid sequence shown as any one of SEQ ID NO. 5 to SEQ ID NO. 35 provided above is the amino acid sequence of the variable region of the heavy chain (VH) or the variable region of the light chain (VL) of the exemplary antibody provided in the "Best Mode for Carrying Out the Invention" section of the present application. Using the definition tools for antibody heavy chain or light chain complementarity determining regions well known in the art (e.g. Chothia, Kabat, IMGT, Contact, AbM, etc.), one skilled in the art can readily determine the respective contained heavy chain CDRs and light chain CDRs. The heavy chain CDRs and light chain CDRs combinations can be obtained according to the definition tools well known or conventional in the art, and the antibodies or fragments thereof containing each of these heavy chain CDRs and light chain CDRs combinations are within the scope of the present application.

[0021] Preferably, the anti-ADAM9 antibody or antigen-binding fragment thereof provided by the present application comprises the heavy chain CDRs and light chain CDRs from the following pairs of heavy chain variable region and light chain variable region:

[0022] (1) SEQ ID NO. 5 + SEQ ID NO. 6;

[0023] (2) SEQ ID NO. 7 + SEQ ID NO. 8;

[0024] (3) SEQ ID NO. 9 + SEQ ID NO. 8;

[0025] (4) SEQ ID NO. 10 + SEQ ID NO. 8;

[0026] (5) SEQ ID NO. 11 + SEQ ID NO. 8;

[0027] (6) SEQ ID NO. 12 + SEQ ID NO. 25;

[0028] (7) SEQ ID NO. 13 + SEQ ID NO. 25;

[0029] (8) SEQ ID NO. 14 + SEQ ID NO. 25;

[0030] (9) SEQ ID NO. 15 + SEQ ID NO. 25;

[0031] (10) SEQ ID NO. 16 + SEQ ID NO. 25;

[0032] (11) SEQ ID NO. 17 + SEQ ID NO. 25;

[0033] (12) SEQ ID NO. 7+SEQ ID NO. 18;

[0034] (13) SEQ ID NO. 7+SEQ ID NO. 19;

[0035] (14) SEQ ID NO. 7+SEQ ID NO. 20;

[0036] (15) SEQ ID NO. 7+SEQ ID NO. 21;

[0037] (16) SEQ ID NO. 7+SEQ ID NO. 22;

[0038] (17) SEQ ID NO. 7+SEQ ID NO. 23;

[0039] (18) SEQ ID NO. 7+SEQ ID NO. 24;

[0040] (19) SEQ ID NO. 7+SEQ ID NO. 25;

[0041] (20) SEQ ID NO. 7+SEQ ID NO. 26;

[0042] (21) SEQ ID NO. 7+SEQ ID NO. 27;

[0043] (22) SEQ ID NO. 7+SEQ ID NO. 28;

[0044] (23) SEQ ID NO. 7+SEQ ID NO. 29;

[0045] (24) SEQ ID NO. 7+SEQ ID NO. 30;

[0046] (25) SEQ ID NO. 7+SEQ ID NO. 31;

[0047] (26) SEQ ID NO. 7+SEQ ID NO. 32;

[0048] (27) SEQ ID NO. 7+SEQ ID NO. 33;

[0049] (28) SEQ ID NO. 7+SEQ ID NO. 34;

[0050] (29) SEQ ID NO. 7+SEQ ID NO. 35;

[0051] (30) SEQ ID NO. 14 + SEQ ID NO. 29;

[0052] (31) SEQ ID NO. 16 + SEQ ID NO. 29;

[0053] (32) SEQ ID NO. 17 + SEQ ID NO. 29;

[0054] (33) SEQ ID NO. 14 + SEQ ID NO. 31;

[0055] (34) SEQ ID NO. 16 + SEQ ID NO. 31.

[0056] As described above, the CDRs in the above amino acid sequence pairs can be divided according to Kabat, for example, see the examples of the present application.

[0057] Correspondingly, in the anti-ADAM9 antibody or antigen binding fragment thereof provided by the present application, the heavy chain CDRs and light chain CDRs are as follows:

[0058] (1) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences shown in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38 in turn; and L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences shown in SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47 in turn;

[0059] (2) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences shown in SEQ ID NO. 36, SEQ ID NO. 39, SEQ ID NO. 38 in turn; and L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences shown in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47 in turn;

[0060] (3) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences shown in SEQ ID NO. 36, SEQ ID NO. 40, SEQ ID NO. 38 in turn; and L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences shown in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47 in turn;

[0061] (4) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 38, respectively; and, L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47, respectively;

[0062] (5) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 42, respectively; and, L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47, respectively;

[0063] (6) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 43, respectively; and, L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47, respectively;

[0064] (7) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 44, respectively; and, L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47, respectively;

[0065] (8) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, respectively; and, L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 48, SEQ ID NO. 47, respectively;

[0066] (9) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, respectively; and, L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47, respectively;

[0067] (10) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, respectively; and, L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 50, SEQ ID NO. 47, respectively;

[0068] (11) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, respectively; and, L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 51, SEQ ID NO. 49, SEQ ID NO. 47, respectively;

[0069] (12) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, respectively; and, L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 52, respectively;

[0070] (13) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, respectively; and, L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 53, respectively;

[0071] (14) H-CDR1, H-CDR2, H-CDR3, which successively comprise the amino acid sequences represented by SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38; and L-CDR1, L-CDR2, L-CDR3, which successively comprise the amino acid sequences represented by SEQ ID NO. 51, SEQ ID NO. 49, SEQ ID NO. 53;

[0072] (15) H-CDR1, H-CDR2, H-CDR3, which successively comprise the amino acid sequences represented by SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38; and L-CDR1, L-CDR2, L-CDR3, which successively comprise the amino acid sequences represented by SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 54;

[0073] (16) H-CDR1, H-CDR2, H-CDR3, which successively comprise the amino acid sequences represented by SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38; and L-CDR1, L-CDR2, L-CDR3, which successively comprise the amino acid sequences represented by SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 55;

[0074] (17) H-CDR1, H-CDR2, H-CDR3, which successively comprise the amino acid sequences represented by SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38; and L-CDR1, L-CDR2, L-CDR3, which successively comprise the amino acid sequences represented by SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 56;

[0075] (18) H-CDR1, H-CDR2, H-CDR3, which successively comprise the amino acid sequences represented by SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 38; and L-CDR1, L-CDR2, L-CDR3, which successively comprise the amino acid sequences represented by SEQ ID NO. 51, SEQ ID NO. 49, SEQ ID NO. 47;

[0076] (19) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 43, respectively; and L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 51, SEQ ID NO. 49, SEQ ID NO. 47, respectively;

[0077] (20) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 44, respectively; and L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 51, SEQ ID NO. 49, SEQ ID NO. 47, respectively;

[0078] (21) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 38, respectively; and L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 53, respectively; and

[0079] (22) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 43, respectively; and L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 53, respectively.

[0080] As described above, the anti-ADAM9 antibody or antigen-binding fragment thereof provided by the present application specifically binds to disintegrin and metalloproteinase 9 (ADAM9), preferably human ADAM9. Alternatively, the antibody or antigen-binding fragment thereof provided by the present application has or does not have species cross-binding activity to human, cyno, mouse ADAM9. Alternatively, the antibody or antigen-binding fragment thereof provided by the present application is capable of or incapable of blocking the metalloproteinase activity of ADAM9, which can be ADAM9 in membrane-bound or secreted form, such as human ADAM9.

[0081] Preferably, the anti-ADAM9 antibody or antigen-binding fragment thereof provided in the present application comprises a heavy chain variable region and a light chain variable region, both of which comprise the above-mentioned CDRs and the framework regions (FRs) therebetween, and the arrangement of each region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from N-terminal to C-terminal.

[0082] Further preferably, in the anti-ADAM9 antibody or antigen-binding fragment thereof provided in the present application, the heavy chain variable region can comprise the amino acid sequence shown in any one of SEQ ID NO. 5, SEQ ID NO. 7, and SEQ ID NO. 9 to SEQ ID NO. 17, or an amino acid sequence having at least 75% identity to the amino acid sequence; and / or, the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO. 6, SEQ ID NO. 8, and SEQ ID NO. 18 to SEQ ID NO. 35, or an amino acid sequence having at least 75% identity to the amino acid sequence.

[0083] In the context of the present application, the term "at least 75% identity" resulting in at most 25% difference in amino acid sequence can exist in any framework region in the heavy chain variable region or the light chain variable region, or in any domain or sequence other than the heavy chain variable region and the light chain variable region in the antibody or antigen-binding fragment thereof of the present application. The difference can be caused by deletion, addition, or substitution of amino acids at any position, wherein the substitution can be conservative substitution or non-conservative substitution. The "at least 75% identity" covers any percentage of identity between at least 75% identity and 100% identity, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity.

[0084] According to the specific embodiments of the present application, the anti-ADAM9 antibody or antigen-binding fragment thereof provided in the present application comprises a heavy chain variable region and a light chain variable region, which comprises a combination selected from the following amino acid sequences:

[0085] (1) the amino acid sequence shown in SEQ ID NO. 5, or an amino acid sequence having at least 75% identity to the amino acid sequence; and the amino acid sequence shown in SEQ ID NO. 6, or an amino acid sequence having at least 75% identity to the amino acid sequence;

[0086] (2) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence having at least 75% identity to the amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence having at least 75% identity to the amino acid sequence;

[0087] (3) the amino acid sequence set forth in SEQ ID NO. 9, or an amino acid sequence having at least 75% identity to the amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence having at least 75% identity to the amino acid sequence;

[0088] (4) the amino acid sequence set forth in SEQ ID NO. 10, or an amino acid sequence having at least 75% identity to the amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence having at least 75% identity to the amino acid sequence;

[0089] (5) the amino acid sequence set forth in SEQ ID NO. 11, or an amino acid sequence having at least 75% identity to the amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence having at least 75% identity to the amino acid sequence;

[0090] (6) the amino acid sequence set forth in SEQ ID NO. 12, or an amino acid sequence having at least 75% identity to the amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence having at least 75% identity to the amino acid sequence;

[0091] (7) the amino acid sequence set forth in SEQ ID NO. 13, or an amino acid sequence having at least 75% identity to the amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence having at least 75% identity to the amino acid sequence;

[0092] (8) the amino acid sequence set forth in SEQ ID NO. 14, or an amino acid sequence having at least 75% identity to the amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence having at least 75% identity to the amino acid sequence;

[0093] (9) the amino acid sequence set forth in SEQ ID NO. 15, or an amino acid sequence having at least 75% identity to the amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence having at least 75% identity to the amino acid sequence;

[0094] (10) the amino acid sequence set forth in SEQ ID NO. 16, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence at least 75% identical thereto;

[0095] (11) the amino acid sequence set forth in SEQ ID NO. 17, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence at least 75% identical thereto;

[0096] (12) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 18, or an amino acid sequence at least 75% identical thereto;

[0097] (13) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 19, or an amino acid sequence at least 75% identical thereto;

[0098] (14) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 20, or an amino acid sequence at least 75% identical thereto;

[0099] (15) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 21, or an amino acid sequence at least 75% identical thereto;

[0100] (16) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 22, or an amino acid sequence at least 75% identical thereto;

[0101] (17) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 23, or an amino acid sequence at least 75% identical thereto;

[0102] (18) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 24, or an amino acid sequence at least 75% identical thereto;

[0103] (19) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence at least 75% identical thereto;

[0104] (20) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 26, or an amino acid sequence at least 75% identical thereto;

[0105] (21) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 27, or an amino acid sequence at least 75% identical thereto;

[0106] (22) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 28, or an amino acid sequence at least 75% identical thereto;

[0107] (23) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 29, or an amino acid sequence at least 75% identical thereto;

[0108] (24) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 30, or an amino acid sequence at least 75% identical thereto;

[0109] (25) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 31, or an amino acid sequence at least 75% identical thereto;

[0110] (26) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 32, or an amino acid sequence at least 75% identical thereto;

[0111] (27) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 33, or an amino acid sequence at least 75% identical thereto;

[0112] (28) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 34, or an amino acid sequence at least 75% identical thereto;

[0113] (29) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 35, or an amino acid sequence at least 75% identical thereto;

[0114] (30) the amino acid sequence set forth in SEQ ID NO. 14, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 29, or an amino acid sequence at least 75% identical thereto;

[0115] (31) the amino acid sequence set forth in SEQ ID NO. 16, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 29, or an amino acid sequence at least 75% identical thereto;

[0116] (32) the amino acid sequence set forth in SEQ ID NO. 17, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 29, or an amino acid sequence at least 75% identical thereto;

[0117] (33) the amino acid sequence set forth in SEQ ID NO. 14, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 31, or an amino acid sequence at least 75% identical thereto;

[0118] (34) the amino acid sequence set forth in SEQ ID NO. 16, or an amino acid sequence having at least 75% identity to the amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 31, or an amino acid sequence having at least 75% identity to the amino acid sequence.

[0119] Preferably, the anti-ADAM9 antibody provided by the present application can be a murine, rabbit, human antibody, and can also be a murine antibody, a chimeric antibody, or a fully or partially humanized antibody. The anti-ADAM9 antibody can also be referred to as a derivatized antibody, such as an antibody obtained by CDR grafting, affinity maturation, point mutation modification, chemical modification, etc. based on an original murine monoclonal antibody, wherein the chemical modification includes glycosylation, acetylation, pegylation, phosphorylation, amidation, protease cleavage, linkage with a cell ligand or effector molecule, protection and / or blocking of active reaction groups, etc. Preferably, the antigen-binding fragment of the antibody can be any form of fragment of the antibody, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv, etc.

[0120] In addition to the heavy chain and / or light chain variable region, the anti-ADAM9 antibody or antigen-binding fragment thereof provided by the present application further comprises a heavy chain constant region (CH) and / or a light chain constant region (CL), preferably a human or murine heavy chain constant region and / or a light chain constant region. Preferably, the anti-ADAM9 antibody or fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE, and / or a kappa or lambda type light chain constant region.

[0121] According to the specific embodiments of the present application, the anti-ADAM9 antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody. According to the specific embodiments of the present application, the monoclonal antibody comprises a heavy chain constant region sequence of IgG1, such as the human IgG1 heavy chain constant region set forth in SEQ ID NO. 3 or SEQ ID NO. 57; and / or comprises a kappa light chain constant region, such as the human kappa light chain constant region set forth in SEQ ID NO. 4.

[0122] According to the specific embodiments of the present application, the anti-ADAM9 antibody of the present application is a monoclonal antibody. Preferably, the anti-ADAM9 antibody provided by the present application is an immunoglobulin, such as a human IgA, IgD, IgE, IgG or IgM. Further preferably, the antibody is of the human IgG1 subtype or a variant thereof, such as the IgG1-LALA (L234A / L235A) subtype, or the IgG4 subtype or a variant thereof, such as the IgG4-L235E or F234A / L235A subtype.

[0123] The second aspect

[0124] The present application also provides a nucleic acid molecule comprising a nucleotide sequence encoding the anti-ADAM9 antibody or antigen-binding fragment thereof of the present application.

[0125] The "nucleotide sequence encoding the anti-ADAM9 antibody or antigen-binding fragment thereof of the present application" refers to a nucleotide sequence encoding the heavy chain CDRs, light chain CDRs, light chain variable region, heavy chain variable region, heavy chain and / or light chain contained in the antibody or antigen-binding fragment thereof. For example, the nucleic acid molecule provided by the present application comprises a nucleotide sequence encoding each of the heavy chain CDRs and light chain CDRs contained in the above-mentioned antibody or antigen-binding fragment thereof; a nucleotide sequence encoding the heavy chain variable region and light chain variable region contained in the above-mentioned antibody or antigen-binding fragment thereof; or a nucleotide sequence encoding the heavy chain and light chain contained in the above-mentioned antibody or antigen-binding fragment thereof.

[0126] Third aspect

[0127] The nucleic acid molecule of the present application can be cloned into a vector, which in turn transforms or transfects a host cell. Therefore, in a third aspect, the present application also provides a vector comprising the nucleic acid molecule of the present application. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a bacteriophage vector, etc. The vector or nucleic acid molecule of the present application can be used to transform or transfect a host cell for the purpose of preservation or expression of the antibody, etc.

[0128] Fourth aspect

[0129] The present application also provides a host cell comprising the nucleic acid molecule and / or vector of the present application, or the host cell is transformed or transfected by the nucleic acid molecule and / or vector of the present application. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal or animal cell.

[0130] Fifth aspect

[0131] The anti-ADAM9 antibody or antigen-binding fragment thereof provided by the present application can be obtained by any method known in the art. For example, the present application also provides a method for preparing the anti-ADAM9 antibody or antigen-binding fragment thereof, which comprises culturing the host cell provided by the present application under conditions allowing the host cell to express the heavy chain and light chain of the antibody. Optionally, the method further comprises the step of recovering the produced anti-ADAM9 antibody.

[0132] Sixth aspect

[0133] The anti-ADAM9 antibody or antigen-binding fragment thereof provided by the present application can also be directly or indirectly linked to other moieties, such as, for example, heavy chain CDRs, light chain CDRs, heavy chain variable regions, light chain variable regions, heavy chains, light chains of other antibodies; or, for example, small molecule compounds, such as cytotoxic compounds used in antibody drug conjugates; or, for example, moieties that modify the antibody or antigen-binding fragment thereof, such as cell surface receptors, sugars, polymers, and the like.

[0134] Accordingly, the present application provides an antibody drug conjugate targeting ADAM9 or a salt thereof, comprising the anti-ADAM9 antibody or antigen-binding fragment thereof provided by the present application. The antibody drug conjugate can be formed by conjugating the anti-ADAM9 antibody or antigen-binding fragment thereof provided by the present application to a small molecule cytotoxic compound, which can be, for example, a microtubulin inhibitor, a topoisomerase inhibitor, or a DNA binding agent. Preferably, the microtubulin inhibitor is selected from the group consisting of maytansinoid derivatives, Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), Monomethyl Dolastatin 10, Tubulysin derivatives, Cryptophycin derivatives, and Taltobulin. Preferably, the topoisomerase inhibitor is selected from the group consisting of camptothecin compounds such as exatecan and derivatives thereof, and the like, the Doxorubicin metabolite PNU-159682 derivative, and the irinotecan (CPT-11) metabolite SN38 derivative. Preferably, the DNA binding agent is selected from the group consisting of PBD derivatives and Duocarmycin derivatives.

[0135] Further, the present application provides the following preferred embodiments for the antibody drug conjugate.

[0136] First, the present application provides an antibody drug conjugate targeting ADAM9 or a salt thereof, having the structure shown in the general formula In the context of the present application, each of the groups or structures in the general formula is as follows, unless otherwise specified:

[0137] Ab represents the anti-ADAM9 antibody or antigen-binding fragment thereof provided by the present application.

[0138] E L is selected from the group consisting of representing a linkage to Ab via the thiol of a cysteine:

[0139] E L -1a and / or EL -1b:

[0140] M is phenylene or phenylene substituted with one or more substituents, or a chemical bond; in substituted phenylene, the substituents are selected from the group consisting of alkyl (e.g. Ci-6alkyl, preferably Ci-4alkyl), haloalkyl (e.g. haloCi-6alkyl, preferably haloCi-4alkyl, e.g. trifluoromethyl), alkoxy (e.g. Ci-6alkoxy, preferably Ci-4alkoxy, preferably methoxy), halogen, ester, amide and cyano; preferably, M is halogen-substituted phenylene.

[0141] SP1 is selected from Ci-8alkylene, Ci-8cycloalkylene or Ci-21 (preferably Ci-16, more preferably Ci-n, more preferably C5-9) straight chain heteroalkylene comprising 1-11 (preferably 1-6, more preferably 3-5) heteroatoms selected from N, O or S, wherein each of said Ci-8alkylene, Ci-8cycloalkylene and Ci-21 straight chain heteroalkylene is independently optionally substituted with one or more substituents selected from hydroxyl, amino, sulfonic acid and cyano.

[0142] SP2 is selected from -NH(CH2CH2O) a CH2CH2CO-, -NH(CH2CH2O) a CH2CO-, -S(CH2) a CO- or a chemical bond, wherein a is an integer from 1 to 20, preferably an integer from 1 to 10, more preferably an integer from 1 to 6.

[0143] A represents a short peptide structure of 2-4 amino acids. When A represents a short peptide structure of 2 amino acids, it can be NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO, NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO or NH-Val-Cit-CO; when A represents a short peptide structure of 3 amino acids, it can be NH-Glu-Val-Ala-CO, NH-Glu-Val-Cit-CO or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO; when A represents a short peptide structure of 4 amino acids, it can be NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-Gly-Gly-CO, preferably NH-Gly-Gly-Phe-Gly-CO. Preferably, A is NH-Val-Ala-CO, NH-Gly-Gly-Phe-Gly-CO or NH-Ala-Ala-Ala-CO, NH represents the amino terminus of group A, and CO represents the carboxyl terminus of group A. Group A can be linked to SP2 via the amino group at the amino terminus of its short peptide structure.

[0144] In this formula, preferably M can be a halogen-substituted phenylene, in particular a fluorine-substituted phenylene. In this formula, preferably SP1 can be a C1-11, preferably C5-9, more preferably C7 straight chain heteroalkylene comprising 1-6, preferably 3-5, more preferably 4 heteroatoms selected from N, O or S. In this formula, preferably SP2 can be a chemical bond.

[0145] m can be 1-10, preferably 1-8 (e.g. 1-5), more preferably 3-8; and m can be an integer or a non-integer.

[0146] D represents a small molecule cytotoxic compound, as defined above.

[0147] In this formula, group may be selected from the following structures, in which the wavy line indicates attachment to a cysteine in the antibody or attachment to a small molecule cytotoxic compound D:

[0148] Further, the present application provides an antibody drug conjugate targeting ADAM9 or a salt thereof, which is formed by conjugating an anti-ADAM9 antibody or an antigen binding fragment thereof provided by the present application with a camptothecin compound. Accordingly, in the general formula, D represents a camptothecin compound.

[0149] The structure of the camptothecin compound can be shown as the structural formula I:

[0150] In the structural formula I, R1, R2, R3, R4 are independently hydrogen, halogen, hydroxyl, C1-6alkoxy, amino or substituted amino, C1-7alkyl or substituted C1-7alkyl, or any two of R1, R2, R3, R4 together with the carbon atom to which they are attached form a C3-6 (preferably C3-5) cyclic alkyl group. When R1, R2, R3, R4 are independently C1-6alkoxy, the C1-6alkoxy includes straight chain or branched C1-6alkoxy, preferably straight chain or branched C1-3alkoxy, more preferably methoxy. When R1, R2, R3, R4 are independently substituted amino, the substituted amino is amino substituted with one or more substituents selected from methyl and ethyl. When R1, R2, R3, R4 are independently C1-7alkyl or substituted C1-7alkyl, the C1-7alkyl or substituted C1-7alkyl includes straight chain or branched C1-7 (preferably C3-5, more preferably C4) alkyl or substituted C1-7 (preferably C3-5, more preferably C4) alkyl, and the substituted C1-7alkyl is C1-7alkyl substituted with one or more substituents selected from cyclopropyl and cyclobutyl; or the straight chain or branched C1-7alkyl or substituted C1-7alkyl is preferably C1-3alkyl or substituted C1-3alkyl, such as methyl, halomethyl (preferably trifluoromethyl).

[0151] In the structural formula I, G is hydrogen, halogen, methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.

[0152] In the structural formula I, Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene. The substituted methylene can be substituted with one hydrogen or both hydrogens, and the substituents can be benzyl or alkyl; when the substituents are alkyl, the alkyl together with R3and / or R4and the carbon atom to which they are attached can form a C3-6membered fused or spiro ring structure; or when the substituents are 2 alkyl groups, the 2 alkyl groups together with the group Y can form a C3-6membered spiro ring structure. When Y is substituted methylene, the substituents of the substituted methylene are preferably alkyl, more preferably straight chain or branched C1-4alkyl.

[0153] Preferably, Y is oxygen, sulfur, sulfone, sulfoxide or methylene; or, preferably, Y is oxygen, sulfur or methylene.

[0154] In Structural Formula I, X is oxygen or sulfur.

[0155] In Structural Formula I, n = 0 or 1.

[0156] In Structural Formula I, where R1, R2, R3, R4 are all hydrogen, X is oxygen, and n = 0, G cannot be hydrogen or fluorine when Y is methylene; and G cannot be hydrogen when Y is oxygen or sulfur.

[0157] Preferably, R1, R2, R3, R4 are independently hydrogen, halogen (e.g., fluorine), C1-7alkyl, or substituted C1-7alkyl, or any two of R1, R2, R3, R4 together with the carbon atom to which they are attached form a C3-6cyclic alkyl group (e.g., a C3-5cyclic alkyl group). Further, R1, R2 can be the same; and / or, R3, R4 can be the same.

[0158] Preferably, Y is methylene substituted with an alkyl group that can form a C3-6membered annular or spirocyclic structure with R3and / or R4and the carbon atom to which they are attached.

[0159] Preferably, X can be oxygen.

[0160] Preferably, X is oxygen, G is hydrogen, halogen (e.g., fluorine or chlorine), methyl, or methoxy, and Y and R1, R2, R3, R4 are as defined above.

[0161] Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen, sulfur, sulfoxide, or sulfone, and R1, R2, R3, R4 are as defined above.

[0162] Preferably, X is oxygen, G is fluorine, Y is methylene or substituted methylene, oxygen, or sulfur, and R1, R2, R3, R4 are as defined above.

[0163] Preferably, X is oxygen, G is chlorine, Y is methylene or substituted methylene, oxygen, or sulfur, and R1, R2, R3, R4 are as defined above.

[0164] Preferably, X is oxygen, G is methyl, Y is methylene or substituted methylene, oxygen, or sulfur, and R1, R2, R3, R4 are as defined above.

[0165] Preferably, X is oxygen, G is methoxy, Y is methylene or substituted methylene, oxygen, or sulfur, and R1, R2, R3, R4 are as defined above.

[0166] Preferably, X is oxygen, G is hydrogen, Y is methylene, sulfoxide, sulfone, oxygen, or sulfur, and R1, R2 are independently hydrogen, fluorine, or methyl, and R3, R4 are independently hydrogen.

[0167] Preferably, X is oxygen, G is fluorine, Y is methylene, sulfoxide, sulfone, oxygen or sulfur, R1, R2 are independently hydrogen, fluorine or methyl, R3, R4 are independently hydrogen.

[0168] Preferably, n = 0.

[0169] According to the specific embodiment of the present application, in structural formula I:

[0170] G is hydrogen, Y is methylene, R1 and R2 are methyl, R3 and R4 are hydrogen, X is oxygen, n = 0;

[0171] G is hydrogen, Y is methylene, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, n = 0;

[0172] G is hydrogen, Y is methylene, one of R1 and R2 together with one of R3 and R4 along with the carbon atom to which they are attached forms a C3 cyclic alkyl group, the other of R1 and R2 is hydrogen, the other of R3 and R4 is hydrogen, X is oxygen, n = 0;

[0173] G is hydrogen, Y is sulfur, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, n = 0;

[0174] G is hydrogen, Y is sulfoxide, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, n = 0;

[0175] G is hydrogen, Y is sulfur, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, n = 0;

[0176] G is hydrogen, Y is sulfone, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, n = 0;

[0177] G is hydrogen, Y is methylene, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, n = 1;

[0178] G is fluorine, Y is oxygen, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, n = 0;

[0179] G is fluorine, Y is sulfur, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, n = 0;

[0180] G is fluorine, Y is oxygen, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, n = 0;

[0181] G is fluorine, Y is methylene, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, n = 0;

[0182] G is hydrogen, Y is oxygen, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, n = 0; or

[0183] G is fluorine, Y is sulfur, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, n = 0.

[0184] In the antibody drug conjugate targeting ADAM9 or salt thereof provided in the present application, the camptothecin compound shown in structural formula I is connected to the carboxyl in group A in the general formula by an amide bond, preferably the amino group adjacent to group G in structural formula I is connected to the carboxyl in group A in the general formula by an amide bond.

[0185] Preferably, the structure of the camptothecin compound can be shown in structural formula IA:

[0186] In structural formula IA, groups R1, R2, R3, R4 are the same as the definitions of groups R1, R2, R3, R4 in structural formula I above, but R1, R2, R3, R4 are not simultaneously hydrogen.

[0187] In the antibody drug conjugate targeting ADAM9 or salt thereof provided in the present application, the camptothecin compound shown in structural formula IA is connected to the carboxyl in group A in the general formula by an amide bond, preferably the amino group on the left benzene ring in structural formula IA is connected to the carboxyl in group A in the general formula by an amide bond.

[0188] Alternatively, the structure of the camptothecin compound can be shown in structural formula II:

[0189] In structural formula II, R5 is C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, phenyl or substituted phenyl. When R5 is C1-5 alkyl or substituted C1-5 alkyl, the C1-5 alkyl includes straight chain or branched C1-5 alkyl. Further, R5 is C1-4 straight chain alkyl. When R5 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl, the substituents are selected from halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino is amino substituted by one or more substituents selected from methyl and ethyl. When R5 is substituted phenyl, the substituents are selected from alkyl (e.g. C1-6 alkyl, preferably C1-3) or halogen.

[0190] In structural formula II, G is hydrogen, halogen (e.g. fluorine), methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.

[0191] In structural formula II, X is oxygen or sulfur.

[0192] In structural formula II, n = 0 or 1.

[0193] In structural formula II, when X is oxygen, G is hydrogen and n = 0, R5 cannot be n-butyl.

[0194] In the antibody drug conjugate targeting ADAM9 or salt thereof provided in the present application, the camptothecin compound shown in structural formula II is connected to the carboxyl group in the group A in the general formula by an amide bond, and preferably the amino group adjacent to the group G in structural formula II is connected to the carboxyl group in the group A in the general formula by an amide bond.

[0195] Preferably, the structure of the camptothecin compound can be shown in structural formula IIA:

[0196] In structural formula IIA, the group R5 is the same as the definition of the group R5 in structural formula II above, but R5 cannot be n-butyl.

[0197] In the antibody drug conjugate targeting ADAM9 or salt thereof provided in the present application, the camptothecin compound shown in structural formula IIA is connected to the carboxyl group in the group A in the general formula by an amide bond, and preferably the amino group on the left benzene ring in structural formula IIA is connected to the carboxyl group in the group A in the general formula by an amide bond.

[0198] According to the specific embodiments of the present application, the structure of the camptothecin compound is as follows:

[0199] In the antibody drug conjugate targeting ADAM9 or salt thereof provided in the present application, the camptothecin compound shown in each of the above structural formulas is connected to the carboxyl group in the group A in the general formula by an amide bond, and preferably the amino group on the left benzene ring in each of the structural formulas is connected to the carboxyl group in the group A in the general formula by an amide bond.

[0200] Alternatively, the structure of the camptothecin compound can be shown in structural formula IV:

[0201] In structural formula IV, R8 is hydrogen, trifluoromethyl, C1-5 alkyl, or C1-5 alkyl substituted with one or more substituents, C3-6 cyclic alkyl, or C3-6 cyclic alkyl substituted with one or more substituents, or halogen.

[0202] When R8 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl, the substituents are selected from halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl, and C3-6 cyclic alkyl; and wherein the substituted amino is amino substituted with one or more substituents selected from methyl and ethyl.

[0203] In the antibody drug conjugate targeting ADAM9 or salt thereof provided by the present application, the hydroxyl group in the camptothecin compound shown in structural formula IV, which is connected to the same carbon as R8, is connected to the carboxyl group of group A in the general formula through a self-releasing structure, for example The solid line indicates the site connected to the carboxyl group of group A in the general formula, and the wavy line indicates the site connected to the hydroxyl group in structural formula IV.

[0204] Further, the antibody drug conjugate or salt thereof provided by the present application has a structure as shown in structural formula Ia (bridging site-specific conjugation structure general formula) and / or Ib (bridging site-specific conjugation open-loop structure general formula):

[0205] and / or

[0206] In structural formula Ia and / or Ib, Ab, m, group M, SP1, SP2, A, and D are the same as the definitions of Ab, m, group M, SP1, SP2, A, and D in the general formula above.

[0207] Further, the antibody drug conjugate or salt thereof provided by the present application has a structure as shown in structural formula Ic and / or Id:

[0208] and / or

[0209] In structural formula Ic and / or Id, Ab, m, group A, and D are the same as the definitions of Ab, m, group A, and D in the general formula above.

[0210] According to the specific embodiments of the present application, the antibody drug conjugate or salt thereof provided by the present application has a structure as shown below:

[0211] Seventh aspect

[0212] The anti-ADAM9 antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, or antibody drug conjugate or salt thereof provided by the present application can be included in a composition, more particularly a pharmaceutical composition, e.g., a pharmaceutical preparation, for various purposes as needed.

[0213] Therefore, the present application also provides a composition comprising the anti-ADAM9 antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, or antibody drug conjugate or salt thereof provided by the present application. Preferably, the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable excipient. The pharmaceutical composition provided by the present application can be prepared into various dosage forms known in the medical or pharmaceutical field, and is administered in a suitable manner.

[0214] Eighth aspect

[0215] The present application also provides use of the anti-ADAM9 antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, antibody drug conjugate or salt thereof, or composition in the preparation of a medicament for preventing, treating and / or ameliorating a disease or disorder, which can be associated with ADAM9 expression (including overexpression), such as a blood tumor or solid tumor positive for ADAM9 expression. The anti-ADAM9 antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, antibody drug conjugate or salt thereof, or composition can function by binding ADAM9 to exert ADCC, block ADAM9 metalloprotease activity, or cell-killing toxicity of a small molecule cytotoxic compound in the antibody drug conjugate, etc., but is not limited thereto. For example, the disease or disorder can be non-small cell lung cancer, large cell lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma, and lymphoma.

[0216] Ninth aspect

[0217] The present application also provides a method for preventing, treating and / or ameliorating a disease or disorder, which can be associated with ADAM9 expression (including overexpression), such as a blood tumor or solid tumor positive for ADAM9 expression, comprising administering to a subject in need thereof the anti-ADAM9 antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, antibody drug conjugate or salt thereof, or composition of the present application. For example, the disease or disorder can be non-small cell lung cancer, large cell lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma, and lymphoma. The subject can be a mammal; preferably, the subject is a human.

[0218] The above method for preventing, treating and / or ameliorating a disease or disorder provided by the present application depends on various factors when applied, including the specific active ingredient of the pharmaceutical composition administered, the age, weight, sex or physical and medical condition of the patient, the severity of the condition to be treated, the route of administration, etc.

[0219] The above-mentioned method provided by the present application can also be used in combination with other drugs or means. The other drugs or means refer to other drugs or means which can be administered in combination with the anti-ADAM9 antibody or antigen binding fragment thereof, nucleic acid molecule, vector, host cell, antibody drug conjugate or salt thereof or composition of the present application, such as small molecule chemical drugs, targeted drugs, antibody and other recombinant protein drugs, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapeutic drugs and radiotherapy. The combined administration of the two can be carried out in any form, such as simultaneously, consecutively or at intervals.

[0220] Tenth aspect

[0221] The present application also provides the use of the anti-ADAM9 antibody or antigen binding fragment thereof in the preparation of an antibody drug conjugate for preventing, treating and / or ameliorating a disease or disorder, which can be associated with ADAM9 expression (including overexpression), such as an ADAM9 expression positive hematological or solid tumor. For example, the disease or disorder can be non-small cell lung cancer, large cell lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma or lymphoma.

[0222] Eleventh aspect

[0223] The present application also provides the use of the anti-ADAM9 antibody or antigen binding fragment thereof, nucleic acid molecule, vector, host cell, antibody drug conjugate or salt thereof or composition in the preparation of a reagent for diagnosing a disease or disorder, which can be associated with ADAM9 expression (including overexpression), such as an ADAM9 expression positive hematological or solid tumor. For example, the disease or disorder can be non-small cell lung cancer, large cell lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma and lymphoma.

[0224] Twelfth aspect

[0225] The present application also provides a method for diagnosing a disease or disorder, which can be associated with ADAM9 expression (including overexpression), such as ADAM9 expression-positive hematological or solid tumors, comprising contacting an anti-ADAM9 antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, antibody drug conjugate or salt thereof or composition of the present application with a sample from a subject. For example, the disease or disorder can be non-small cell lung cancer, large cell lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma and lymphoma.

[0226] The subject can be a mammal; preferably, the subject is a human.

[0227] Thirteenth aspect

[0228] The present application provides a kit comprising an anti-ADAM9 antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, antibody drug conjugate or salt thereof or composition of the present application. The kit can be used for the above-mentioned prevention, treatment and / or improvement, or for the above-mentioned diagnosis. Depending on the intended mode of application, the kit further comprises other reagents. For example, the kit is a kit for detecting ADAM9 expression (including overexpression) in any biological sample using ELISA.

[0229] Compared with the prior art, the present application provides a series of murine antibody sequences, humanized sequences and corresponding antibody molecules against human ADAM9. The antibodies provided by the present application can specifically bind to human ADAM9, can be rapidly internalized into tumor cells, and can effectively block the metalloproteinase activity of ADAM9, and can be advantageously coupled with small molecule cytotoxic compounds to prepare targeted killing antibody drug conjugates, etc. Experiments have shown that, taking humanized monoclonal antibody Mab-A as a control antibody, the antibodies provided by the present application have the following advantages:

[0230] Firstly, the affinities of some of the antibodies provided by the present application to human ADAM9 are weaker than those of the control antibody molecule Mab-A, but the binding and internalization activities of the antibodies to tumor cells are comparable to or stronger than those of the control molecule Mab-A. It is worth noting that the binding of the antibodies of the present application to ADAM9 on the surface of normal tissues and cells is weaker than that of Mab-A, so the antibodies of the present application have lower target toxicity and better safety potential.

[0231] Secondly, the antibodies provided by the present application specifically bind to human ADAM9 recombinant protein and cell surface ADAM9, and have a significant ADAM9 enzyme activity inhibition effect, and are stronger than the control molecule Mab-A.

[0232] Thirdly, the distribution of different anti-ADAM9 antibodies in the tumor microenvironment of a mouse model was also detected in the present application. It was found that the antibody provided by the present application was more likely to accumulate in the tumor after administration to tumor-bearing mice compared to Mab-A, suggesting that it had a stronger tumor inhibition effect.

[0233] Fourthly, after the antibody of the present application and the control molecule Mab-A were coupled with the same small molecule cytotoxic compound to form ADC, it was found that the ADC obtained from the antibody of the present application retained the binding and internalization characteristics of the antibody and showed a significantly better anti-tumor effect than the ADC obtained from the control molecule Mab-A in various tumor-bearing mouse models. Further, the antibody provided by the present application was coupled with small molecule cytotoxic compounds of different structures, and high DAR value, high purity ADC products were obtained, and the obtained ADC products showed high in vitro cell killing activity, stronger than IMGC936.

[0234] Fifthly, compared with IMGC936, the antibody drug conjugate provided by the present application also showed less P-glycoprotein (p-gp) mediated efflux, and potentially had lower tumor drug resistance possibility.

[0235] Therefore, the antibody provided by the present application has important application potential in tumor targeted therapy, targeted killing ADC drug development, targeted and immune combined therapy, etc. BRIEF DESCRIPTION OF DRAWINGS

[0236] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0237] FIG. 1 is a structural schematic diagram of ADAM9.

[0238] FIG. 2 shows the binding activity of the chimeric antibody to human ADAM9 recombinant expression cells.

[0239] FIG. 3 shows the binding activity of the chimeric antibody to monkey ADAM9 recombinant expression cells.

[0240] FIG. 4 shows the binding activity of the chimeric antibody to tumor cell A431.

[0241] FIG. 5 shows the binding activity of the chimeric antibody to tumor cell MDA-MB-468.

[0242] FIG. 6 shows the binding activity of the chimeric antibody to tumor cell SKBR3.

[0243] FIG. 7 shows the binding activity of the chimeric antibody to tumor cell SKOV3.

[0244] FIG. 8 shows the binding activity of the chimeric antibody to ADAM9 family members.

[0245] Figure 9 shows the internalization activity of the chimeric antibody on tumor cell A549.

[0246] Figure 10 shows the internalization activity of the chimeric antibody on tumor cell MDA-MB-231.

[0247] Figure 11 shows the enzyme inhibition activity (inhibition rate) of the chimeric antibody.

[0248] Figure 12 shows the enzyme inhibition activity (relative fluorescence intensity) of the chimeric antibody.

[0249] Figure 13 shows the binding activity of the humanized antibody to MDA-MB-231 cells.

[0250] Figure 14 shows the binding activity of the humanized antibody to A2780 cells.

[0251] Figure 15 shows the binding activity of the humanized antibody to NCI-H1975 cells.

[0252] Figure 16 shows the binding activity of the humanized antibody to NCI-H460 cells.

[0253] Figure 17 shows the binding activity of the humanized antibody to A549 cells.

[0254] Figure 18 shows the internalization activity of the humanized antibody on tumor cell MDA-MB-231.

[0255] Figure 19 shows the internalization activity of the humanized antibody on tumor cell A2780.

[0256] Figure 20 shows the internalization activity of the humanized antibody on tumor cell NCI-H1975.

[0257] Figure 21 shows the internalization activity of the humanized antibody on tumor cell MDA-MB-231.

[0258] Figure 22 shows the internalization activity of the humanized antibody on tumor cell A549.

[0259] Figure 23 shows the internalization activity of the humanized antibody on tumor cell Hela.

[0260] Figure 24 shows the killing activity of the antibody drug conjugate on tumor cell MDA-MB-231.

[0261] Figure 25 shows the killing activity of the antibody drug conjugate on tumor cell SKOV3.

[0262] Figures 26 and 27 show the killing activity of the antibody drug conjugate on tumor cell A2780.

[0263] Figure 28 shows the tumor growth curves of each group in the mouse xenograft model of human ovarian cancer A2780.

[0264] Figure 29 shows the tumor weight data of each group in the mouse xenograft model of human ovarian cancer A2780.

[0265] Figure 30 shows the tumor growth curves of each group in the mouse xenograft model of human breast cancer MDA-MB-231.

[0266] Figure 31 shows the tumor weight data of each group in the mouse xenograft model of human breast cancer MDA-MB-231.

[0267] Figure 32 shows the tumor growth curves of each group in the mouse xenograft model of human colorectal cancer RKO.

[0268] Figure 33 shows the tumor weight data of each group in the mouse xenograft model of human colorectal cancer RKO.

[0269] Figure 34 shows the staining results of the antibody on human normal stomach tissue frozen sections.

[0270] Figure 35 shows the staining results of the antibody on human normal kidney tissue frozen sections.

[0271] Figure 36 shows the staining results of the antibody on human normal intestinal tissue frozen sections.

[0272] Figure 37 shows the binding activity of the antibody with human PBMC (CD14+).

[0273] Figure 38 shows the binding activity of the antibody on tumor cells NCI-H460.

[0274] Figure 39 shows the binding activity of the antibody on tumor cells DLD-1.

[0275] Figure 40 shows the internalization activity of the antibody on tumor cells NCI-H460.

[0276] Figure 41 shows the internalization activity of the antibody on tumor cells DLD-1.

[0277] Figure 42 shows the tumor growth curves of each group in the mouse xenograft model of human large cell lung cancer NCI-H460.

[0278] Figure 43 shows the tissue distribution of the targeted ADAM9 antibody detected in the mouse xenograft model of human pancreatic cancer AsPC-1.

[0279] Figure 44 shows the killing activity of the ADC targeting ADAM9 on HCT15 cells.

[0280] Figure 45 shows the drug resistance characteristics of the ADC targeting ADAM9 on HCT15 cells.

[0281] Figure 46 shows the tumor growth curve of each group of mice bearing human colorectal cancer RKO model.

[0282] Best Mode for Carrying Out the Invention

[0283] The present application will be described with respect to the following examples. It will be apparent to those skilled in the art that the application is not limited to the following examples, which are provided to illustrate the application.

[0284] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents and materials used in the following examples are commercially available unless otherwise specified.

[0285] Example 1: Preparation of Anti-human ADAM9 Hybridoma Antibodies

[0286] Balb / c mice were immunized with human ADAM9 ECD-mFc recombinant protein (UniprotKB sequence number: Q13443, 29-697aa), and the serum titer was detected using human ADAM9 ECD-his recombinant protein coating. After reaching the fusion requirements, hybridoma cell fusion and positive clone screening were performed. Positive clones that bind to the human ADAM9-his recombinant protein were obtained. Total RNA was extracted from the hybridoma cells of the positive clones using Trizol, and cDNA was obtained by reverse transcription. Amplification primers for mouse-derived antibodies were obtained from relevant literature and conventional databases, and the obtained cDNA was used as a template for PCR amplification and purification of the amplification product. Then, the amplification PCR product was ligated to a T vector, and E. coli competent cells were transformed. After strain amplification and plasmid extraction, DNA sequencing was performed to determine the heavy chain and light chain variable region sequences of the hybridoma antibodies.

[0287] Through the above experimental operations, the light and heavy chain variable region sequences of six specific hybridoma antibodies were obtained from hybridoma clones 5H6, 5H1, 27F8, 10E5, 26B1, and 20H1.

[0288] Example 2: Preparation of Anti-human ADAM9 Chimeric Antibodies and Control Antibodies

[0289] Humanized monoclonal antibody Mab-A was used as a positive control antibody of the same target. The coding gene synthesis of the light and heavy chain variable region sequences of the control antibody was performed, and then cloned into eukaryotic transient expression vector PTT5 containing human kappa and IgG1 light and heavy chain constant regions, respectively, to obtain light and heavy chain expression plasmids of the control antibody. The expression plasmids were transformed into E. coli for amplification, and a large amount of light and heavy chain expression plasmids were obtained by isolation. Then, according to the operation instruction of transfection reagent 293fectin (Cat: 12347019, Gibco), the expression plasmids were transformed into HEK293 cells for recombinant expression of the antibody. Five to six days after cell transfection, the culture supernatant was taken, and the expression supernatant was purified by ProA affinity chromatography column to obtain the control antibody Mab-A.

[0290] In the same way as described above, the coding genes of the light and heavy chain variable region sequences of the six specific hybridoma antibodies obtained in Example 1 were cloned into eukaryotic transient expression vector PPT5 containing human kappa and IgG1 light and heavy chain constant regions (SEQ ID NO. 3 and SEQ ID NO. 4), respectively, to finally obtain six chimeric antibodies. According to the naming of hybridoma clones, they were named as chimeric antibodies ch5H6, ch5H1, ch27F8, ch10E5, ch26B1, and ch20H1, respectively.

[0291] The heavy and light chain variable region sequences of the control antibody Mab-A and the chimeric antibody ch5H6 are as follows, and the antigenic determinant CDR is shown in bold underlined part, which is obtained by Kabat definition method.

[0292] SEQ ID NO. 1: Mab-A heavy chain variable region amino acid sequence

[0293] SEQ ID NO. 2: Mab-A light chain variable region amino acid sequence

[0294] SEQ ID NO. 3: IgG1 heavy chain constant region amino acid sequence

[0295] SEQ ID NO. 4: kappa light chain constant region amino acid sequence

[0296] SEQ ID NO. 5 (H-CDR1 / H-CDR2 / H-CDR3: SEQ ID NO. 36 / SEQ ID NO. 37 / SEQ ID NO. 38): ch5H6 heavy chain variable region amino acid sequence

[0297] SEQ ID NO. 6 (L-CDR1 / L-CDR2 / L-CDR3: SEQ ID NO. 45 / SEQ ID NO. 46 / SEQ ID NO. 47): ch5H6 light chain variable region amino acid sequence

[0298] Example 3: Detection of the affinity of chimeric antibodies to human ADAM9 recombinant protein

[0299] The antibody affinity was determined by the method of capturing the Fc segment of the antibody with the Fortebio Octet QKe system instrument using the capture antibody (AHC) biological probe of the Fc segment of the anti-human antibody. In the determination, the chimeric antibodies ch5H6, ch5H1, ch27F8, ch10E5, ch26B1, ch20H1 and the control antibody Mab-A were diluted to 4 μg / ml with PBS buffer and flowed through the surface of the AHC probe (Cat: 18-0015, PALL) for 120 s. The human ADAM9 ECD-His recombinant protein was diluted to 60 nM as the mobile phase, the binding time was 300 s, and the dissociation time was 300 s. After the experiment was completed, the blank control response value was deducted, and the 1:1 Langmuir binding mode fitting was performed to calculate the kinetic constant of the antigen-antibody binding.

[0300] The kinetic parameters are shown in Table 1. The results show that the chimeric antibodies all specifically bind to the human ADAM9 recombinant protein, and the affinities are all weaker than that of the control antibody Mab-A to varying degrees.

[0301] Table 1. Affinity determination results of chimeric antibodies to human ADAM9 recombinant protein

[0302] Example 4: Detection of the affinity of chimeric antibodies to human ADAM9 different truncations and mouse ADAM9

[0303] The extracellular region of ADAM9 mainly includes the following domains: zinc esterase domain, disintegrin domain, Cys-rich region and EGF-like domain. Different lengths of ADAM9 truncations were expressed to analyze the binding of chimeric antibodies to the truncations to preliminarily determine the binding epitope region; at the same time, it was analyzed whether the chimeric antibodies had species cross-binding activity with mouse ADAM9.

[0304] Affinity detection was performed in the same way as described in Example 3, using the following recombinant proteins: human ADAM929-643-His recombinant protein (UniprotKB sequence number: Q13443, 29-643 aa, without EGF-like domain), human ADAM9s-His recombinant protein (UniprotKB sequence number: Q13443, 29-576 aa), murine ADAM9 ECD-His recombinant protein (UniprotKB sequence number: Q61072, 29-697 aa).

[0305] The results of affinity (KD(M)) detection of 6 chimeric antibodies and control antibody Mab-A to each of the above proteins are shown in Table 2. The results show that chimeric antibody ch27F8 binds to human ADAM9s-His, i.e. the secreted form of ADAM9, and chimeric antibody ch10E5 has cross-binding activity to murine ADAM9, suggesting that ch27F8 has different binding epitopes to other molecules, while ch5H6, ch5H1, ch10E5, ch26B1 and ch20H1 have similar binding epitopes to control antibody Mab-A, and none of them binds to human secreted ADAM9 (ADAM9s-His).

[0306] Table 2. Affinity (KD(M)) detection results of chimeric antibodies to human ADAM9 and its different truncated forms and murine ADAM9

[0307] Example 5: Detection of binding activity of chimeric antibodies to human ADAM9 recombinant expression cells

[0308] CHO cells stably expressing human ADAM9 (UniprotKB sequence number: Q13443, 1-819 aa) were constructed.

[0309] 2E5 cells were taken. Chimeric antibodies ch5H6, ch5H1, ch27F8, ch10E5, ch26B1 and control antibody Mab-A and isotype control were diluted in 3-fold gradient from 10 μg / mL, and the dilutions were added to the cells. After incubation at 4°C in the dark for 60 min, the cells were washed with PBS, and then FITC-labeled goat anti-human antibody (F9512, Sigma) was added at a dilution of 1:200, and incubated at 4°C in the dark for 30 min, followed by washing with PBS. The cells were resuspended in 200 μl of PBS, and then detected using a flow cytometer.

[0310] The results are shown in Figure 2. The results show that chimeric antibodies ch5H6, ch5H1, ch27F8, ch10E5, ch26B1 can bind to human ADAM9 recombinant expression cells in a dose-dependent manner. The EC50 values are shown in Table 3.

[0311] Table 3. EC50 values of chimeric antibodies binding to human ADAM9 recombinant expression cells

[0312] Example 6: Detection of binding activity of chimeric antibodies to monkey ADAM9 recombinant expression cells

[0313] CHO cells stably expressing monkey ADAM9 (UniprotKB sequence number: A0A2K5X4X8, 1-819aa) were constructed.

[0314] The binding of antibodies to cells was detected by flow cytometry in the same manner as described in Example 5.

[0315] The results are shown in Figure 3. The results show that chimeric antibodies ch5H6, ch5H1, ch27F8, ch10E5 can bind to monkey ADAM9 recombinant expression cells in a dose-dependent manner, ch26B1 does not bind to monkey ADAM9 recombinant expression cells; ch10E5 weakly binds to monkey ADAM9 recombinant expression cells. The EC50 values are shown in Table 4.

[0316] Table 4. EC50 values of chimeric antibodies binding to monkey ADAM9 recombinant expression cells

[0317] Example 7: Detection of binding activity of chimeric antibodies to A431 tumor cells

[0318] 2E5 A431 cells were taken. The binding of antibodies to cells was detected by flow cytometry in the same manner as described in Example 5.

[0319] The results are shown in Figure 4. The results show that chimeric antibodies ch5H6, ch5H1, ch27F8, ch10E5 can bind to tumor cells A431 in a dose-dependent manner. Among them, ch5H1 and ch5H6 have binding ability comparable to that of positive control antibody Mab-A, and the binding of other molecules to tumor cells is weaker than that of Mab-A to varying degrees.

[0320] Example 8: Detection of binding activity of chimeric antibodies to MDA-MB-468, SKBR3 and SKOV3 tumor cells

[0321] Take 2E5 MDA-MB-468, SKBR3 and SKOV3 cells respectively. Dilute chimeric antibodies ch5H6, ch5H1, ch27F8 and ch10E5 and positive control antibody Mab-A and isotype control to 3.3 ug / ml and 0.37 ug / ml, and add the dilutions to the cells. Incubate at 4°C in the dark for 60 min, and then wash thoroughly with PBS. Add 1:200 diluted FITC-labeled goat anti-human antibody (F9512, Sigma), and incubate at 4°C in the dark for 30 min. Then wash thoroughly with PBS. Resuspend the cells in 200 ul of PBS, and detect using a flow cytometer.

[0322] The results are shown in Figures 5, 6 and 7. The results show that chimeric antibodies ch5H6, ch5H1, ch27F8 and ch10E5 specifically bind to different tumor cells, and the binding activity of ch5H6 and ch5H1 is basically comparable to that of the control antibody.

[0323] Example 9: Detection of cross-binding activity of chimeric antibodies to ADAM9 family members

[0324] Transiently transfect HEK293 cells with human ADAM9 family members human ADAM8 (UniprotKB sequence number: P78325), human ADAM10 (UniprotKB sequence number: O14672), and human ADAM17 (UniprotKB sequence number: P78536) full-length expression vectors; at the same time, transiently transfect human ADAM9 full-length expression vector, and 36 h later, take 2E5 human ADAM9, human ADAM8, human ADAM10 and human ADAM17 recombinant expression cells respectively. Dilute chimeric antibodies ch5H6, ch5H1, ch27F8 and ch10E5 and positive control antibody Mab-A and isotype control to 10 ug / ml, and add the dilutions to the cells. Incubate at 4°C in the dark for 60 min, and then wash thoroughly with PBS. Add 1:200 diluted FITC-labeled goat anti-human antibody (F9512, Sigma), and incubate at 4°C in the dark for 30 min. Then wash thoroughly with PBS. Resuspend the cells in 200 ul of PBS, and detect using a flow cytometer.

[0325] The results are shown in Figure 8. The results show that chimeric antibodies ch5H6, ch5H1, ch27F8, ch10E5 and control antibody Mab-A specifically bind to human ADAM9, but do not bind to other family members.

[0326] Example 10: Detection of internalization activity of chimeric antibodies on different tumor cells

[0327] Take 2E5 MDA-MB-231 and A549 cells respectively. Dilute the chimeric antibodies ch5H6, ch5H1, ch27F8, ch10E5 and the positive control antibody Mab-A and the isotype control to 1 ug / ml, 0.5 ug / ml and 0.25 ug / ml three concentrations, and add them to the cells together with the internalization reagent Antibody Internalization human Reagent (Cat. 90565, Sartorius) according to the instructions of the reagent. Incubate at 37°C in the dark for 120 min, and use a flow cytometer to detect the immunofluorescence signal internalized into the cells.

[0328] The results are shown in Figures 9 and 10. The results show that the chimeric antibodies ch5H6, ch5H1, ch27F8 and ch10E5 all have strong internalization in different tumor cells, and the internalization activity of ch5H1 and ch5H6 is significantly better than that of the control antibody Mab-A.

[0329] Example 11: Enzyme inhibition activity of chimeric antibodies

[0330] ADAM9 has strong metalloprotease activity, and its enzyme activity is an important pathway for its participation in physiological and pathological processes such as inflammation, tumor, injury, etc., and anti-ADAM9 antibodies can affect the above pathological processes by blocking enzyme activity. The purpose of this embodiment is to analyze the inhibition of different antibodies on ADAM9 enzyme activity through enzyme activity experiments.

[0331] Dilute the recombinant human ADAM9-His protein to 80 ug / mL with the reaction solution (25 mM Tris, 2.5 μM ZnC12, 0.005% Brij 35, pH 9.0) and add 25 uL / well to the 96-well plate; the antibodies to be tested are also diluted to 2400 ug / mL with the reaction solution, then 4-fold gradient dilution for a total of 8 gradients, and the dilution solution is added to the 96-well plate at 25 uL / well, mixed well, and then reacted at 37°C for 15 min; then add 50 uL / well of 5 μM enzyme fluorescence substrate, so that the total volume in each well of the plate is 100 uL, and react at 37°C for 4 h. Then use an enzyme marker to read the fluorescence value and record (EX: 320 nm; Em: 405 nm), and calculate the inhibition rate according to the following formula:

[0332] Inhibition rate % = (relative fluorescence intensity hIgG1 - relative fluorescence intensity Sample) / (relative fluorescence intensity hIgG1 - relative fluorescence intensity background) x 100%; wherein the relative fluorescence intensity hIgG1 is the relative fluorescence intensity of the isotype control.

[0333] The results are shown in Figures 11 and 12. The results show that the chimeric antibodies can all inhibit the metalloprotease activity of ADAM9 in a dose-dependent manner, with ch5H6 having the strongest inhibitory activity. The IC50values are shown in Table 5.

[0334] Table 5. IC50values of chimeric antibodies for inhibiting the metalloprotease activity of human ADAM9

[0335] Example 12: Humanization and recombinant expression of murine-derived antibodies

[0336] A comprehensive analysis of the murine-derived antibody variable region sequences was performed to determine the antigen-complementary determining region (CDR) region of the antibody binding to the antigen and the framework region (FR) supporting the conserved three-dimensional conformation of the antibody. Based on the homology alignment results, the closest human antibody template in the human antibody germline library was selected as the basic template, and the CDR grafting was performed in combination with the full-sequence blast results, thereby humanizing the light and heavy chain variable regions.

[0337] The murine anti-humanized heavy and light chain variable region sequences derived from the hybridoma clone 5H6 obtained above were subjected to gene synthesis encoding the light and heavy chain variable region sequences, and were cloned into eukaryotic transient expression vectors containing human kappa and IgG1 light and heavy chain constant regions, respectively, to obtain light and heavy chain expression plasmids, and were recombinantly expressed in HEK293 cells. After 5-6 days of cell transfection, the culture supernatant was collected, and the expression supernatant was purified using a ProA affinity chromatography column to obtain the CDR-grafted humanized antibody. According to the naming of the hybridoma clone, it was named humanized antibody hz5H6.

[0338] The heavy and light chain variable region sequences of the humanized antibody hz5H6 are as follows, wherein the underlined bold part shows the antigenic determinant CDR, which is obtained by the Kabat definition method.

[0339] SEQ ID NO. 7 (H-CDR1 / H-CDR2 / H-CDR3: SEQ ID NO. 36 / SEQ ID NO. 37 / SEQ ID NO. 38): hz5H6 heavy chain variable region amino acid sequence

[0340] SEQ ID NO. 8 (L-CDR1 / L-CDR2 / L-CDR3: SEQ ID NO. 45 / SEQ ID NO. 46 / SEQ ID NO. 47): hz5H6 light chain variable region amino acid sequence

[0341] Further, the heavy and light chain variable regions of hz5H6 were subjected to point mutations in the CDR regions, and the specific mutation sites are shown in Table 6.

[0342] Table 6. Point mutations of hz5H6 antibody in CDR regions

[0343] Note: Take E65Q as an example, which means that the 65th amino acid E is mutated to Q

[0344] The amino acid sequences of the heavy chain and light chain variable regions after point mutation are as follows, wherein the underlined bold part shows the antigenic determinant CDR, which is obtained by Kabat definition.

[0345] Using the light and heavy chain variable region sequences obtained after the above point mutation, different hz5H6 mutants were obtained by recombinant expression in the same way as above.

[0346] Example 13: Affinity detection of humanized antibody

[0347] Using the Octet QKe system instrument of Fortebio Company, the method of capturing antibody Fc fragment by using anti-human antibody Fc fragment capture antibody (AHC) bioprobe was used to detect the affinity of the antibody. When detecting, the humanized antibody hz5H6 and its mutants and the control antibody Mab-A were diluted to 4 ug / ml with PBS buffer, and flowed through the surface of AHC probe (Cat: 18-0015, PALL) for 120 s. Human ADAM9 ECD-His recombinant protein was diluted to 60 nM as the mobile phase, and the binding time was 300 s and the dissociation time was 300 s. After the experiment was completed, the blank control response value was deducted, and the 1:1 Langmuir Global binding mode fitting was performed to calculate the kinetic constant of antigen-antibody binding.

[0348] The kinetic parameters of hz5H6 and its mutants and the control antibody Mab-A are shown in Table 7. The results show that the affinities of hz5H6 and most of its mutants are weaker than that of the control antibody Mab-A to varying degrees.

[0349] Table 7. Affinity determination results

[0350] Example 14: Detection of binding activity of humanized antibody to different tumor cells

[0351] Using tumor cells with different ADAM9 expression abundances, the binding strength of the humanized antibody to the cell surface antigen was detected.

[0352] Take 2E5 MDA-MB-231, A2780, NCI-H1975 and NCI-H460 cells respectively. Dilute the humanized antibody hz5H6Lm8 and the positive control antibody Mab-A and the isotype control to 10 ug / ml, 3 times gradient dilution, 8 gradients in total, add the dilution to the cells. Incubate at 4°C in the dark for 60 min, wash with PBS, add 1:200 diluted FITC labeled goat anti-human antibody (F9512, Sigma), incubate at 4°C in the dark for 30 min, then wash with PBS. Resuspend the cells in 200ul PBS, and use flow cytometry to detect.

[0353] The results are shown in Figures 13, 14, 15 and 16. The results show that the humanized antibody hz5H6Lm8 and the control antibody Mab-A can specifically bind to different tumor cells in a dose-dependent manner, and the binding ability of hz5H6Lm8 is basically equivalent to that of the control antibody Mab-A.

[0354] Different hz5H6 mutant assay: Take 2E5 A549 cells, and use flow cytometry to detect the binding of different hz5H6 mutants to the cells in the same way as described above.

[0355] The binding curve of different hz5H6 mutants to A549 cells is shown in Figure 17, and the results show that there is no significant difference in binding activity between the mutants.

[0356] Example 15: Detection of internalization activity of humanized antibody on different tumor cells

[0357] Different ADAM9 expression abundance tumor cells were used to detect the internalization of the humanized antibody.

[0358] Take 2E5 MDA-MB-231, A2780 and NCI-H1975 cells respectively. Dilute the humanized antibody hz5H6Lm8 and the control antibody Mab-A and the isotype control to 1 ug / ml, 0.5 ug / ml, 0.25 ug / ml and 0.125 ug / ml, and use the internalization reagent Antibody Internalization human Reagent (Cat. 90565, Sartorius) to add the dilution to the cells according to the instructions of the reagent. Incubate at 37°C in the dark for 120 min, and use flow cytometry to detect the internalization of the immunofluorescence signal into the cells.

[0359] The results are shown in Figures 18, 19 and 20. The results show that the humanized antibody hz5H6Lm8 and the control antibody Mab-A are strongly internalized in different tumor cells.

[0360] Different hz5H6 mutant assay: 2E5 MDA-MB-231, A549 and Hela cells were taken, and the internalization activity of different hz5H6 mutants was detected by flow cytometry in the same way as described above.

[0361] The results are shown in Figures 21, 22 and 23. The results show that the humanized antibody hz5H6 mutants and the control antibody Mab-A all have strong internalization in different tumor cells.

[0362] Example 16: Detection of the killing activity of ADCs obtained by coupling humanized antibodies with small molecule cytotoxic compounds on tumor cells

[0363] The small molecule cytotoxic compound monomethyl auristatin E (MMAE) was coupled with different antibodies through a drug-containing linker BL20E to prepare ADC molecules, named "antibody-MMAE (BL20E)". The structure of BL20E is shown below in the Example.

[0364] The killing activity of the prepared ADC molecules on MDA-MB-231, SKOV3 and A2780 tumor cells was evaluated.

[0365] The different tumor cells described above were inoculated into 96-well culture plates, and after overnight adhesion, each ADC was diluted to 100 μg / ml, then 3-fold gradient dilution was performed to obtain 9 gradients, and the dilutions were added to the cells, which were incubated at 37°C for 72 h. Then CCK8 kit was used for detection.

[0366] The results are shown in Figures 24 and 25. The results show that the killing activity of each ADC on MDA-MB-231 and SKOV3 cells has the same trend.

[0367] Different hz5H6 mutant ADC killing activity assay: A2780 cells were plated and adhered overnight, and each hz5H6 mutant and / or Mab-A ADC was diluted to 10 μg / ml, then 3-fold gradient dilution was performed, and the cells were incubated at 37°C for 96 h, and then CCK8 kit was used to detect the killing activity of each mutant.

[0368] The results are shown in Figures 26 and 27. The results show that the killing activity of ADCs prepared from different humanized antibody hz5H6 mutants is similar, and the killing activity is similar to that of ADC prepared from Mab-A.

[0369] Example 17: Pharmacodynamic evaluation of ADCs obtained by coupling humanized antibodies with small molecule cytotoxic compounds in NCG mice bearing human ovarian cancer A2780 model

[0370] To evaluate the anti-tumor therapeutic effect of ADC in NCG mice bearing human ovarian cancer A2780 model.

[0371] Human ovarian cancer A2780 cells were inoculated subcutaneously in the right anterior flank of female NCG mice, and when the tumor grew to 100 mm 3 The mice were grouped and administered at a dose of 3 mg / kg, i.p., single administration, when the tumor grew to 100 mm

[0372] The tumor growth inhibition is shown in Table 8, Figure 28 and Figure 29. The results show that each group has a clear anti-tumor effect compared with the NC group. The efficacy of hz5H6Lm8-MMAE(BL20E) (TGI TV and TGI Tw were 86% and 84%, respectively) was significantly better than that of the control antibody Mab-A-MMAE(BL20E) (TGI TV and TGI Tw were 55% and 53%, respectively).

[0373] Table 8. Tumor growth inhibition of each group

[0374] Example 18: Pharmacodynamic evaluation of ADC conjugated with humanized antibody in M-NSG mice bearing human breast cancer MDA-MB-231 model

[0375] In addition, the anti-tumor therapeutic effect of ADC (mutant-MMAE(BL20E)) was evaluated in M-NSG mice bearing human breast cancer MDA-MB-231 model.

[0376] Human breast cancer MDA-MB-231 cells were inoculated subcutaneously in the right anterior flank of NSG mice, and when the tumor grew to 180 mm 3 The mice were grouped and administered at a dose of 5 mg / kg, i.p., single administration, when the tumor grew to 180 mm

[0377] The tumor growth inhibition is shown in Table 9, Figure 30 and Figure 31. The results show that each group has a clear anti-tumor effect compared with the NC control group. The tumor volume and tumor weight were compared at the end of the experiment on D45. The results show that the tumor inhibition effect of hz5H6Lm8-MMAE(BL20E), hz5H6Hm6Lm14-MMAE(BL20E) and hz5H6Hm8Lm14-MMAE(BL20E) is better than that of the control antibody Mab-A-MMAE(BL20E); among them, the tumor inhibition effect of hz5H6Hm6Lm14-MMAE(BL20E) is the best.

[0378] Table 9. Tumor growth inhibition of each group

[0379] Example 19: Pharmacodynamic evaluation of ADC coupled with humanized antibody in Balb / C-nude mice bearing human colorectal cancer RKO model

[0380] In addition, the anti-tumor therapeutic effect of the ADC targeting ADAM9 of the application and the control ADC IMGC936 (Mab-A-DM21, DAR value of 2; wherein the compound is DM21-C, from patent application publication document WO2022192134A1) was evaluated in Balb / C-nude mice bearing human colorectal cancer RKO model.

[0381] Human colorectal cancer RKO cells were inoculated subcutaneously in the right anterior flank of Balb / C-nude mice, and when the tumors grew to 300mm 3 The mice were grouped and dosed when the tumors grew to 300mm

[0382] The tumor growth inhibition is shown in Figures 32 and 33. The results show that the hz5H6Hm6Lm14-ADC has better efficacy, and the tumor inhibition effect of hz5H6Hm6Lm14-MF-L6 is better than that of Mab-A-MF-L6 with the same toxin, and significantly better than the control molecule IMGC936. MF-L6, i.e. MWF-L6, is used interchangeably in this application, and the structure is shown below in the example.

[0383] Example 20: Binding of anti-ADAM9 antibody to ADAM9+ human normal tissues or cells

[0384] ADAM9 is expressed in a variety of normal tissues, including kidney, gastrointestinal tract, etc. Using frozen sections of human kidney, intestine, and stomach tissues (3 donors each), hz5H6Hm6Lm14 and positive control antibody Mab-A and isotype control antibody NC-IgG1 were used as primary antibodies at a concentration of 10 μg / mL for immunohistochemical detection of frozen sections. The binding of each antibody molecule to normal ADAM9+ tissues was evaluated.

[0385] In addition, ADAM9 is expressed on the surface of monocytes. The binding characteristics of hz5H6Hm6Lm14, hz5H6Hm8Lm14 and Mab-A to ADAM9+ human PBMC cells were analyzed by FACS method. After PBMC (derived from healthy volunteers) were added to 20 μg / ml of NC-hIgG1 and blocked at room temperature for 30 min, CF488 (Cat. MX488AS100-1KT, Sigma)-labeled hz5H6Hm6Lm14, hz5H6Hm8Lm14 and control antibody Mab-A and isotype control antibody NC-hIgG1 (starting concentration of 20 μg / mL, 2-fold gradient dilution of 6 concentrations) were added, and APC Anti-Human CD14 Antibody (Cat. E-AB-F1209E, Elabscience) was also added to each well, and incubated at 4°C in the dark for 30 min. After washing with pre-cooled PBS and resuspending, the signal values of FITC and APC were detected by flow cytometry. The CD14+ subpopulation (APC) was circled from the total cells, and the FITC signal values of each antibody were analyzed and compared in the CD14+ subpopulation.

[0386] The results are shown in Figures 34, 35, 36, 37 and Table 10. The results show that, compared with the control antibody Mab-A, the binding of hz5H6Hm6Lm14 to normal human positive tissues is significantly weaker than that of Mab-A; the binding ability of hz5H6Hm6Lm14 and hz5H6Hm8Lm14 to human PBMC-derived monocytes (CD14+) is also significantly weaker than that of Mab-A, suggesting that ADC drugs based on hz5H6 have lower toxicity risk to normal tissues and better safety.

[0387] Table 10. Summary of results of frozen section immunohistochemical staining

[0388] Note: -, negative; +, weakly positive; ++, positive.

[0389] Example 21: Effect of Fc variants on the activity of anti-ADAM9 antibodies

[0390] The human IgGl variant L234A / L235A ("LALA"; Xu et al., Cell Immunol 2000 Feb 25; 200(1): 16-26) can eliminate the binding of FcyRs without affecting the binding of FcRn, thereby eliminating Fc-mediated effector functions. To further confirm the effect of FcyRs on the activity of the anti-ADAM9 antibody of the present application, the heavy chain variable region encoding gene sequence of hz5H6Hm6Lm14 was cloned into a eukaryotic transient expression vector containing the human IgGl-LALA (human IgGl variant L234A / L235A) heavy chain constant region (SEQ ID NO. 57) encoding gene sequence to obtain a heavy chain expression plasmid; the light chain variable region encoding gene sequence of hz5H6Hm6Lm14 was cloned into a eukaryotic transient expression plasmid containing a human kappa light chain constant region (SEQ ID NO. 4) to obtain a light chain expression vector. The two expression vectors were recombinantly expressed in HEK293 cells to obtain a human IgGl-LALA subtype recombinant protein, designated "hz5H6Hm6Lm14-LALA".

[0391] SEQ ID NO. 57: IgGl-LALA heavy chain constant region amino acid sequence

[0392] The 2E5 human large cell lung cancer NCI-H460 and human colorectal cancer DLD-1 cells were used to detect the binding and internalization of hz5H6Hm6Lm14-LALA to the cells by flow cytometry in the same manner as in Examples 14 and 15. The results are shown in Figures 38 to 41, and the activity of hz5H6Hm6Lm14-LALA in binding and internalization was consistent with that of hz5H6Hm6Lm14.

[0393] Further, the anti-tumor therapeutic effect of the ADC (IgGl variant) was evaluated in a Balb / C-nude mouse model bearing human large cell lung cancer NCI-H460.

[0394] Human large cell lung cancer NCI-H460 cells were inoculated subcutaneously on the right anterior flank of Balb / C-nude mice, and when the tumors grew to 200 mm3, the mice were grouped and administered at a dose of 10 mg / kg, i.p., a single administration. 3

[0395] The tumor growth inhibition is shown in Figure 42. The results showed that hz5H6Hm6Lm14-LALA-MF-L6 and hz5H6Hm6Lm14-MF-L6 had comparable tumor inhibition effects, and the use of the human IgGl-LALA subtype did not affect the anti-tumor activity of ADAM9.

[0396] ​Example 22 Distribution of Anti-ADAM9 Antibodies in Tumor Microenvironment in AsPC-1 Cell CDX Subcutaneous Tumor Mouse Model

[0397] ADAM9 expression positive tumor AsPC-1 cells were inoculated into nude mice, 5x10 6 / each, divided into 3 groups, 3 each, and when the tumor grew to 400-600mm 3 , respectively, CY7 (MCE, HY-D0824A) labeled antibodies: hz5H6Hm6Lm14, Mab-A and isotype control antibody NC were given intraperitoneally, at a dose of 10mg / kg, 5 days later the mice were heart perfused, then the whole body and heart, liver, spleen, lung, kidney and tumor site of the mice were imaged respectively using PerkinElmer IVIS Lumina III instrument, the fluorescence signal values of each tissue were obtained, then the signal ratio of each tissue to the corresponding whole body signal of the mouse was calculated to confirm the tissue distribution of the antibody, and the statistical difference was analyzed by T test.

[0398] The tissue distribution of the antibody is shown in Figure 43. The results showed that the detection amount of hz5H6Hm6Lm14 in the tumor was higher than that of Mab-A, and there was a statistical difference in the detection amount of hz5H6Hm6Lm14 and NC control group in the tumor (P<0.05), suggesting that hz5H6Hm6Lm14 has better tumor accumulation.

[0399] Example 23: Preparation of Drug Linker Containing

[0400] 1. Synthesis of compound of formula A

[0401] According to the method described in patent application publication WO2023109965A1, the compounds of formula A shown in Table 11 were synthesized.

[0402] Table 11. Compounds of formula A

[0403] 2. Synthesis of camptothecin compounds

[0404] According to the method described in patent application publication WO2023109965A1, the camptothecin compounds shown in Table 12 were synthesized.

[0405] Table 12. Camptothecin compounds

[0406] 3. Synthesis of drug-linker conjugates

[0407] According to the method described in patent application publication WO2023109965A1 or WO2018095422A1, the drug-linker conjugates shown in Table 13 were synthesized.

[0408] Table 13. Drug-linker conjugates

[0409] Preparation and characterization of antibody-drug conjugates targeting ADAM9

[0410] 1. General method for preparing antibody-drug conjugates

[0411] 1.1 General method for site-directed conjugation

[0412] Reduction of antibody: 120 mg of antibody sample was replaced into a Sephadex G25 carrier NAP-25 column, into a pH 7.0 buffer solution containing 50 mM sodium chloride, 50 mM sodium phosphate dibasic-sodium phosphate monobasic buffer solution, and the antibody concentration was diluted to 10 mg / ml. 10 ml of a total of 100 mg of antibody sample was added with 10 mg / ml TCEP (Sigma-Aldrich) aqueous solution in an antibody-TCEP molar ratio of 1 to 10 equivalents, and the volume of TCEP aqueous solution added was 2.1 ml. After 2 hours of incubation, the reaction solution was replaced using a Sephadex G25 column, into a pH 6.5 buffer solution containing 50 mM sodium chloride, 50 mM sodium phosphate dibasic-sodium phosphate monobasic buffer.

[0413] Coupling of antibody with drug-linker and hydrolysis: the above reduced antibody was diluted to 5 mg / mL, and 0.38 ml of N,N-dimethylacetamide (DMA) was added as a pre-solvent, followed by adding 0.67 ml of a DMA-drug-linker mixed solution containing 10 mg / mL drug-linker as a reaction solution in an antibody-small molecule drug molar ratio of 1 to 5.5 times the equivalent amount. Stir at room temperature for 30 minutes. The reaction solution was replaced using a Sephadex G25 carrier NAP-25 column into a pH 8.0 sodium phosphate dibasic-sodium phosphate monobasic buffer solution to remove excess drug-linker, and heated at 37°C for 3 hours.

[0414] Purification of antibody drug conjugate: The above sample was concentrated using AMICOM ultrafiltration centrifuge tubes to about 15 mg / mL. 50 mM Na2HPO4-NaH2PO4 + 3 M ammonium phosphate buffer solution was added to a conductivity of 100 ms / cm. Loaded onto a TOYOPEAL Butyl-650M packing (purchased from TOSOH) hydrophobic column, A phase is 50 mm Na2HPO4-NaH2PO4 + 0.6 M ammonium sulfate, B phase is 50 mm Na2HPO4-NaH2PO4 buffer solution. Eluted with a B phase 0-100% gradient of 8 column volumes, and the main peak was collected.

[0415] The final sample was replaced into 50 mM Na2HPO4-NaH2PO4 buffer salt at pH 7.4 using AMICOM ultrafiltration centrifuge tubes, and filtered using a 0.22 um filter membrane (Sartorius stedim Ministart).

[0416] 1.2 General preparation method of random conjugation

[0417] Prepared according to the preparation method disclosed in patent CN105849126A.

[0418] 1.3 Preparation method of control ADC IMGC936

[0419] The control ADC IMGC936 was prepared according to the method described in patent application publication CN112543770A (CN201980049672.2).

[0420] 2. General analysis method of antibody drug conjugate

[0421] 2.1 Determination of drug antibody conjugation ratio (UV-DAR method) and concentration by ultraviolet spectrophotometry

[0422] According to the method described in patent application publication WO2023109965A1, the drug antibody conjugation ratio (DAR value) and concentration were determined by ultraviolet spectrophotometry. The molar absorption coefficient of each drug-containing linker at 280 nm and its characteristic wavelength is shown below, wherein is the molar absorption coefficient of the drug-containing linker at its characteristic absorption wavelength Z nm, is the molar absorption coefficient of the drug-containing linker at 280 nm.

[0423] 2.2 Hydrophobic chromatography

[0424] a. Determination of DAR value by hydrophobic chromatography HIC-HPLC of antibody drug conjugate

[0425] Sample preparation: the sample was diluted to 2.0 mg / ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was used for HPLC analysis;

[0426] Column: Sepax Proteomix HIC Butyl-NP5, 5 μm, 4.6 mm*35 mm;

[0427] Mobile phase A: 1.5 M (NH4)2SO4+25 mM PB, pH 7.0

[0428] Mobile phase B: 25 mM PB+20% IPA, pH 7.0

[0429] Flow rate: 0.6 mL / min;

[0430] Detection wavelength: 280 nm;

[0431] Column temperature: 30°C;

[0432] Injection volume: 10 μL;

[0433] HIC chromatographic gradient:

[0434] DAR calculation formula:

[0435] DAR = ∑(weighted peak area) / 100, i.e. DAR = (D0 peak area ratio*0+D1 peak area ratio*1+D2 peak area ratio*2+D3 peak area ratio*3+D4 peak area ratio*4+D5 peak area ratio*5+D6 peak area ratio*6+D7 peak area ratio*7+D8 peak area ratio*8) / 100.

[0436] b. Determination of DAR value of antibody drug conjugate by hydrophobic chromatography HIC-HPLC

[0437] Sample preparation: the sample was diluted to 2.0 mg / ml with mobile phase B, centrifuged at 12000 rpm for 10 min, and the supernatant was used for HPLC analysis;

[0438] Column: Sepax Proteomix HIC Butyl-NP5, 5 μm, 4.6 mm*35 mm;

[0439] Mobile phase A: 1.5 M (NH4)2SO4+25 mM PB, pH 7.0

[0440] Mobile phase B: 25 mM PB+20% IPA, pH 7.0

[0441] Flow rate: 0.6 mL / min;

[0442] Detection wavelength: 280 nm;

[0443] Column temperature: 30℃;

[0444] Injection volume: 10 μL;

[0445] HIC chromatography gradient

[0446] DAR calculation formula: same as a.

[0447] 2.3 Determination of molecular size heterogeneity by size-exclusion chromatography SEC-HPLC

[0448] Sample treatment: after the sample was diluted with mobile phase to about 1.0 mg / ml, centrifuged at 12000 rpm for 10 min, the supernatant was taken for sample analysis.

[0449] Chromatographic column: TOSOH, TSKgel G3000SWXL, 5 μm, 7.8 mm*300 mm;

[0450] Mobile phase: 100 mM PB + 200 mM arginine hydrochloride, 5% isopropanol (pH 6.8);

[0451] Flow rate: 0.6 mL / min;

[0452] Detection wavelength: 280 nm;

[0453] Column temperature: 30℃;

[0454] Injection volume: 20 uL;

[0455] Elution time: 20 min;

[0456] Elution gradient: isocratic elution.

[0457] 3. Preparation and characterization of antibody drug conjugates targeting ADAM9

[0458] According to the above general preparation method in this embodiment, the anti-ADAM9 antibodies hz5H6Lm1, hz5H6Lm3, Mab-A, hz5H6, hz5H6Hm6Lm14, hz5H6Hm6Lm14-LALA and hz5H6Lm8 were coupled with the drug linker containing to prepare ADC.

[0459] According to the preparation method of the control ADC IMGC936 in this embodiment, the anti-ADAM9 antibody Mab-A was coupled with DM21-C to prepare the control ADC IMGC936 (i.e. ADAM9-ADC-14 in Table 14).

[0460] The DAR value, concentration and purity of the site-directed conjugated ADC were determined by the above ultraviolet spectrophotometry, hydrophobic chromatography and molecular exclusion chromatography, respectively, in this example.

[0461] The DAR and purity of the control ADC IMGC936 were determined by the molecular exclusion chromatography (SEC-HPLC) in this example; the concentration of the control ADC IMGC936 was determined according to the "Fourth Method 2,2'-Biquinoline-4,4'-dicarboxylic Acid Method (BCA Method)" in Chinese Pharmacopoeia 2015 Edition General 0731 "Protein Content Determination Method".

[0462] The results are shown in Table 14.

[0463] Table 14. Characterization results of antibody drug conjugates

[0464] Example 25 Activity evaluation of antibody drug conjugate targeting ADAM9

[0465] The cell density of the ADAM9 high-expression cell line HCT15 (purchased from: ATCC) was adjusted to 1.5 x 10 4 6 / ml with complete medium, 100 μl / well was added to a 96-well cell culture plate, and incubated overnight.

[0466] The ADC targeting ADAM9 was diluted to 20 μg / ml using complete medium, and then diluted by 4-fold gradient, with 8 gradients in total. 50 μL of the above diluted ADC was added to the 96-well cell culture plate containing HCT15 cells, and then 150 μL of complete medium was added to each well, and mixed well. Two replicates were set for all samples; at the same time, negative control wells (cells + medium) and blank control wells (no cells, only medium) were set in the cell culture plate. After incubation for 168 hours, the cell culture plate was taken out, the supernatant was discarded, 100 μl / well of CCK-8 was added, and the reaction was carried out at 37°C for 1-4 h. The cell culture plate was taken out, and the OD value was read at 450 nm.

[0467] The results are shown in Table 15 and Figure 44. The results show that the killing effect of the ADC molecule ADAM9-ADC-8 targeting ADAM9 on ADAM9 high-expression HCT15 cells is significantly better than that of the control ADC IMGC936 (ADAM9-ADC-14).

[0468] Table 15. In vitro cell killing activity of ADC targeting ADAM9

[0469] Example 26 Evaluation of drug resistance characteristics of antibody drug conjugate targeting ADAM9

[0470] Multidrug resistance (MDR) was first discovered in tumor cells. This phenomenon refers to that after long-term treatment of tumor cells sensitive to a drug with an anti-tumor drug, the sensitivity of the cells to the drug decreases, drug resistance is generated, and the sensitivity to other structural types of anti-tumor drugs also decreases. Determining whether tumor cells are resistant to a drug is particularly important for evaluating the efficacy of the drug.

[0471] P-glycoprotein (p-gp), also known as ABCB1, is a classical MDR pathway mediated by MDR known in the art, and it is generally believed that the drug efflux mediated by this protein leads to drug resistance of the body to the drug. In this embodiment, the p-gp inhibitor Tariquidar is used to detect whether the ADC of the application is prone to p-gp-mediated efflux, so as to understand its drug resistance characteristics.

[0472] The cell density of the ADAM9 high-expression cell line HCT15 was adjusted to 1.5x10 4 The cells were cultured overnight in a 96-well cell culture plate. The ADC targeting ADAM9 was diluted to 20 μg / ml using complete medium, and then diluted by 4-fold gradient, and diluted by 8 gradients in succession. The following experimental groups were set up:

[0473] The inhibitor-free group: complete medium was added to the above-mentioned 96-well cell culture plate containing HCT15 cells at 50 μl / well, and incubated at 37°C for 1 hour, and then the diluted ADC was added to the 96-well cell culture plate at 50 μl / well, and mixed well; all samples were set in duplicate.

[0474] The inhibitor group: the drug resistance protein p-gp inhibitor Tariquidar was added to the above-mentioned 96-well cell culture plate containing HCT15 cells at 50 μl / well, and the concentration in a final volume of 200 μl was 200 nM, and incubated at 37°C for 1 hour, and then the diluted ADC was added to the 96-well cell culture plate at 50 μl / well, and mixed well; all samples were set in duplicate.

[0475] At the same time, negative control wells (cells + medium) and blank control wells (no cells, only medium) were set up in the cell culture plate. After incubation for 168 hours, the cell culture plate was taken out, the supernatant was discarded, 100 μl / well of CCK-8 was added, and the reaction was carried out at 37°C for 1-4 h. The cell culture plate was taken out, and the OD value was read at 450 nm.

[0476] The results are shown in Table 16 and Figure 45. The results show that the activity of the control ADC ADAM9-ADC-14 is affected by p-gp expression in the cell line HCT15 with high p-gp expression: without the addition of a p-gp inhibitor, the control ADC has no killing activity, but after the addition of a p-gp inhibitor, the killing activity of the control ADC is significantly increased, with an EC50 of 184.3 ng / mL; in contrast, the killing activity of the ADC ADAM9-ADC-8 of the application is not affected by p-gp protein expression. Therefore, it can be known that ADAM9-ADC-8 is not a substrate for p-gp, and ADAM9-ADC-14 is a substrate for p-gp, and is more likely to produce drug resistance.

[0477] Table 16. Drug resistance characteristics of ADCs targeting ADAM9

[0478] Example 27 Pharmacodynamic evaluation of antibody drug conjugates targeting ADAM9 in a Balb / C-nude mouse model of human colorectal cancer

[0479] A small molecule cytotoxic compound DXD was coupled with hz5H6Hm6Lm14 via a drug-containing linker MC-GGFG-DXD to prepare an ADC molecule, designated as "hz5H6Hm6Lm14-GGFG-DXD (ADAM9-ADC-17)". The pharmacodynamic evaluation of hz5H6Hm6Lm14-GGFG-DXD (ADAM9-ADC-17) and hz5H6Hm6Lm14-MF-L6 (ADAM9-ADC-16) was performed.

[0480] Human colorectal cancer RKO cells were inoculated subcutaneously in the right anterior flank of Balb / C-nude mice, and when the tumors grew to 200 mm 3 The mice were grouped and dosed when the tumors grew to 200 mm

[0481] The tumor growth inhibition is shown in Figure 46. The results show that different toxin ADCs have a clear anti-tumor effect in the RKO colorectal cancer model. hz5H6Hm6Lm14-MF-L6 has a better anti-tumor effect.

[0482] The above description of specific embodiments of the application does not limit the application, and those skilled in the art can make various changes or modifications to the application according to the application, as long as they do not deviate from the spirit of the application, and should belong to the scope of the claims attached to the application.

Claims

1. An antibody or antigen-binding fragment thereof against a disintegrin and metalloproteinase 9 (ADAM9), the antibody or antigen-binding fragment thereof comprising heavy chain CDRs, i.e. heavy chain CDR1 (H-CDR1), heavy chain CDR2 (H-CDR2), heavy chain CDR3 (H-CDR3), and light chain CDRs, i.e. light chain CDR1 (L-CDR1), light chain CDR2 (L-CDR2), light chain CDR3 (L-CDR3), wherein, The heavy chain CDRs and light chain CDRs are shown as follows: (1) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, respectively; and L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, respectively; (2) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 39, SEQ ID NO. 38, respectively; and L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47, respectively; (3) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 40, SEQ ID NO. 38, respectively; and L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47, respectively; (4) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 38, respectively; and L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47, respectively; (5) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 42, respectively; and L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47, respectively; (6) H-CDR1, H-CDR2, H-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 43, respectively; and L-CDR1, L-CDR2, L-CDR3 comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47, respectively; (7) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 44, in sequence; and L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47, in sequence; (8) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, in sequence; and L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 48, SEQ ID NO. 47, in sequence; (9) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, in sequence; and L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 47, in sequence; (10) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, in sequence; and L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 50, SEQ ID NO. 47, in sequence; (11) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, in sequence; and L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 51, SEQ ID NO. 49, SEQ ID NO. 47, in sequence; (12) H-CDR1, H-CDR2, H-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, in sequence; and L-CDR1, L-CDR2, L-CDR3, comprising the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 52, in sequence; (13) H-CDR1, H-CDR2, H-CDR3, which consist of amino acid sequences shown in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, respectively; and L-CDR1, L-CDR2, L-CDR3, which consist of amino acid sequences shown in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 53, respectively; (14) H-CDR1, H-CDR2, H-CDR3, which consist of amino acid sequences shown in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, respectively; and L-CDR1, L-CDR2, L-CDR3, which consist of amino acid sequences shown in SEQ ID NO. 51, SEQ ID NO. 49, SEQ ID NO. 53, respectively; (15) H-CDR1, H-CDR2, H-CDR3, which consist of amino acid sequences shown in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, respectively; and L-CDR1, L-CDR2, L-CDR3, which consist of amino acid sequences shown in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 54, respectively; (16) H-CDR1, H-CDR2, H-CDR3, which consist of amino acid sequences shown in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, respectively; and L-CDR1, L-CDR2, L-CDR3, which consist of amino acid sequences shown in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 55, respectively; (17) H-CDR1, H-CDR2, H-CDR3, which consist of amino acid sequences shown in SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, respectively; and L-CDR1, L-CDR2, L-CDR3, which consist of amino acid sequences shown in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 56, respectively; (18) H-CDR1, H-CDR2, H-CDR3, which consist of amino acid sequences shown in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 38, respectively; and L-CDR1, L-CDR2, L-CDR3, which consist of amino acid sequences shown in SEQ ID NO. 51, SEQ ID NO. 49, SEQ ID NO. 47, respectively; (19) H-CDRs of the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 43, in order; and L-CDRs of the amino acid sequences set forth in SEQ ID NO. 51, SEQ ID NO. 49, SEQ ID NO. 47, in order; (20) H-CDRs of the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 44, in order; and L-CDRs of the amino acid sequences set forth in SEQ ID NO. 51, SEQ ID NO. 49, SEQ ID NO. 47, in order; (21) H-CDRs of the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 38, in order; and L-CDRs of the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 53, in order; and (22) H-CDRs of the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 43, in order; and L-CDRs of the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 53, in order.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, the antibody or antigen-binding fragment thereof specifically targets a disintegrin and metalloproteinase 9 (ADAM9), preferably a human ADAM9; optionally, the antibody or antigen-binding fragment thereof has or does not have species cross- binding activity to human, cyno, mouse ADAM9; optionally, the antibody or antigen-binding fragment thereof is capable of or incapable of blocking metalloproteinase activity of ADAM9, which can be ADAM9 in membrane-bound or secreted form, e.g., human ADAM9.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region comprising a combination selected from the group consisting of: (1) the amino acid sequence set forth in SEQ ID NO. 5, or an amino acid sequence having at least 75% identity to said amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 6, or an amino acid sequence having at least 75% identity to said amino acid sequence; (2) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence at least 75% identical thereto; (3) the amino acid sequence set forth in SEQ ID NO. 9, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence at least 75% identical thereto; (4) the amino acid sequence set forth in SEQ ID NO. 10, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence at least 75% identical thereto; (5) the amino acid sequence set forth in SEQ ID NO. 11, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence at least 75% identical thereto; (6) the amino acid sequence set forth in SEQ ID NO. 12, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence at least 75% identical thereto; (7) the amino acid sequence set forth in SEQ ID NO. 13, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence at least 75% identical thereto; (8) the amino acid sequence set forth in SEQ ID NO. 14, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence at least 75% identical thereto; (9) the amino acid sequence set forth in SEQ ID NO. 15, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence at least 75% identical thereto; (10) the amino acid sequence set forth in SEQ ID NO. 16, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence at least 75% identical thereto; (11) the amino acid sequence set forth in SEQ ID NO. 17, or an amino acid sequence at least 75% identical thereto; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence at least 75% identical thereto; (12) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to said amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 18, or an amino acid sequence at least 75% identical to said amino acid sequence; (13) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to said amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 19, or an amino acid sequence at least 75% identical to said amino acid sequence; (14) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to said amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 20, or an amino acid sequence at least 75% identical to said amino acid sequence; (15) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to said amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 21, or an amino acid sequence at least 75% identical to said amino acid sequence; (16) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to said amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 22, or an amino acid sequence at least 75% identical to said amino acid sequence; (17) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to said amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 23, or an amino acid sequence at least 75% identical to said amino acid sequence; (18) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to said amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 24, or an amino acid sequence at least 75% identical to said amino acid sequence; (19) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to said amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence at least 75% identical to said amino acid sequence; (20) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to said amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 26, or an amino acid sequence at least 75% identical to said amino acid sequence; (21) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to said amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 27, or an amino acid sequence at least 75% identical to said amino acid sequence; (22) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to that amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 28, or an amino acid sequence at least 75% identical to that amino acid sequence; (23) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to that amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 29, or an amino acid sequence at least 75% identical to that amino acid sequence; (24) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to that amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 30, or an amino acid sequence at least 75% identical to that amino acid sequence; (25) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to that amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 31, or an amino acid sequence at least 75% identical to that amino acid sequence; (26) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to that amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 32, or an amino acid sequence at least 75% identical to that amino acid sequence; (27) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to that amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 33, or an amino acid sequence at least 75% identical to that amino acid sequence; (28) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to that amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 34, or an amino acid sequence at least 75% identical to that amino acid sequence; (29) the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence at least 75% identical to that amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 35, or an amino acid sequence at least 75% identical to that amino acid sequence; (30) the amino acid sequence set forth in SEQ ID NO. 14, or an amino acid sequence at least 75% identical to that amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 29, or an amino acid sequence at least 75% identical to that amino acid sequence; (31) the amino acid sequence set forth in SEQ ID NO. 16, or an amino acid sequence at least 75% identical to that amino acid sequence; and the amino acid sequence set forth in SEQ ID NO. 29, or an amino acid sequence at least 75% identical to that amino acid sequence; (32) the amino acid sequence represented by SEQ ID NO. 17, or an amino acid sequence having at least 75% identity to the amino acid sequence; and the amino acid sequence represented by SEQ ID NO. 29, or an amino acid sequence having at least 75% identity to the amino acid sequence; (33) the amino acid sequence represented by SEQ ID NO. 14, or an amino acid sequence having at least 75% identity to the amino acid sequence; and the amino acid sequence represented by SEQ ID NO. 31, or an amino acid sequence having at least 75% identity to the amino acid sequence; (34) the amino acid sequence represented by SEQ ID NO. 16, or an amino acid sequence having at least 75% identity to the amino acid sequence; and the amino acid sequence represented by SEQ ID NO. 31, or an amino acid sequence having at least 75% identity to the amino acid sequence.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, characterized in that, The antibody is a murine, rabbit, human antibody, or a murine antibody, a chimeric antibody, a fully or partially humanized antibody, or a derivatized antibody; Alternatively, the antigen-binding fragment of the antibody is any form of fragment of the antibody, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv. Optionally, the antibody or the antigen-binding fragment thereof further comprises a heavy chain constant region (CH) and / or a light chain constant region (CL), preferably a human or murine heavy chain constant region and / or a light chain constant region; preferably, the antibody or the antigen-binding fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE, and / or a kappa or lambda type light chain constant region.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, characterized in that, The antibody is a monoclonal antibody; preferably, the antibody is an immunoglobulin, for example, the type of the immunoglobulin is human IgA, IgD, IgE, IgG, or IgM; preferably, the antibody is human IgG1 and variants thereof, such as IgG1-LALA (L234A / L235A) subtype, or IgG4 and variants thereof, such as IgG4-L235E or F234A / L235A subtype.

6. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or the antigen-binding fragment thereof of any one of claims 1 to 5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A host cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7.

9. An antibody drug conjugate targeting ADAM9 or a salt thereof, comprising the anti-ADAM9 antibody or the antigen-binding fragment thereof of any one of claims 1 to 5; Preferably, the antibody drug conjugate or the salt thereof is formed by conjugating the antibody or the antigen-binding fragment thereof of any one of claims 1 to 5 with a small molecule cytotoxic compound; wherein preferably, the small molecule cytotoxic compound is selected from a tubulin inhibitor, a topoisomerase inhibitor, or a DNA binding agent; Preferably, the antibody drug conjugate or the salt thereof is formed by conjugating the antibody or the antigen-binding fragment thereof of any one of claims 1 to 5 with a small molecule cytotoxic compound; wherein preferably, the small molecule cytotoxic compound is selected from a tubulin inhibitor, a topoisomerase inhibitor, or a DNA binding agent; More preferably, the tubulin inhibitor is selected from the group consisting of a maytansine derivative, Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), Monomethyl Dolastatin 10, a Tubulysin derivative, a Cryptophycin derivative, and Taltobulin; the topoisomerase inhibitor is selected from the group consisting of a camptothecin compound such as exatecan and derivatives thereof, and the like, the Doxorubicin metabolite PNU-159682 derivative, and the irinotecan (CPT-11) metabolite SN38 derivative; the DNA binding agent is selected from the group consisting of a PBD derivative and a Duocarmycin derivative.

10. The antibody drug conjugate or salt thereof according to claim 9, characterized by, The antibody drug conjugate or salt thereof has a structure represented by the general formula wherein: Ab represents an anti-ADAM9 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; E L selected from the group consisting of represents a linkage to Ab via a thiol of a cysteine: E L -1a and / or E L -1b: and / or E L -2： E L -3： E L -4： E L -5： E L -6： M is phenylene or phenylene substituted with one or more substituents, or a bond; in substituted phenylene, the substituents are selected from the group consisting of alkyl (e.g. C1-6alkyl, preferably C1-4alkyl), haloalkyl (e.g. haloC1-6alkyl, preferably haloC1-4alkyl, e.g. trifluoromethyl), alkoxy (e.g. C1-6alkoxy, preferably C1-4alkoxy, preferably methoxy), halogen, ester, amide, and cyano; preferably, M is halogen-substituted phenylene; further preferably, M is fluoro-substituted phenylene; SP1 is selected from the group consisting of C1-8alkylene, C1-8cycloalkylene, or C1-21 (preferably C1-16, more preferably C1-11, more preferably C5-9, more preferably C7) straight chain heteroalkylene comprising 1-11 (preferably 1-6, more preferably 3-5, more preferably 4) heteroatoms selected from N, O, or S, wherein each of said C1-8alkylene, C1-8cycloalkylene, and C1-21straight chain heteroalkylene is independently optionally substituted with one or more substituents selected from the group consisting of hydroxyl, amino, sulfonic acid, and cyano; SP2is selected from -NH(CH2CH2O) a CH2CH2CO-, -NH(CH2CH2O) a CH2CO-, -S(CH2) a CO- or a chemical bond, wherein a is an integer from 1 to 20, preferably an integer from 1 to 10, more preferably an integer from 1 to 6; A represents a short peptide structure of 2-4 amino acids, wherein A represents a short peptide structure of 2 amino acids selected from NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO, NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO or NH-Val-Cit-CO; A represents a short peptide structure of 3 amino acids selected from NH-Glu-Val-Ala-CO, NH-Glu-Val-Cit-CO or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO; A represents a short peptide structure of 4 amino acids selected from NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-Gly-Gly-CO, preferably NH-Gly-Gly-Phe-Gly-CO; preferably A is NH-Val-Ala-CO, NH-Gly-Gly-Phe-Gly-CO or NH-Ala-Ala-Ala-CO, NH represents the amino terminus of the group A, CO represents the carboxyl terminus of the group A, and the group A can be linked to SP2 via the amino group at the amino terminus of the short peptide structure of the group A; m is 1 to 10, preferably 1 to 8 (e.g. 1 to 5), more preferably 3 to 8; and m can be an integer or a non-integer; and D represents the small molecule cytotoxic compound.

11. The antibody drug conjugate or salt thereof according to claim 10, characterized by In the general formula the group is selected from the following structures, where the wavy line indicates attachment to a cysteine in the antibody or to a small molecule cytotoxic compound D:

12. The antibody drug conjugate or salt thereof according to any one of claims 9 to 11, characterized in that, The antibody drug conjugate or salt thereof is formed by conjugating the antibody or antigen binding fragment thereof of any one of claims 1 to 5 with a camptothecin compound, and has a general structure as shown, wherein D represents a camptothecin compound.

13. The antibody drug conjugate or salt thereof according to any one of claims 9 to 12, characterized in that, The structure of the camptothecin compound is shown in structural formula I: In structural formula I, R1, R2, R3, R4are independently hydrogen, halogen, hydroxyl, C1-6alkoxy, amino or substituted amino, C1-7alkyl or substituted C1-7alkyl, or any two of R1, R2, R3, R4together with the carbon atom to which they are attached form a C3-6 (preferably C3-5) cyclic alkyl group; wherein, when R1, R2, R3, R4 are independently C1-6alkoxy, the C1-6alkoxy includes linear or branched C1-6alkoxy, preferably linear or branched C1-3alkoxy, more preferably methoxy; when R1, R2, R3, R4 are independently substituted amino, the substituted amino is amino substituted with one or more substituents selected from methyl and ethyl; when R1, R2, R3, R4 are independently C1-7alkyl or substituted C1-7alkyl, the C1-7alkyl or substituted C1-7alkyl includes linear or branched C1-7 (preferably C3-5, more preferably C4) alkyl or substituted C1-7 (preferably C3-5, more preferably C4) alkyl, and the substituted C1-7alkyl is C1-7alkyl substituted with one or more substituents selected from cyclopropyl and cyclobutyl; or, the linear or branched C1-7alkyl or substituted C1-7alkyl is preferably C1-3alkyl or substituted C1-3alkyl, such as methyl, halomethyl (preferably trifluoromethyl); in structural formula I, G is hydrogen, halogen, methyl or methoxy; further preferably, G is hydrogen, fluorine or chlorine; in structural formula I, Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene; wherein substituted methylene is methylene substituted with one hydrogen or both hydrogens, the substituent being benzyl or alkyl; wherein, when the substituent is alkyl, the alkyl together with R3and / or R4and the carbon atoms to which they are attached forms a C3-6membered fused or spirocyclic ring; or, when the substituent is 2 alkyl groups, the 2 alkyl groups together with the group Y form a C3-6membered spirocyclic ring; and, wherein, when Y is substituted methylene, the substituent of substituted methylene is preferably alkyl, more preferably linear or branched C1-4alkyl; preferably, Y is oxygen, sulfur, sulfone, sulfoxide or methylene; or, preferably, Y is oxygen, sulfur or methylene; in structural formula I, X is oxygen or sulfur; in structural formula I, n = 0 or 1 ; further, in structural formula I, when R1, R2, R3, R4 are all hydrogen, X is oxygen, and n = 0, G cannot be hydrogen or fluorine when Y is methylene; and G cannot be hydrogen when Y is oxygen or sulfur; preferably, the camptothecin class compound is connected to the carboxyl group in group A through an amide bond, preferably the amino group adjacent to group G in structural formula I is connected to the carboxyl group in group A through an amide bond.

14. The antibody drug conjugate or salt thereof according to claim 13, characterized by R1, R2, R3, R4 are independently hydrogen, halogen (e.g. fluorine), C1-7alkyl or substituted C1-7alkyl, or any two of R1, R2, R3, R4 together with the carbon atoms to which they are attached form a C3-6cyclic alkyl (e.g. C3-5cyclic alkyl); preferably, R1, R2 are the same; and / or, R3, R4 are the same; preferably, Y is methylene substituted with alkyl, the alkyl together with R3and / or R4and the carbon atoms to which they are attached forms a C3-6membered fused or spirocyclic ring; preferably, X is oxygen; Preferably, X is oxygen, G is hydrogen, halogen (e.g. fluorine or chlorine), methyl or methoxy, Y and R1, R2, R3, R4 are as defined above; Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen, sulfur, sulfoxide or sulfone, R1, R2, R3, R4 are as defined above; Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen, sulfur, sulfoxide or sulfone, R1, R2, R3, R4 are as defined above; Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen, sulfur, sulfoxide or sulfone, R1, R2, R3, R4 are as defined above; Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen, sulfur, sulfoxide or sulfone, R1, R2, R3, R4 are as defined above; Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen, sulfur, sulfoxide or sulfone, R1, R2, R3, R4 are as defined above; Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen, sulfur, sulfoxide or sulfone, R1, R2, R3, R4 are as defined above; Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen, sulfur, sulfoxide or sulfone, R1, R2, R3, R4 are as defined above; Preferably, n = 0.

15. The antibody drug conjugate or salt thereof according to claim 13 or 14, characterized in that, In structural formula I: G is hydrogen, Y is methylene, R1 and R2 are methyl, R3 and R4 are hydrogen, X is oxygen, n = 0; G is hydrogen, Y is methylene, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, n = 0; G is hydrogen, Y is methylene, one of R1 and R2 together with one of R3 and R4 along with the carbon atoms to which they are attached form a C3 cyclic alkyl group, the other of R1 and R2 together with the other of R3 and R4 is hydrogen, X is oxygen, n = 0; G is hydrogen, Y is sulfur, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, n = 0; G is hydrogen, Y is sulfoxide, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, n = 0; G is hydrogen, Y is sulfur, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, n = 0; G is hydrogen, Y is sulfone, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, n = 0; G is hydrogen, Y is methylene, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, n = 1; G is fluorine, Y is oxygen, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, n = 0; G is fluorine, Y is sulfur, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, n = 0; G is fluorine, Y is oxygen, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, n = 0; G is fluorine, Y is methylene, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, n = 0; G is hydrogen, Y is oxygen, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, n = 0; or G is fluorine, Y is sulfur, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, n = 0.

16. The antibody drug conjugate or salt thereof according to any one of claims 9 to 15, characterized in that, The structure of the camptothecin compound is shown in structural formula IA: In structural formula IA, the groups R1, R2, R3, R4 are the same as defined for the groups R1, R2, R3, R4 in structural formula I in any one of claims 13 to 15, but R1, R2, R3, R4 are not simultaneously hydrogen; In structural formula IA, the groups R1, R2, R3, R4 are the same as defined for the groups R1, R2, R3, R4 in structural formula I in any one of claims 13 to 15, but R1, R2, R3, R4 are not simultaneously hydrogen; Preferably, the camptothecin compound is linked to the carboxyl group of group A by an amide bond, preferably the amino group on the left-hand phenyl ring in structural formula I is linked to the carboxyl group of group A by an amide bond.

17. The antibody drug conjugate or salt thereof according to any one of claims 9 to 12, characterized in that, The structure of the camptothecin compound is shown in structural formula II: In structural formula II, R5 is C1-5 alkyl or C1-5 alkyl substituted with one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted with one or more substituents, phenyl or substituted phenyl; wherein, when R5 is C1-5 alkyl or substituted C1-5 alkyl, the C1-5 alkyl includes straight chain or branched C1-5 alkyl, preferably R5 is C1-4 straight chain alkyl; when R5 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl, the substituents are selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl, and wherein the substituted amino is amino substituted with one or more substituents selected from methyl and ethyl; when R5 is substituted phenyl, the substituents are selected from alkyl (e.g. C1-6 alkyl, preferably C1-3) or halogen; In structural formula II, G is hydrogen, halogen (e.g. fluorine), methyl or methoxy; preferably G is hydrogen, fluorine or chlorine; In structural formula II, X is oxygen or sulfur; In structural formula II, n = 0 or 1 ; In structural formula II, when X is oxygen, G is hydrogen and n = 0, R5 cannot be n-butyl; Preferably, the camptothecin compound is linked to the carboxyl group of group A by an amide bond, preferably the amino group on the left-hand phenyl ring in structural formula II is linked to the carboxyl group of group A by an amide bond.

18. The antibody drug conjugate or salt thereof according to any one of claims 9 to 12 or 17, characterized in that, The structure of the camptothecin compound is shown in structural formula IIA: In structural formula IIA, group R5 is the same as the definition of group R5 in structural formula II in claim 17, except that R5 cannot be n-butyl; Preferably, the camptothecin compound is linked to the carboxyl group of group A by an amide bond, preferably the amino group on the left-hand phenyl ring in structural formula IIA is linked to the carboxyl group of group A by an amide bond.

19. The antibody drug conjugate or salt thereof according to any one of claims 9 to 18, characterized in that, The structure of the camptothecin compound is as follows: Preferably, the camptothecin compound is linked to the carboxyl group of group A by an amide bond, preferably the amino group on the left-hand phenyl ring in each of the structural formulae is linked to the carboxyl group of group A by an amide bond.

20. The antibody drug conjugate or salt thereof according to any one of claims 9 to 12, characterized in that, The structure of the camptothecin compound is shown in structural formula IV: In structural formula IV, R8 is hydrogen, trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted with one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted with one or more substituents, or halogen; wherein, when R8 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl, the substituents are selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino is amino substituted with one or more substituents selected from methyl and ethyl; Preferably, the camptothecin compound is linked via its hydroxyl group on the same carbon to which R8is attached to the carboxyl group of the group A via a self- releasing structure, for example The solid line indicates the site of attachment to the carboxyl group of group A, and the wavy line indicates the site of attachment to the hydroxyl group in structural formula IV.

21. The antibody drug conjugate or salt thereof according to any one of claims 9 to 20, characterized in that, The antibody drug conjugate or salt thereof has a structure as shown in structural formula Ia and / or Ib: and / or In structural formulae Ia and / or Ib, Ab, m, group M, SP1, SP2, A and D are the same as the definitions of Ab, m, group M, SP1, SP2, A and D in any one of claims 10 to 20; Preferably, the antibody drug conjugate or salt thereof has a structure as shown in structural formula Ic and / or Id: and / or In structural formula Ic and / or Id, Ab, m, group A and D are the same as the definition of Ab, m, group A and D in any one of claims 10 to 20.

22. The antibody drug conjugate or salt thereof according to any one of claims 9 to 21, characterized in that, The antibody drug conjugate or salt thereof has a structure as shown below:

23. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, or the antibody-drug conjugate of any one of claims 9 to 22, or a salt thereof, and optionally a pharmaceutically acceptable excipient.

24. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the antibody-drug conjugate of any one of claims 9 to 22, or a salt thereof, and / or the composition of claim 23, in the manufacture of a medicament for preventing, treating and / or ameliorating a disease or disorder.

25. Use according to claim 24, characterized in that, the disease or disorder is associated with ADAM9 expression, including overexpression; preferably, the disease or disorder is a hematological or solid tumor positive for ADAM9 expression; preferably, the disease or disorder is non-small cell lung cancer, large cell lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, renal cell cancer, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma or lymphoma.

26. A method of preventing, treating and / or ameliorating a disease or disorder, the method comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the antibody-drug conjugate of any one of claims 9 to 22, or a salt thereof, and / or the composition of claim 23.

27. The method of claim 26, wherein, the disease or disorder is associated with ADAM9 expression, including overexpression; preferably, the disease or disorder is a hematological or solid tumor positive for ADAM9 expression; preferably, the disease or disorder is non-small cell lung cancer, large cell lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, renal cell cancer, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma or lymphoma; preferably, the subject is a mammal; further preferably, the subject is a human.

28. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 5 in the manufacture of an antibody-drug conjugate for preventing, treating and / or ameliorating a disease or disorder.

29. Use according to claim 28, characterized in that, the disease or disorder is associated with ADAM9 expression, including overexpression; preferably, the disease or disorder is a hematological or solid tumor positive for ADAM9 expression; preferably, the disease or disorder is non-small cell lung cancer, large cell lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, renal cell cancer, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma or lymphoma.

30. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the antibody drug conjugate of any one of claims 9 to 22, or a salt thereof, and / or the composition of claim 23 for the manufacture of a reagent for the diagnosis of a disease or disorder.

31. Use according to claim 30, characterized in that, the disease or disorder is associated with ADAM9 expression, including overexpression; preferably, the disease or disorder is an ADAM9 expression-positive hematological or solid tumor; preferably, the disease or disorder is non-small cell lung cancer, large cell lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, renal cell cancer, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma, or lymphoma.

32. A method of diagnosing a disease or disorder, the method comprising contacting the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the antibody drug conjugate of any one of claims 9 to 22, or a salt thereof, and / or the composition of claim 23 with a sample from a subject.

33. The method of claim 32, wherein, the disease or disorder is associated with ADAM9 expression, including overexpression; preferably, the disease or disorder is an ADAM9 expression-positive hematological or solid tumor; preferably, the disease or disorder is non-small cell lung cancer, large cell lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, renal cell cancer, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma, or lymphoma; preferably, the subject is a mammal; further preferably, the subject is a human.

34. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the antibody drug conjugate of any one of claims 9 to 22, or a salt thereof, and / or the composition of claim 23.

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