Anti-human disintegrin and metalloproteinase 9 antibody and application thereof

Abstract

The invention provides an antibody for resisting human disintegrin and metalloproteinase 9 (ADAM9) and application of the antibody. The antibody provided by the invention can be specifically combined with human ADAM9, can rapidly internalize into tumor cells, can effectively block the metalloproteinase activity of ADAM9, and is coupled with a small-molecule cytotoxic compound to prepare a targeted killing antibody drug conjugate; the invention has important application potential in the aspects of tumor targeted therapy, targeted killing ADC drug development, targeted and immune combined therapy, bispecific or multispecific antibody development and the like.

Description

[0001] Cross-references to related applications

[0002] This patent application claims priority to Chinese invention patent application No. CN202411380427.9, filed on September 30, 2024, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This invention belongs to the field of antibody drugs. Specifically, this invention relates to antibodies against human ADAM9 and their use in the preparation of drugs, as well as antibody-drug conjugates targeting human ADAM9. Background Technology

[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 plays an important role in various diseases, including neurodegenerative diseases, retinal diseases, inflammation, and tumors.

[0005] Overexpression of ADAM9 has been found in various cancers. For example, immunohistochemical analysis using commercial antibodies indicates that ADAM9 is expressed in most tumors, with some showing higher expression levels. Specifically, its expression is significantly higher in lung cancer, triple-negative breast cancer, colorectal cancer, cholangiocarcinoma, esophageal cancer, glioblastoma, glioma, and pancreatic cancer than in normal tissues (GEPIA2 Database). Furthermore, ADAM9 has been 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 widely expressed in various normal human tissues and exists in multiple structural forms. Therefore, the development of anti-ADAM9 antibodies with tumor differential selection capabilities is of great significance for improving anti-tumor effects and reducing the impact on normal tissues.

[0007] Furthermore, given ADAM9's crucial role in tumorigenesis and progression, it has become a hot target for developing antibody-drug conjugates (ADCs). Currently, several ADAM9-targeting ADCs are in clinical trials, including IMGC936, a drug developed by MacroGenics in collaboration with ImmunoGen (now acquired by AbbVie). IMGC936 consists of three parts: a high-affinity humanized monoclonal antibody, a maytansine alkaloid microtubule inhibitor payload, and a stable tripeptide linker with a DAR of 2. A Phase I / II clinical trial (NCT04622774) of IMGC936 for advanced malignant solid tumors has been completed, but development has been terminated due to the trial's failure to meet the expected safety and efficacy targets. Therefore, there is still a need to develop more effective and safer ADAM9-targeting ADCs. Summary of the Invention

[0008] The technical problem to be solved by this invention is to provide a novel anti-ADAM9 antibody. This novel antibody should be able to specifically bind to ADAM9 and have a high tumor selectivity, that is, it should have a strong binding ability to ADAM9 expressed in tumor cells and a weak binding ability to ADAM9 expressed in normal tissues, ensuring that drugs prepared based on this antibody have better safety. Furthermore, the novel antibody should also have a strong ability to internalize into ADAM9-expressing tumor cells, thus making it suitable for preparation into antibody-drug conjugates, providing an antibody-drug conjugate with better targeting and killing effect on ADAM9-expressing tumor cells.

[0009] To address the aforementioned technical problems, the inventors of this application have provided a series of novel antibodies capable of specifically binding to human ADAM9 through hybridoma screening, characterization of the biological activity of hybridoma antibodies, and humanization modification of antibodies. These novel antibodies can efficiently internalize and enter tumor cells by binding to ADAM9. In particular, compared with the humanized monoclonal antibody Mab-A (WO2020 / 005945A1, molecular code hMAB-A(2I.2)) in IMGC936, the world's first antibody-drug conjugate targeting ADAM9, this antibody showed better in vivo antitumor activity after being conjugated with the same toxin compound to prepare an ADC. Furthermore, this antibody has a moderate affinity for human ADAM9, and its binding to human ADAM9 in normal tissues is significantly lower than that of Mab-A, suggesting better safety potential. In addition, it was found that the anti-ADAM9 antibody provided by this invention can effectively block the metalloproteinase activity of ADAM9, and its activity is stronger than that of Mab-A. Furthermore, the inventors of this application conjugated the new series of antibodies with small molecule cytotoxic compounds with specific structures to obtain a new set of antibody-drug conjugates, and selected antibody-drug conjugates with strong targeting and killing effects.

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

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

[0012] In the context of this invention, the terms "connector" and "connector compound" are used interchangeably.

[0013] In the context of this invention, the term "drug-containing linker" refers to a compound in which a drug (e.g., a small molecule drug, such as camptothecin compounds) is directly or indirectly covalently bonded to a linker.

[0014] The present invention provides the following technical solution.

[0015] First aspect

[0016] The present invention provides an antibody or antigen-binding fragment thereof against deintegrin and metalloproteinase 9 (ADAM9), wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to ADAM9, particularly human ADAM9.

[0017] In the context of this invention, unless otherwise stated, the term "ADAM9" covers any form of ADAM9, such as active and inactive forms or membrane-bound and soluble forms; and covers any structural region of ADAM9, such as the extracellular region (ECD) and its contained domains.

[0018] In the context of this invention, the term "antigen-binding fragment" encompasses various functional fragments of the antibody that specifically binds to ADAM9, which retain the antibody's ability to bind to the antigen and the corresponding biological activity. It is well known in the art that the antibody's ability to bind to the antigen and the corresponding biological activity can be achieved from fragments of the intact antibody, which can be obtained using conventional techniques known to those skilled in the art and screened for functionality in the same manner as for the intact antibody. For example, antigen-binding fragments of the antibody can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody.

[0019] Specifically, the anti-ADAM9 antibody or its antigen-binding fragment provided by the present invention comprises complementarity-determining regions (CDRs) of the heavy chain, namely heavy chain CDR1 (H-CDR1), heavy chain CDR2 (H-CDR2), and heavy chain CDR3 (H-CDR3), and complementarity-determining regions (CDRs) of the light chain, namely light chain CDR1 (L-CDR1), light chain CDR2 (L-CDR2), and light chain CDR3 (L-CDR3). According to a specific embodiment of the present invention, the heavy chain CDRs comprised in the anti-ADAM9 antibody or its antigen-binding fragment are derived from the amino acid sequences 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 light chain CDRs comprised in the anti-ADAM9 antibody or its antigen-binding fragment are derived from the amino acid sequences 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 sequences shown in any one of SEQ ID NO. 5 to SEQ ID NO. 35 provided above are the amino acid sequences of the heavy chain variable region (VH) or light chain variable region (VL) of the exemplary antibody provided in the "Detailed Description" section of this application. Using antibody heavy chain or light chain complementarity-determining region definition tools known in the art (e.g., Chothia, Kabat, IMGT, Contact, AbM, etc.), those skilled in the art can easily determine the heavy chain CDRs and light chain CDRs contained therein. Combinations of heavy chain CDRs and light chain CDRs can be obtained according to definition tools known or conventional in the art, and antibodies or fragments thereof containing each of these combinations of heavy chain CDRs and light chain CDRs are within the protection scope of this invention.

[0021] Preferably, the anti-ADAM9 antibody or its antigen-binding fragment provided by the present invention comprises heavy chain CDRs and light chain CDRs from the heavy chain variable region and light chain variable region shown in the following amino acid sequence pairings:

[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, Kabat can be used, for example, to divide the CDRs in the above amino acid sequence pairings, as shown in the embodiments of the present invention.

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

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

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

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

[0061] (4) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.41, and SEQ ID NO.38; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.45, SEQ ID NO.49, and SEQ ID NO.47;

[0062] (5) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37, and SEQ ID NO.42; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.45, SEQ ID NO.49, and SEQ ID NO.47;

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

[0064] (7) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.41, and SEQ ID NO.44; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.45, SEQ ID NO.49, and SEQ ID NO.47;

[0065] (8) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37, and SEQ ID NO.38; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.45, SEQ ID NO.48, and SEQ ID NO.47;

[0066] (9) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37, and SEQ ID NO.38; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.45, SEQ ID NO.49, and SEQ ID NO.47;

[0067] (10) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37, and SEQ ID NO.38; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.45, SEQ ID NO.50, and SEQ ID NO.47;

[0068] (11) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37, and SEQ ID NO.38; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.51, SEQ ID NO.49, and SEQ ID NO.47;

[0069] (12) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37, and SEQ ID NO.38; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.45, SEQ ID NO.49, and SEQ ID NO.52;

[0070] (13) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37, and SEQ ID NO.38; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.45, SEQ ID NO.49, and SEQ ID NO.53;

[0071] (14) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37, and SEQ ID NO.38; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.51, SEQ ID NO.49, and SEQ ID NO.53;

[0072] (15) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37, and SEQ ID NO.38; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.45, SEQ ID NO.49, and SEQ ID NO.54;

[0073] (16) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37, and SEQ ID NO.38; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.45, SEQ ID NO.49, and SEQ ID NO.55;

[0074] (17) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.37, and SEQ ID NO.38; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.45, SEQ ID NO.49, and SEQ ID NO.56;

[0075] (18) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.41, and SEQ ID NO.38; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.51, SEQ ID NO.49, and SEQ ID NO.47;

[0076] (19) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.41, and SEQ ID NO.43; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.51, SEQ ID NO.49, and SEQ ID NO.47;

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

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

[0079] (22) H-CDR1, H-CDR2, and H-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.36, SEQ ID NO.41, and SEQ ID NO.43; and L-CDR1, L-CDR2, and L-CDR3, which sequentially contain the amino acid sequences shown in SEQ ID NO.45, SEQ ID NO.49, and SEQ ID NO.53.

[0080] As described above, the anti-ADAM9 antibody or its antigen-binding fragment provided by the present invention specifically binds to detegrin and metalloproteinase 9 (ADAM9), preferably human ADAM9. Optionally, the antibody or its antigen-binding fragment provided by the present invention may or may not have cross-species binding activity with human, cyno, or mouse ADAM9. Optionally, the antibody or its antigen-binding fragment provided by the present invention may or may not block the metalloproteinase activity of ADAM9, wherein ADAM9 may be in a membrane-bound or secreted form, such as human ADAM9.

[0081] Preferably, the anti-ADAM9 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region and a light chain variable region, both of which include the aforementioned CDRs and the framework region (FR) therebetween, and the arrangement of each region from the N-terminus to the C-terminus is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0082] Further preferably, in the anti-ADAM9 antibody or its antigen-binding fragment provided by the present invention, the heavy chain variable region may contain an 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 contain an amino acid sequence having at least 75% identity with said amino acid sequence; and / or, the light chain variable region contains an 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 contain an amino acid sequence having at least 75% identity with said amino acid sequence.

[0083] In the context of this invention, the term "at least 75% identity" refers to a maximum of 25% difference in the amino acid sequence that may exist in any frame region within 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 its antigen-binding fragment of this invention. This difference may result from amino acid deletions, additions, or substitutions at any position, wherein the substitutions may be conservative or non-conservative. The term "at least 75% identity" encompasses any percentage of identity between at least 75% 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 a specific embodiment of the present invention, the anti-ADAM9 antibody or its antigen-binding fragment provided by the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise 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 with the amino acid sequence shown in SEQ ID NO.6, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.6;

[0086] (2) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.8, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.8;

[0087] (3) The amino acid sequence shown in SEQ ID NO.9, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.8, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.8;

[0088] (4) The amino acid sequence shown in SEQ ID NO.10, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.8, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.8;

[0089] (5) The amino acid sequence shown in SEQ ID NO.11, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.8, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.8;

[0090] (6) The amino acid sequence shown in SEQ ID NO.12, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25;

[0091] (7) The amino acid sequence shown in SEQ ID NO.13, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25;

[0092] (8) The amino acid sequence shown in SEQ ID NO.14, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25;

[0093] (9) The amino acid sequence shown in SEQ ID NO.15, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25;

[0094] (10) The amino acid sequence shown in SEQ ID NO.16, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25;

[0095] (11) The amino acid sequence shown in SEQ ID NO.17, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25;

[0096] (12) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.18, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.18;

[0097] (13) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.19, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.19;

[0098] (14) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.20, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.20;

[0099] (15) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.21, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.21;

[0100] (16) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.22, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.22;

[0101] (17) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.23, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.23;

[0102] (18) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.24, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.24;

[0103] (19) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.25;

[0104] (20) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.26, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.26;

[0105] (21) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.27, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.27;

[0106] (22) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.28, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.28;

[0107] (23) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.29, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.29;

[0108] (24) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.30, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.30;

[0109] (25) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.31, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.31;

[0110] (26) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.32, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.32;

[0111] (27) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.33, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.33;

[0112] (28) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.34, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.34;

[0113] (29) The amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.35, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.35;

[0114] (30) The amino acid sequence shown in SEQ ID NO.14, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.29, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.29;

[0115] (31) The amino acid sequence shown in SEQ ID NO.16, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.29, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.29;

[0116] (32) The amino acid sequence shown in SEQ ID NO.17, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.29, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.29;

[0117] (33) The amino acid sequence shown in SEQ ID NO.14, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.31, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.31;

[0118] (34) The amino acid sequence shown in SEQ ID NO.16, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.31, or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.31.

[0119] Preferably, the anti-ADAM9 antibody provided by the present invention can be a mouse antibody, rabbit antibody, or human antibody, or it can be a mouse antibody, a chimeric antibody, or a fully or partially humanized antibody. The anti-ADAM9 antibody can also be called a derivatized antibody, for example, an antibody obtained by CDR transplantation, affinity maturation, point mutation modification, or chemical modification based on an initial mouse monoclonal antibody. The chemical modification includes glycosylation, acetylation, polyethylene glycol modification, phosphorylation, amidation, protease cleavage, linkage with cellular ligands or effector molecules, protection of active reactive groups, and / or blocking. Preferably, the antigen-binding fragment of the antibody can be any form of fragment such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv.

[0120] In addition to the heavy chain and / or light chain variable regions, the anti-ADAM9 antibody or its antigen-binding fragment provided by the present invention further comprises a heavy chain constant region (CH) and / or a light chain constant region (CL), preferably comprising a human or mouse heavy chain constant region and / or a light chain constant region. Preferably, the anti-ADAM9 antibody or its fragment comprises a heavy chain constant region of IgG, IgA, IgM, IgD, or IgE and / or a κ or λ type light chain constant region.

[0121] According to a specific embodiment of the present invention, the anti-ADAM9 antibody is a monoclonal antibody, preferably a mouse, chimeric, or humanized monoclonal antibody. According to a specific embodiment of the present invention, the monoclonal antibody comprises a heavy chain constant region sequence of IgG1, such as the human IgG1 heavy chain constant region shown 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 shown in SEQ ID NO.4.

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

[0123] Second aspect

[0124] The present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the anti-ADAM9 antibody or its antigen-binding fragment described herein.

[0125] The term "nucleotide sequence encoding the anti-ADAM9 antibody or its antigen-binding fragment according to the present invention" refers to a nucleotide sequence encoding the heavy chain CDRs, light chain CDRs, light chain variable regions, heavy chain variable regions, heavy chains, and / or light chains contained in the antibody or its antigen-binding fragment. For example, the nucleic acid molecule provided by the present invention contains nucleotide sequences encoding each of the heavy chain CDRs and light chain CDRs contained in the aforementioned antibody or its antigen-binding fragment; contains nucleotide sequences encoding the heavy chain variable regions and light chain variable regions contained in the aforementioned antibody or its antigen-binding fragment; or contains nucleotide sequences encoding the heavy chains and light chains contained in the aforementioned antibody or its antigen-binding fragment.

[0126] Third aspect

[0127] The nucleic acid molecules of this invention can be cloned into a vector, and then transformed or transfected into host cells. Therefore, in a third aspect, this invention also provides a vector containing the nucleic acid molecules of this invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a phage vector, etc. The vectors or nucleic acid molecules of this invention can be used to transform or transfect host cells, for purposes such as preserving or expressing antibodies.

[0128] Fourth aspect

[0129] The present invention also provides a host cell comprising the nucleic acid molecules and / or vectors of the present invention, or the host cell being transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, or animal cells.

[0130] Fifth aspect

[0131] The anti-ADAM9 antibody or its antigen-binding fragment provided by this invention can be obtained using any method known in the art. For example, this invention also provides a method for preparing the anti-ADAM9 antibody or its antigen-binding fragment, the method comprising culturing the host cells provided by this invention while allowing the host cells to express the heavy and light chains of the antibody. Optionally, the method further includes a step of recovering the generated anti-ADAM9 antibody.

[0132] Sixth aspect

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

[0134] Therefore, the present invention provides an antibody-drug conjugate targeting ADAM9 or a salt thereof, comprising the anti-ADAM9 antibody or its antigen-binding fragment provided by the present invention. The antibody-drug conjugate may be formed by conjugating the anti-ADAM9 antibody or its antigen-binding fragment provided by the present invention with a small molecule cytotoxic compound, wherein the small molecule cytotoxic compound may be, for example, a tubulin inhibitor, a topoisomerase inhibitor, or a DNA binder. Preferably, the tubulin inhibitor is selected from maytansine 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 camptothecin compounds such as exatecan and its derivatives, doxorubicin metabolite PNU-159682 derivative, and irinotecan (CPT-11) metabolite SN38 derivative. Preferably, the DNA binding agent is selected from PBD derivatives and Duocarmycin derivatives.

[0135] Furthermore, the present invention also provides the following preferred embodiments for the antibody-drug conjugate.

[0136] First, the present invention provides an antibody-drug conjugate or a salt thereof targeting ADAM9, wherein the antibody-drug conjugate or the salt thereof has a general formula. The structure is shown. In the context of this invention, unless otherwise specified, the various groups or structures in this general formula are as follows:

[0137] Ab represents the anti-ADAM9 antibody or its antigen-binding fragment provided by this invention.

[0138] E L Selected from the following groups ( (This indicates that Ab is linked to the sulfhydryl group of cysteine):

[0139] 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:

[0140] M is a phenylene or a phenylene substituted with one or more substituents, or a chemical bond; in the substituted phenylene, the substituent is selected from alkyl (e.g., C1-6 alkyl, preferably C1-4 alkyl), haloalkyl (e.g., haloC1-6 alkyl, preferably haloC1-4 alkyl, such as trifluoromethyl), alkoxy (e.g., C1-6 alkoxy, preferably C1-4 alkoxy, preferably methoxy), halogen, ester, amide, and cyano; preferably, M is a halogen-substituted phenylene.

[0141] SP1 is selected from C1-8 alkylene, C1-8 cycloalkylene, or C1-21 (preferably C1-16, more preferably C1-11, more preferably C5-9) straight-chain heteroalkylene, wherein the C1-21 straight-chain heteroalkylene comprises 1-11 (preferably 1-6, more preferably 3-5) heteroatoms selected from N, O, or S, wherein each of the C1-8 alkylene, C1-8 cycloalkylene, and C1-21 straight-chain heteroalkylene is independently and optionally substituted by one or more substituents selected from hydroxyl, amino, sulfonic acid, and cyano groups.

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

[0143] A represents a short peptide structure formed by 2-4 amino acids. When A represents a short peptide structure formed by 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; A represents a short peptide structure formed by 3 amino acids. When A represents a short peptide structure formed by 1 amino acid, 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 formed by 4 amino acids, it can be NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-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, where NH represents the amino terminus of group A and CO represents the carboxyl terminus of group A. Group A can be linked to SP2 through the amino group at the amino terminus of its short peptide structure.

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

[0145] m can be 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.

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

[0147] In this general formula, the group The following structures can be selected, where the wavy line indicates a link to a cysteine ​​residue in the antibody or to a small molecule cytotoxic compound D:

[0148]

[0149]

[0150] Furthermore, the present invention provides an antibody-drug conjugate targeting ADAM9 or a salt thereof, wherein the antibody-drug conjugate is formed by conjugating an anti-ADAM9 antibody or its antigen-binding fragment provided by the present invention with a camptothecin-like compound. Accordingly, in this general formula, D represents a camptothecin-like compound.

[0151] The structure of the camptothecin-like compound can be as shown in structural formula I:

[0152]

[0153] In structural formula I, R1, R2, R3, and R4 are independently hydrogen, halogen, hydroxyl, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, or any two of R1, R2, R3, and R4 together with the carbon atoms they are attached to form a C3-6 (preferably C3-5) cyclic alkyl group. When R1, R2, R3, and R4 are independently C1-6 alkoxy, the C1-6 alkoxy includes straight-chain or branched C1-6 alkoxy, preferably straight-chain or branched C1-3 alkoxy, more preferably methoxy. When R1, R2, R3, and R4 are independently substituted amino, the substituted amino is an amino group substituted by one or more substituents selected from methyl and ethyl. When R1, R2, R3, and R4 are independently C1-7 alkyl or substituted C1-7 alkyl, the C1-7 alkyl or substituted C1-7 alkyl 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 C1-7 alkyl is a C1-7 alkyl substituted by one or more substituents selected from cyclopropyl and cyclobutyl; or, the straight-chain or branched C1-7 alkyl or substituted C1-7 alkyl is preferably a C1-3 alkyl or substituted C1-3 alkyl, such as methyl, halomethyl (preferably trifluoromethyl).

[0154] In structural formula I, G is hydrogen, halogen, methyl, or methoxy. Preferably, G is hydrogen, fluorine, or chlorine.

[0155] In structural formula I, Y is oxygen, sulfur, sulfone, sulfoxide, methylene, or a substituted methylene group. A substituted methylene group may be formed by replacing one hydrogen atom or both hydrogen atoms of the methylene group. The substituent may be benzyl or alkyl. When the substituent is alkyl, the alkyl group, together with R3 and / or R4 and the carbon atoms attached to them, can form a C3-6 membered fused or spirocyclic structure. Alternatively, when there are two alkyl groups, the two alkyl groups can form a C3-6 membered spirocyclic structure together with group Y. When Y is a substituted methylene group, the substituent is preferably an alkyl group, more preferably a straight-chain or branched C1-4 alkyl group.

[0156] Preferably, Y is oxygen, sulfur, sulfone, sulfoxide, or methylene; or, preferably, Y is oxygen, sulfur, or methylene.

[0157] In structural formula I, X is oxygen or sulfur.

[0158] In structural formula I, n = 0 or 1.

[0159] In structural formula I, when R1, R2, R3, and R4 are all hydrogen, X is oxygen, and n = 0, when Y is methylene, G cannot be hydrogen or fluorine; and when Y is oxygen or sulfur, G cannot be hydrogen.

[0160] Preferably, R1, R2, R3, and R4 are independently hydrogen, halogen (e.g., fluorine), C1-7 alkyl, or substituted C1-7 alkyl, or any two of R1, R2, R3, and R4 together with the carbon atoms they are attached to form a C3-6 cyclic alkyl group (e.g., a C3-5 cyclic alkyl group). Further, R1 and R2 can be the same; and / or, R3 and R4 can be the same.

[0161] Preferably, Y is a methylene group substituted with an alkyl group, and the alkyl group, together with R3 and / or R4 and the carbon atoms attached thereto, can form a C3-6 fused or spirocyclic structure.

[0162] Preferably, X can be oxygen.

[0163] 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.

[0164] 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.

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

[0166] Preferably, X is oxygen, G is chlorine, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, and R4 are as defined above.

[0167] 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.

[0168] Preferably, X is oxygen, G is methoxy, Y is methylene or substituted methylene, oxygen or sulfur, and R1, R2, R3, and R4 are as defined above.

[0169] Preferably, X is oxygen, G is hydrogen, Y is methylene, sulfoxide, sulfone, oxygen or sulfur, R1 and R2 are independently hydrogen, fluorine or methyl, and R3 and R4 are independently hydrogen.

[0170] Preferably, X is oxygen, G is fluorine, Y is methylene, sulfoxide, sulfone, oxygen or sulfur, R1 and R2 are independently hydrogen, fluorine or methyl, and R3 and R4 are independently hydrogen.

[0171] Preferably, n = 0.

[0172] According to a specific embodiment of the present invention, in structural formula I:

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

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

[0175] G is hydrogen, Y is methylene, one of R1 and R2 together with one of R3 and R4, along with the carbon atoms they are attached to, forms 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;

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

[0177] G is hydrogen, Y is sulfoxide, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n = 0;

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

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

[0180] G is hydrogen, Y is methylene, R1 and R2 are hydrogen, R3 and R4 are hydrogen, X is oxygen, and n = 1;

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

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

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

[0184] G is fluorine, Y is methylene, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n = 0;

[0185] G is hydrogen, Y is oxygen, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, n = 0; or

[0186] G is fluorine, Y is sulfur, R1 and R2 are fluorine, R3 and R4 are hydrogen, X is oxygen, and n = 0.

[0187] In the antibody-drug conjugate targeting ADAM9 or its salt provided by the present invention, the camptothecin compound represented by structural formula I is connected to the carboxyl group in group A of the general formula via an amide bond, preferably the amino group adjacent to group G in structural formula I is connected to the carboxyl group in group A of the general formula via an amide bond.

[0188] Preferably, the structure of the camptothecin-like compound can be as shown in structural formula IA:

[0189]

[0190] In structural formula IA, groups R1, R2, R3, and R4 are defined the same as those in structural formula I above, but R1, R2, R3, and R4 are not all hydrogen.

[0191] In the antibody-drug conjugate targeting ADAM9 or its salt provided by the present invention, the camptothecin compound represented by structural formula IA is connected to the carboxyl group in group A of the general formula via an amide bond, preferably with the amino group on the left benzene ring of structural formula IA connected to the carboxyl group in group A of the general formula via an amide bond.

[0192] Alternatively, the structure of the camptothecin-like compound can be as shown in structural formula II:

[0193]

[0194] In structural formula II, R5 is a C1-5 alkyl group or a C1-5 alkyl group substituted with one or more substituents, a C3-6 cyclic alkyl group or a C3-6 cyclic alkyl group substituted with one or more substituents, a phenyl group, or a substituted phenyl group. When R5 is a C1-5 alkyl group or a substituted C1-5 alkyl group, the C1-5 alkyl group includes straight-chain or branched C1-5 alkyl groups. Further, R5 is a C1-4 straight-chain alkyl group. When R5 is a substituted C1-5 alkyl group or a substituted C3-6 cyclic alkyl group, the substituent is selected from halogens, hydroxyl groups, methoxy groups, trifluoromethyl groups, amino groups or substituted amino groups, methanesulfonyl groups, and C3-6 cyclic alkyl groups; and wherein the substituted amino group is an amino group substituted with one or more substituents selected from methyl and ethyl groups. When R5 is a substituted phenyl group, the substituent is selected from alkyl groups (e.g., C1-6 alkyl groups, preferably C1-3) or halogens.

[0195] In structural formula II, G is hydrogen, halogen (e.g., fluorine), methyl, or methoxy. Preferably, G is hydrogen, fluorine, or chlorine.

[0196] In structural formula II, X is oxygen or sulfur.

[0197] In structural formula II, n = 0 or 1.

[0198] In structural formula II, when X is oxygen, G is hydrogen, and n = 0, R5 cannot be n-butyl.

[0199] In the antibody-drug conjugate targeting ADAM9 or its salt provided by the present invention, the camptothecin compound represented by structural formula II is connected to the carboxyl group in group A of the general formula via an amide bond, preferably the amino group adjacent to group G in structural formula II is connected to the carboxyl group in group A of the general formula via an amide bond.

[0200] Preferably, the structure of the camptothecin-like compound can be as shown in structural formula IIA:

[0201]

[0202] In structural formula IIA, the group R5 is defined the same as the group R5 in structural formula II above, but R5 cannot be n-butyl.

[0203] In the antibody-drug conjugate targeting ADAM9 or its salt provided by the present invention, the camptothecin compound represented by structural formula IIA is connected to the carboxyl group in group A of the general formula via an amide bond, preferably the amino group on the left benzene ring of structural formula IIA is connected to the carboxyl group in group A of the general formula via an amide bond.

[0204] According to a specific embodiment of the present invention, the structure of the camptothecin-like compound is as follows:

[0205]

[0206]

[0207]

[0208]

[0209] In the antibody-drug conjugate targeting ADAM9 or its salt provided by the present invention, the camptothecin-like compounds shown in the above structural formulas are connected to the carboxyl group in group A of the general formula via an amide bond, preferably with the amino group on the left benzene ring in each structural formula connected to the carboxyl group in group A of the general formula via an amide bond.

[0210] Alternatively, the structure of the camptothecin-like compound can be as shown in structural formula IV:

[0211]

[0212] 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.

[0213] When R8 is a substituted C1-5 alkyl or a substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, methanesulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino is an amino group substituted by one or more substituents selected from methyl and ethyl.

[0214] In the antibody-drug conjugate targeting ADAM9 or its salt provided by this invention, the hydroxyl group of the camptothecin compound represented by structural formula IV, which is attached to the same carbon atom as R8, is linked to the carboxyl group of group A in the general formula via a self-releasing structure. This self-releasing structure is, for example... Solid lines represent sites that are linked to the carboxyl group of group A in the general formula, and wavy lines represent sites that are linked to the hydroxyl group in structural formula IV.

[0215] Furthermore, the antibody-drug conjugate or its salt provided by the present invention has a structure as shown in structural formula Ia (general formula for bridged site-directed conjugation structure) and / or Ib (general formula for bridged site-directed open-ring conjugation structure):

[0216]

[0217] and / or

[0218]

[0219] In structural formulas Ia and / or Ib, Ab, m, groups M, SP1, SP2, A, and D are consistent with the general formula above. The definitions of Ab, m, group M, SP1, SP2, A, and D are the same.

[0220] Furthermore, the antibody-drug conjugate or its salt provided by the present invention has a structure as shown in structural formulas Ic and / or Id:

[0221]

[0222] and / or

[0223]

[0224] In structural formulas Ic and / or Id, Ab, m, groups A and D are consistent with the general formula above. The definitions of Ab, m, and groups A and D are the same.

[0225] According to specific embodiments of the present invention, the antibody-drug conjugate or its salt provided by the present invention has the following structure:

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234] Seventh aspect

[0235] The anti-ADAM9 antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, or antibody-drug conjugate or its salt provided by the present invention can be included in a composition, more particularly in a pharmaceutical composition, such as a pharmaceutical formulation, for use in various purposes as needed.

[0236] Therefore, the present invention also provides a composition comprising the anti-ADAM9 antibody or its antigen-binding fragment provided by the present invention, a nucleic acid molecule, a carrier, a host cell, or an antibody-drug conjugate or its salt. Preferably, the composition is a pharmaceutical composition, which optionally further comprises pharmaceutically acceptable excipients. The pharmaceutical compositions provided by the present invention can be formulated into various dosage forms known in the medical or pharmaceutical fields and administered in an applicable manner.

[0237] Eighth aspect

[0238] This invention also provides the use of the anti-ADAM9 antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, antibody-drug conjugate or its salt or composition in the preparation of a medicament for the prevention, treatment and / or improvement of a disease or condition that may be associated with ADAM9 expression (including overexpression), such as ADAM9-positive hematologic malignancies or solid tumors. The anti-ADAM9 antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, antibody-drug conjugate or its salt or composition may exert their effects by binding ADAM9 to exert ADCC, blocking ADAM9 metalloproteinase activity, or through the cytotoxic effects of small molecule cytotoxic compounds in the antibody-drug conjugate, but are not limited thereto. For example, the disease or condition may 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.

[0239] Ninth aspect

[0240] This invention also provides a method for preventing, treating, and / or improving a disease or condition, the method comprising administering to a subject in need an anti-ADAM9 antibody of the present invention or an antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, an antibody-drug conjugate or a salt thereof, or a combination thereof, wherein the disease or condition may be associated with ADAM9 expression (including overexpression), such as ADAM9-positive hematologic malignancies or solid tumors. For example, the disease or condition may 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 may be a mammal; preferably, the subject is a human.

[0241] The methods for preventing, treating and / or improving diseases or symptoms provided by the present invention depend on a variety of factors when applied, including the specific active ingredient of the pharmaceutical composition applied, the patient's age, weight, gender or physical and medical condition, the severity of the condition to be treated, the route of administration, etc.

[0242] The method provided by this invention can also be used in combination with other drugs or methods. These other drugs or methods refer to those that can be administered in combination with the anti-ADAM9 antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, antibody-drug conjugate or its salt or composition described in this invention, such as small molecule chemical drugs, targeted drugs, recombinant protein drugs such as antibodies, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapeutic drugs, and radiotherapy. The combined administration of these two methods can be carried out in any form, such as simultaneously, continuously, or at intervals.

[0243] Tenth aspect

[0244] This invention also provides the use of the anti-ADAM9 antibody or its antigen-binding fragment in the preparation of antibody-drug conjugates for the prevention, treatment, and / or improvement of diseases or conditions that may be associated with ADAM9 expression (including overexpression), such as ADAM9-positive hematologic malignancies or solid tumors. For example, the diseases or conditions may include 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.

[0245] Eleventh aspect

[0246] This invention also provides the use of the aforementioned anti-ADAM9 antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, antibody-drug conjugate or its salt or composition in the preparation of reagents for diagnosing diseases or conditions that may be associated with ADAM9 expression (including overexpression), such as ADAM9-positive hematologic malignancies or solid tumors. For example, the diseases or conditions may include 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.

[0247] Twelfth aspect

[0248] This invention also provides a method for diagnosing a disease or condition, the method comprising contacting an anti-ADAM9 antibody or its antigen-binding fragment, a nucleic acid molecule, a carrier, a host cell, an antibody-drug conjugate or its salt or a combination thereof, with a sample from a subject, wherein the disease or condition may be associated with ADAM9 expression (including overexpression), such as ADAM9-positive hematologic malignancies or solid tumors. For example, the disease or condition may 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.

[0249] The subject may be a mammal; preferably, the subject is a human.

[0250] Thirteenth aspect

[0251] This invention provides a kit comprising the anti-ADAM9 antibody of the present invention or its antigen-binding fragment, a nucleic acid molecule, a vector, a host cell, an antibody-drug conjugate or its salt or a combination thereof. The kit can be used for the above-mentioned prevention, treatment and / or improvement, or for the above-mentioned diagnosis. Depending on the intended application, the kit may also contain other reagents. For example, the kit is for detecting ADAM9 expression (including overexpression) in any biological sample using ELISA.

[0252] Compared with existing technologies, this invention provides a series of murine antibody sequences, humanized sequences, and corresponding antibody molecules against human ADAM9. The antibodies provided by this invention can specifically bind to human ADAM9, rapidly internalize into tumor cells, and effectively block the metalloproteinase activity of ADAM9. They can also be advantageously conjugated with small molecule cytotoxic compounds to prepare targeted cytotoxic antibody-drug conjugates, etc. Experiments have shown that, using the humanized monoclonal antibody Mab-A as a control antibody, the antibodies provided by this invention have the following advantages:

[0253] First, the antibodies provided by this invention exhibit a weaker affinity for human ADAM9 than the control antibody molecule Mab-A, but their binding and internalization activity with tumor cells is comparable to or stronger than that of Mab-A. Of particular note regarding their target protein binding activity, the antibodies of this invention bind less to ADAM9 on the surface of normal tissues and cells than Mab-A; therefore, the antibodies of this invention possess lower target toxicity and better safety potential.

[0254] Second, the antibody provided by this invention specifically binds to human ADAM9 recombinant protein and ADAM9 on the cell surface, and has a significant inhibitory effect on ADAM9 enzyme activity, which is stronger than that of the control molecule Mab-A.

[0255] Third, this invention also examined the distribution of different anti-ADAM9 antibodies in the tumor microenvironment of mouse models. It was found that, compared to Mab-A, the antibody provided by this invention was more likely to accumulate within the tumor after administration to tumor-bearing mice, suggesting a stronger tumor-suppressing effect.

[0256] Fourth, after conjugating the antibody of this invention, the control molecule Mab-A, and the same small molecule cytotoxic compound to form an ADC, it was found that the ADC obtained from the antibody of this invention retained the binding and internalization characteristics of the antibody, and showed significantly better antitumor activity than the ADC obtained from the control molecule Mab-A in various tumor-bearing mouse models. Furthermore, conjugating the antibody provided by this invention with small molecule cytotoxic compounds of different structures can yield ADC products with high DAR values ​​and high purity, and the resulting ADC products exhibit high in vitro cell-killing activity, stronger than IMGC936.

[0257] Fifth, compared to IMGC936, the antibody-drug conjugate provided by this invention is less susceptible to efflux mediated by P-glycoprotein (p-gp), potentially resulting in a lower likelihood of tumor drug resistance.

[0258] Therefore, the antibody provided by this invention has significant application potential in tumor targeted therapy, development of targeted killing ADC drugs, and combined targeted and immunotherapy. Attached Figure Description

[0259] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0260] Figure 1 This is a schematic diagram of the ADAM9 structure.

[0261] Figure 2 The binding activity of the chimeric antibody to human ADAM9 recombinant expression cells was demonstrated.

[0262] Figure 3 The binding activity of the chimeric antibody to recombinant ADAM9-expressing monkey cells was demonstrated.

[0263] Figure 4 The binding activity of the chimeric antibody to tumor cells A431 was demonstrated.

[0264] Figure 5 The binding activity of the chimeric antibody to tumor cells MDA-MB-468 was demonstrated.

[0265] Figure 6 The binding activity of the chimeric antibody to SKBR3 tumor cells was demonstrated.

[0266] Figure 7 The binding activity of the chimeric antibody to SKOV3 tumor cells was demonstrated.

[0267] Figure 8 The binding activity of the chimeric antibody to members of the ADAM9 family was demonstrated.

[0268] Figure 9 The internalization activity of the chimeric antibody on tumor cells A549 was demonstrated.

[0269] Figure 10 The internalization activity of the chimeric antibody on tumor cells MDA-MB-231 was demonstrated.

[0270] Figure 11 The enzyme-inhibiting activity (inhibition rate) of the chimeric antibody is shown.

[0271] Figure 12 The enzyme-inhibiting activity (relative fluorescence intensity) of the chimeric antibody is shown.

[0272] Figure 13 The binding activity of the humanized antibody to MDA-MB-231 cells was demonstrated.

[0273] Figure 14 The binding activity of the humanized antibody to A2780 cells was demonstrated.

[0274] Figure 15 The binding activity of the humanized antibody to NCI-H1975 cells was demonstrated.

[0275] Figure 16 The binding activity of the humanized antibody to NCI-H460 cells was demonstrated.

[0276] Figure 17 The binding activity of the humanized antibody to A549 cells was demonstrated.

[0277] Figure 18 The internalization activity of the humanized antibody on tumor cells MDA-MB-231 was demonstrated.

[0278] Figure 19 The internalization activity of the humanized antibody on tumor cells A2780 was demonstrated.

[0279] Figure 20 The internalization activity of the humanized antibody on NCI-H1975 tumor cells was demonstrated.

[0280] Figure 21 The internalization activity of the humanized antibody on tumor cells MDA-MB-231 was demonstrated.

[0281] Figure 22 The internalization activity of the humanized antibody on tumor cells A549 was demonstrated.

[0282] Figure 23 The internalization activity of the humanized antibody on HeLa tumor cells was demonstrated.

[0283] Figure 24 The antibody-drug conjugate was shown to have cytotoxic activity against MDA-MB-231 tumor cells.

[0284] Figure 25 The antibody-drug conjugate was shown to have cytotoxic activity against SKOV3 tumor cells.

[0285] Figure 26 and Figure 27 The antibody-drug conjugate was shown to have cytotoxic activity against tumor cells A2780.

[0286] Figure 28 Tumor growth curves for each group of the mouse model of human ovarian cancer A2780 are shown.

[0287] Figure 29 Tumor weight data for each group of the mouse model of human ovarian cancer A2780 are shown.

[0288] Figure 30 Tumor growth curves for each group of the mouse model bearing human breast cancer MDA-MB-231 are shown.

[0289] Figure 31 Tumor weight data for each group of the mouse model bearing human breast cancer MDA-MB-231 are shown.

[0290] Figure 32 Tumor growth curves for each group in the mouse RKO model of human colorectal cancer are shown.

[0291] Figure 33 Tumor weight data for each group in the mouse RKO model of human colorectal cancer are shown.

[0292] Figure 34 The staining results of the antibody on frozen sections of normal human gastric tissue are shown.

[0293] Figure 35 The staining results of the antibody on frozen sections of normal human kidney tissue are shown.

[0294] Figure 36 The staining results of the antibody on frozen sections of normal human intestinal tissue are shown.

[0295] Figure 37 The antibody was shown to bind to human PBMCs (CD14+).

[0296] Figure 38 The binding activity of the antibody on NCI-H460 tumor cells was demonstrated.

[0297] Figure 39 The binding activity of the antibody on tumor cells DLD-1 was demonstrated.

[0298] Figure 40 The internalization activity of the antibody on NCI-H460 tumor cells was demonstrated.

[0299] Figure 41 The internalization activity of the antibody on tumor cells DLD-1 was demonstrated.

[0300] Figure 42 Tumor growth curves for each group of the mouse model bearing human large cell lung cancer NCI-H460 are shown.

[0301] Figure 43 The tissue distribution of the ADAM9-targeting antibody detected in a mouse model of human pancreatic cancer AsPC-1 is shown.

[0302] Figure 44The killing activity of the ADAM9-targeting ADC on HCT15 cells was demonstrated.

[0303] Figure 45 The drug resistance characteristics of ADAM9-targeting ADCs on HCT15 cells were demonstrated.

[0304] Figure 46 Tumor growth curves for each group in the mouse RKO model of human colorectal cancer are shown. Detailed Implementation

[0305] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0306] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products.

[0307] Example 1: Preparation of anti-human ADAM9 hybridoma antibody

[0308] Balb / c mice were immunized with human ADAM9 ECD-mFc recombinant protein (UniprotKB sequence number: Q13443, 29-697aa). Serum titers were measured using human ADAM9 ECD-his recombinant protein. After reaching the fusion requirement, hybridoma cell fusion and positive clone screening were performed. Positive clones binding 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 the murine antibody were obtained from relevant literature and conventional databases. Using the obtained cDNA as a template, PCR amplification was performed, and the amplification product was purified. Subsequently, the amplified PCR product was ligated into a T vector, transformed into E. coli competent cells, and the strain was amplified. After plasmid extraction, DNA sequencing was performed to determine the heavy and light chain variable region sequences of the hybridoma antibody.

[0309] Through the above experimental procedures, 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.

[0310] Example 2: Preparation of anti-human ADAM9 chimeric antibody and control antibody

[0311] Humanized monoclonal antibody Mab-A was used as a positive control antibody targeting the same target. The coding gene for the variable regions of the light and heavy chains of this control antibody was synthesized and then cloned into the eukaryotic transient expression vector PTT5 containing the constant regions of the human kappa and IgG1 light and heavy chains, respectively, to obtain light and heavy chain expression plasmids for the control antibody. These plasmids were transformed into *E. coli* for amplification, and a large number of light and heavy chain expression plasmids were isolated. Following the instructions for using the 293fectin transfection reagent (Cat: 12347019, Gibco), the expression plasmids were then transformed into HEK293 cells for recombinant antibody expression. Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column to obtain the control antibody Mab-A.

[0312] Following the same procedure as described above, the coding genes for the light and heavy chain variable regions of the six specific hybridoma antibodies obtained in Example 1 were cloned into the eukaryotic transient expression vector PPT5 containing the light and heavy chain constant regions of human kappa and IgG1 (SEQ ID NO.3 and SEQ ID NO.4), respectively, ultimately obtaining six chimeric antibodies. Based on the naming convention of the hybridoma clones, they were named chimeric antibodies ch5H6, ch5H1, ch27F8, ch10E5, ch26B1, and ch20H1, respectively.

[0313] The heavy and light chain variable region sequences of the control antibody Mab-A and the chimeric antibody ch5H6 are shown below. The underlined bold portion shows the antigen-determining region (CDR), which was obtained using the Kabat definition method.

[0314] SEQ ID NO.1 : Mab-A heavy chain variable region amino acid sequence

[0315] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLEWVGEIIPIFGHT

[0316] NYNEKFKSRFTISLDNSKNTLYLQMGSLRAEDTAVYYCARGGYYYYPRQGFLDYWGQ

[0317] GTTVTVSS SEQ ID NO.2 : Mab-A light chain variable region amino acid sequence

[0318] DIVMTQSPDSLAVSLGERATISCKASQSVDYSGDSYMNWYQQKPGQPPKLLIYAASDLE

[0319] SGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQQSHEDPFTFGQGTKLEIK SEQ ID NO.3 IgG1 heavy chain constant region amino acid sequence

[0320] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

[0321] SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG

[0322] GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE

[0323] QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP

[0324] PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

[0325] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.4 : amino acid sequence of the constant region of the kappa light chain

[0326] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

[0327] DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 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

[0328] 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

[0329]

[0330] Example 3: Affinity detection of chimeric antibodies binding to recombinant human ADAM9 protein

[0331] Antibody affinity was determined using the Fortebio Octet QKe system with an anti-human antibody Fc fragment capture antibody (AHC) bioprobe to capture the Fc fragment of the antibody. For the assay, chimeric antibodies ch5H6, ch5H1, ch27F8, ch10E5, ch26B1, ch20H1 and control antibody Mab-A were diluted to 4 μg / ml with PBS buffer and flowed through the AHC probe (Cat: 18-0015, PALL) for 120 s. Human ADAM9 ECD-His recombinant protein was diluted to 60 nM as the mobile phase, with a binding time of 300 s and a dissociation time of 300 s. After the experiment, the blank control response value was subtracted, and a 1:1 Langmuir binding model was fitted to calculate the kinetic constants of antigen-antibody binding.

[0332] The kinetic parameters are shown in Table 1. The results show that all chimeric antibodies specifically bind to recombinant human ADAM9 protein, and their affinity is weaker than that of the control antibody Mab-A to varying degrees.

[0333] Table 1. Affinity assay results of chimeric antibody and recombinant human ADAM9 protein

[0334] Antibody KD(M) Kon(1 / Ms) Kdis(1 / s) Mab-A 5.09E-10 5.11E+05 2.60E-04 ch5H6 1.40E-09 1.23E+06 1.73E-03 ch5H1 1.60E-09 1.21E+06 1.95E-03 ch27F8 4.97E-09 3.45E+05 1.71E-03 ch10E5 9.02E-10 2.92E+05 2.63E-04 ch26B1 2.81E-10 7.65E+05 2.15E-04 ch20H1 2.38E-09 1.54E+06 3.67E-03

[0335] Example 4: Affinity detection of chimeric antibodies binding to different truncated human ADAM9 variants and mouse ADAM9

[0336] The extracellular region of ADAM9 mainly includes the following domains: zinc esterase domain, detegrin domain, Cys-rich region, and EGF-like domain. Different lengths of ADAM9 truncated variants were expressed, and the binding of chimeric antibodies to the truncated variants was analyzed to preliminarily determine the binding epitope region; simultaneously, it was analyzed whether the chimeric antibodies exhibited species-cross-binding activity with mouse ADAM9.

[0337] Affinity tests were performed using the following recombinant proteins in the same manner as described in Example 3: human ADAM929-643-His recombinant protein (UniprotKB sequence number: Q13443, 29-643aa, without EGF-like domain), human ADAM9s-His recombinant protein (UniprotKB sequence number: Q13443, 29-576aa), and mouse ADAM9 ECD-His recombinant protein (UniprotKB sequence number: Q61072, 29-697aa).

[0338] The affinity (KD(M)) of the six chimeric antibodies and the control antibody Mab-A to the aforementioned proteins is shown in Table 2. The results indicate that the chimeric antibody ch27F8 binds to human ADAM9s-His (the secreted form of ADAM9), and ch10E5 exhibits cross-binding activity with mouse ADAM9, suggesting that ch27F8 has different binding epitopes compared to other molecules. In contrast, ch5H6, ch5H1, ch10E5, ch26B1, and ch20H1 have similar binding epitopes to the control antibody Mab-A and do not bind to the secreted form of human ADAM9 (ADAM9s-His).

[0339] Table 2. Affinity (KD(M)) of chimeric antibodies to human ADAM9 and its different truncated variants, as well as mouse ADAM9.

[0340]

[0341] Example 5: Detection of the binding activity of chimeric antibodies to recombinant human ADAM9-expressing cells

[0342] CHO cells stably expressing human ADAM9 (UniprotKB sequence number: Q13443, 1-819aa) were constructed.

[0343] 2E5 cells were collected. Chimeric antibodies ch5H6, ch5H1, ch27F8, ch10E5, and ch26B1, along with control antibody Mab-A and isotype control, were serially diluted 3-fold (8 steps) starting at 10 μg / mL and added to the cells. The cells were incubated at 4°C in the dark for 60 min. After thorough washing with PBS, FITC-labeled goat anti-human antibody (F9512, Sigma) diluted 1:200 was added, and the cells were incubated at 4°C in the dark for 30 min, followed by thorough washing with PBS. The cells were resuspended in 200 μL of PBS and analyzed by flow cytometry.

[0344] The results are as follows Figure 2As shown in Table 3, the chimeric antibodies ch5H6, ch5H1, ch27F8, ch10E5, and ch26B1 can bind to human ADAM9 recombinant expression cells in a dose-dependent manner. EC50 values ​​are shown in Table 3.

[0345] Table 3. EC50 values ​​of chimeric antibody binding to recombinant human ADAM9-expressing cells

[0346] Mab-A ch27F8 ch5H1 ch10E5 ch26B1 ch5H6 EC50 (μg / ml) 0.8753 0.7895 0.7202 2.758 4.073 0.6942

[0347] Example 6: Detection of the binding activity of chimeric antibodies to recombinant ADAM9-expressing monkey cells

[0348] CHO cells stably expressing monkey ADAM9 (UniprotKB sequence number: A0A2K5X4X8, 1-819aa) were constructed.

[0349] The binding of the antibody to the cell was detected using flow cytometry in the same manner as described in Example 5.

[0350] The results are as follows Figure 3 As shown in Table 4, the chimeric antibodies ch5H6, ch5H1, ch27F8, and ch10E5 bound to monkey ADAM9 recombinant expression cells in a dose-dependent manner, while ch26B1 did not bind to these cells; and ch10E5 bound weakly. EC50 values ​​are shown in Table 4.

[0351] Table 4. EC50 values ​​of chimeric antibody binding to recombinant ADAM9-expressing monkey cells

[0352] Mab-A ch27F8 ch5H6 ch10E5 ch26B1 ch5H1 EC50 (μg / ml) 0.6261 0.3264 0.4529 2.366 / 0.4799

[0353] Example 7: Detection of the binding activity of chimeric antibodies to A431 tumor cells

[0354] 2E5 A431 cells were collected. The binding of the antibody to the cells was detected by flow cytometry in the same manner as described in Example 5.

[0355] The results are as follows Figure 4 As shown in the figure. The results indicate that the chimeric antibodies ch5H6, ch5H1, ch27F8, and ch10E5 can bind to tumor cells A431 in a dose-dependent manner. Among them, ch5H1 and ch5H6 have a binding capacity that is basically equivalent to that of the positive control antibody Mab-A, while the binding of the other molecules to tumor cells is weaker than that of Mab-A to varying degrees.

[0356] Example 8: Detection of the binding activity of chimeric antibodies with MDA-MB-468, SKBR3 and SKOV3 tumor cells

[0357] Two E5 MDA-MB-468, SKBR3, and SKOV3 cells were collected, respectively. Chimeric antibodies ch5H6, ch5H1, ch27F8, and ch10E5, along with the positive control antibody Mab-A and isotype control, were diluted to 3.3 μg / ml and 0.37 μg / ml, respectively, and added to the cells. The cells were incubated at 4°C in the dark for 60 min. After thorough washing with PBS, FITC-labeled goat anti-human antibody (F9512, Sigma) diluted 1:200 was added, and the cells were incubated at 4°C in the dark for 30 min, followed by thorough washing with PBS. The cells were resuspended in 200 μL of PBS and analyzed by flow cytometry.

[0358] The results are as follows Figure 5 , Figure 6 , Figure 7 As shown in the figure. The results indicate that the chimeric antibodies ch5H6, ch5H1, ch27F8, and ch10E5 specifically bind to different tumor cells, with the binding activity of ch5H6 and ch5H1 being essentially equivalent to that of the control antibody.

[0359] Example 9: Cross-binding activity assay of chimeric antibodies with ADAM9 family members

[0360] HEK293 cells were transiently transfected with the full-length expression vectors of human ADAM9 family members: human ADAM8 (UniprotKB sequence number: P78325), human ADAM10 (UniprotKB sequence number: O14672), and human ADAM17 (UniprotKB sequence number: P78536). Simultaneously, the full-length human ADAM9 expression vector was also transiently transfected. After 36 hours, 2E5 cells expressing human ADAM9, human ADAM8, human ADAM10, and human ADAM17 were collected. Chimeric antibodies ch5H6, ch5H1, ch27F8, and ch10E5, along with the positive control antibody Mab-A and isotype control, were diluted to 10 μg / ml and added to the cells. The cells were incubated at 4°C in the dark for 60 min. After thorough washing with PBS, 1:200 diluted FITC-labeled goat anti-human antibody (F9512, Sigma) was added, and the cells were incubated at 4°C in the dark for 30 min, followed by thorough washing with PBS. Cells were resuspended in 200 μL of PBS and analyzed using flow cytometry.

[0361] The results are as follows Figure 8 As shown in the figure. The results indicate that the chimeric antibodies ch5H6, ch5H1, ch27F8, ch10E5 and the control antibody Mab-A all specifically bind to human ADAM9, but do not bind to other members of the same family.

[0362] Example 10:Detection of internalization activity of chimeric antibodies on different tumor cells

[0363] Two E5 MDA-MB-231 and A549 cells were collected, respectively. Chimeric antibodies ch5H6, ch5H1, ch27F8, and ch10E5, along with the positive control antibody Mab-A and isotype control, were diluted to three concentrations: 1 μg / ml, 0.5 μg / ml, and 0.25 μg / ml. Antibody Internalization Human Reagent (Cat. 90565, Sartorius) was added to the cells along with the diluent according to the reagent's instructions. The cells were incubated at 37°C in the dark for 120 min, and the immunofluorescence signal of the internalized cells was detected using flow cytometry.

[0364] The results are as follows Figure 9 , Figure 10 As shown in the figure. The results indicate that the chimeric antibodies ch5H6, ch5H1, ch27F8 and ch10E5 all exhibited strong internalization under different tumor cell conditions, with ch5H1 and ch5H6 showing significantly better internalization activity than the control antibody Mab-A.

[0365] Example 11: Enzyme-inhibiting activity of chimeric antibodies

[0366] ADAM9 possesses strong metalloproteinase activity, and its enzyme activity is a crucial pathway for its participation in physiological and pathological processes such as inflammation, tumors, and injuries. Anti-ADAM9 antibodies can influence these pathological processes by blocking enzyme activity. The purpose of this embodiment is to analyze the inhibitory effects of different antibodies on ADAM9 enzyme activity through enzyme activity experiments.

[0367] Recombinant human ADAM9-His protein was diluted to 80 μg / mL with reaction solution (25 mM Tris, 2.5 μM ZnCl2, 0.005% Brij 35, pH 9.0), and 25 μL / well was added to a 96-well plate. The detection antibody was also diluted to 2400 μg / mL with reaction solution, and then serially diluted 4-fold for a total of 8 steps. 25 μL of the diluted solution was added to each well of the 96-well plate, and after mixing, the plate was incubated at 37°C for 15 min. Then, 5 μM of the enzyme fluorescent substrate was added to each well, making the total volume 100 μL per well, and the plate was incubated at 37°C for 4 h. The fluorescence values ​​were then read and recorded using a microplate reader (EX: 320 nm; Em: 405 nm), and the inhibition rate was calculated using the following formula:

[0368] Inhibition rate % = (relative fluorescence intensity hIgG1 - relative fluorescence intensity Sample) / (relative fluorescence intensity hIgG1 - relative fluorescence intensity background) × 100%; where relative fluorescence intensity hIgG1 is the relative fluorescence intensity of the isotype control.

[0369] The results are as follows Figure 11 and Figure 12 As shown in Table 5, the chimeric antibodies all inhibited the metalloproteinase activity of ADAM9 in a dose-dependent manner, with ch5H6 exhibiting the strongest inhibitory activity. The IC50 values ​​are shown in Table 5.

[0370] Table 5. IC50 values ​​of chimeric antibodies inhibiting the metalloproteinase activity of human ADAM9

[0371] Mab-A ch27F8 ch10E5 ch5H6 IC50 (μg / ml) 10.34 23.48 41.21 4.89

[0372] Example 12: Humanization and recombinant expression of murine antibodies

[0373] A comprehensive analysis of the variable region sequence of murine antibodies was performed to identify the antigen complementarity determinant (CDR) region for antibody-antigen binding and the framework region (FR) supporting the conserved three-dimensional conformation of the antibody. Based on homology alignment results, the template of the most similar human antibody in the human antibody germline library was selected as the basic template. Combined with the full-sequence BLAST results, CDR transplantation was performed, thereby humanizing the light and heavy chain variable regions.

[0374] The humanized heavy and light chain variable region sequences of the mouse antibody derived from hybridoma clone 5H6, obtained through CDR transplantation as described above, were synthesized. The coding genes for these variable regions were then cloned into eukaryotic transient expression vectors containing the light and heavy chain constant regions of human kappa and IgG1, respectively, to obtain light and heavy chain expression plasmids. These plasmids were then recombinantly expressed in HEK293 cells. Five to six days after cell transfection, the culture supernatant was collected and purified using ProA affinity chromatography to obtain the humanized antibody derived from the CDR transplantation. Following the nomenclature of the hybridoma clone, these were named humanized antibody hz5H6.

[0375] The heavy and light chain variable region sequences of the humanized antibody hz5H6 are shown below, where the underlined bold portion indicates the antigen-determining region (CDR), which was obtained using the Kabat definition method.

[0376] 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

[0377] 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

[0378]

[0379] Furthermore, point mutations were performed on the CDR regions of the heavy chain variable region and the light chain variable region of hz5H6. The specific mutation sites are shown in Table 6.

[0380] Table 6. Point mutations in the CDR region of hz5H6 antibody

[0381]

[0382]

[0383] Note: Taking E65Q as an example, it means that the 65th amino acid E is mutated to Q.

[0384] The amino acid sequences of the variable regions of the heavy and light chains after point mutation are shown below, where the underlined bold parts indicate the antigenic determinant region (CDR), which is obtained through the Kabat definition method.

[0385] SEQ ID NO.9 (H-CDR1 / H-CDR2 / H-CDR3: SEQ ID NO.36 / SEQ ID NO.37 / SEQ IDNO.38): hz5H6_Hm1

[0386] SEQ ID NO.10 (H-CDR1 / H-CDR2 / H-CDR3: SEQ ID NO.36 / SEQ ID NO.37 / SEQ IDNO.38): hz5H6_Hm2

[0387] SEQ ID NO.11 (H-CDR1 / H-CDR2 / H-CDR3: SEQ ID NO.36 / SEQ ID NO.37 / SEQ IDNO.38): hz5H6_Hm3

[0388]

[0389] SEQ ID NO.12 (H-CDR1 / H-CDR2 / H-CDR3: SEQ ID NO.36 / SEQ ID NO.39 / SEQ IDNO.38): hz5H6_Hm4

[0390]

[0391] SEQ ID NO.13 (H-CDR1 / H-CDR2 / H-CDR3:SEQ ID NO.36 / SEQ ID NO.40 / SEQ IDNO.38):hz5H6_Hm5

[0392]

[0393] SEQ ID NO.14 (H-CDR1 / H-CDR2 / H-CDR3:SEQ ID NO.36 / SEQ ID NO.41 / SEQ IDNO.38):hz5H6_Hm6

[0394]

[0395] SEQ ID NO.15 (H-CDR1 / H-CDR2 / H-CDR3:SEQ ID NO.36 / SEQ ID NO.37 / SEQ IDNO.42):hz5H6_Hm7

[0396]

[0397] SEQ ID NO.16 (H-CDR1 / H-CDR2 / H-CDR3:SEQ ID NO.36 / SEQ ID NO.41 / SEQ IDNO.43):hz5H6_Hm8

[0398]

[0399] SEQ ID NO.17 (H-CDR1 / H-CDR2 / H-CDR3:SEQ ID NO.36 / SEQ ID NO.41 / SEQ IDNO.44):hz5H6_Hm9

[0400]

[0401] SEQ ID NO.18 (L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.45 / SEQ ID NO.46 / SEQ IDNO.47):hz5H6_Lm1

[0402]

[0403] SEQ ID NO.19(L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.45 / SEQ ID NO.46 / SEQ IDNO.47):hz5H6_Lm2

[0404]

[0405] SEQ ID NO.20 (L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.45 / SEQ ID NO.46 / SEQ IDNO.47):hz5H6_Lm3

[0406]

[0407] SEQ ID NO.21 (L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.45 / SEQ ID NO.46 / SEQ IDNO.47):hz5H6_Lm4

[0408]

[0409] SEQ ID NO.22 (L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.45 / SEQ ID NO.46 / SEQ IDNO.47):hz5H6_Lm5

[0410]

[0411] SEQ ID NO.23 (L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.45 / SEQ ID NO.46 / SEQ IDNO.47):hz5H6_Lm6

[0412]

[0413] SEQ ID NO.24 (L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.45 / SEQ ID NO.48 / SEQ IDNO.47):hz5H6_Lm7

[0414]

[0415] SEQ ID NO. 25 (L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.45 / SEQ ID NO.49 / SEQ IDNO.47):hz5H6_Lm8

[0416]

[0417] SEQ ID NO. 26 (L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.45 / SEQ ID NO.50 / SEQ IDNO.47):hz5H6_Lm9

[0418] SEQ ID NO.27 (L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.45 / SEQ ID NO.46 / SEQ IDNO.47):hz5H6_Lm10

[0419]

[0420] SEQ ID NO.28 (L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.45 / SEQ ID NO.46 / SEQ IDNO.47):hz5H6_Lm11

[0421]

[0422] SEQ ID NO. 29 (L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.51 / SEQ ID NO.49 / SEQ IDNO.47):hz5H6_Lm12

[0423]

[0424] SEQ ID NO. 30 (L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.45 / SEQ ID NO.49 / SEQ IDNO.52):hz5H6_Lm13

[0425]

[0426] SEQ ID NO. 31 (L-CDR1 / L-CDR2 / L-CDR3:SEQ ID NO.45 / SEQ ID NO.49 / SEQ IDNO.53):hz5H6_Lm14

[0427] SEQ ID NO. 32(L-CDR1 / L-CDR2 / L-CDR3: SEQ ID NO.51 / SEQ ID NO.49 / SEQ IDNO.53): hz5H6_Lm15

[0428] SEQ ID NO. 33 (L-CDR1 / L-CDR2 / L-CDR3: SEQ ID NO.45 / SEQ ID NO.49 / SEQ IDNO.54): hz5H6_Lm16

[0429] SEQ ID NO. 34 (L-CDR1 / L-CDR2 / L-CDR3: SEQ ID NO.45 / SEQ ID NO.49 / SEQ IDNO.55): hz5H6_Lm17

[0430] SEQ ID NO. 35 (L-CDR1 / L-CDR2 / L-CDR3: SEQ ID NO.45 / SEQ ID NO.49 / SEQ IDNO.56): hz5H6_Lm18

[0431]

[0432] Using the variable region sequences of the light and heavy chains obtained after the above point mutation, different hz5H6 mutants were obtained through recombination expression in the same manner as above.

[0433] Example 13: Affinity detection of humanized antibodies

[0434] Antibody affinity was determined using the Fortebio Octet QKe system with an antibody capture antibody (AHC) bioprobe to capture the Fc fragment of the anti-human antibody. During the assay, the humanized antibody hz5H6 and its mutant, along with the control antibody Mab-A, were diluted to 4 μg / ml with PBS buffer and flowed through the AHC probe (Cat: 18-0015, PALL) for 120 s. Human ADAM9 ECD-His recombinant protein was diluted to 60 nM as the mobile phase, with a binding time of 300 s and a dissociation time of 300 s. After the experiment, the blank control response value was subtracted, and a 1:1 Langmuir Global binding model was fitted to calculate the kinetic constants of antigen-antibody binding.

[0435] The kinetic parameters of hz5H6, its mutants, and the control antibody Mab-A are shown in Table 7. The results indicate that the affinity of hz5H6 and most of its mutants is weaker than that of the control antibody Mab-A to varying degrees.

[0436] Table 7. Results of Affinity Measurement

[0437]

[0438]

[0439] Example 14: Detection of the binding activity of humanized antibodies to different tumor cells

[0440] The binding strength of humanized antibodies to cell surface antigens was detected using tumor cells with different ADAM9 expression levels.

[0441] 2E5 cells of MDA-MB-231, A2780, NCI-H1975, and NCI-H460 were collected. Humanized antibody hz5H6Lm8, positive control antibody Mab-A, and isotype control were serially diluted 3-fold (8 times) starting at 10 μg / mL and added to the cells. Cells were incubated at 4°C in the dark for 60 min. After thorough washing with PBS, FITC-labeled goat anti-human antibody (F9512, Sigma) diluted 1:200 was added, and the cells were incubated at 4°C in the dark for 30 min, followed by thorough washing with PBS. The cells were resuspended in 200 μL of PBS and analyzed by flow cytometry.

[0442] The results are as follows Figure 13 , Figure 14 , Figure 15 , Figure 16 As shown in the figure. The results indicate that the humanized antibody hz5H6Lm8 and the control antibody Mab-A can bind specifically to different tumor cells in a dose-dependent manner, and the binding ability of hz5H6Lm8 and the control antibody Mab-A is basically equivalent.

[0443] Different hz5H6 mutant assays: 2E5 A549 cells were taken and flow cytometry was used to detect the binding of different hz5H6 mutants to the cells in the same manner as described above.

[0444] The binding curves of different hz5H6 mutants with A549 cells are shown in the figure. Figure 17 As shown, the results indicate that there is no significant difference in binding activity among the multiple mutants.

[0445] Example 15: Detection of internalization activity of humanized antibodies on different tumor cells

[0446] The internalization of humanized antibodies was detected using tumor cells with different ADAM9 expression levels.

[0447] 2E5 MDA-MB-231, A2780, and NCI-H1975 cells were collected. The humanized antibody hz5H6Lm8, control antibody Mab-A, and isotype control were diluted to four concentrations: 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, and 0.125 μg / ml. Antibody Internalization Human Reagent (Cat.90565, Sartorius) was added to the cells along with the diluent according to the reagent's instructions. The cells were incubated at 37°C in the dark for 120 min, and the immunofluorescence signal of the internalized cells was detected using flow cytometry.

[0448] The results are as follows Figure 18 , Figure 19 , Figure 20 As shown in the figure. The results indicate that both the humanized antibody hz5H6Lm8 and the control antibody Mab-A underwent strong internalization in different tumor cells.

[0449] Determination of different hz5H6 mutants: 2E5 MDA-MB-231, A549 and HeLa cells were taken and their internalization activity was detected by flow cytometry in the same manner as described above.

[0450] The results are as follows Figure 21 , Figure 22 , Figure 23 As shown in the figure. The results indicate that both the humanized antibody hz5H6 mutant and the control antibody Mab-A underwent strong internalization in different tumor cells.

[0451] Example 16: Detection of the killing activity of ADCs obtained by conjugating humanized antibodies with small molecule cytotoxic compounds on tumor cells

[0452] The small molecule cytotoxic compound methylaurestatin E (MMAE) was prepared by conjugating different antibodies with the drug-containing linker BL20E to obtain an ADC molecule, named "antibody-MMAE(BL20E)". The structure of BL20E is shown in the examples below.

[0453] The killing activity of the synthesized ADC molecules on these tumor cells was evaluated on MDA-MB-231, SKOV3, and A2780.

[0454] Different tumor cells were seeded into 96-well culture plates. After overnight adhesion, each ADC was diluted to 100 μg / ml, followed by nine 3-fold serial dilutions. The diluted solutions were added to the cells, and the plates were incubated at 37°C for 72 h. The cells were then analyzed using a CCK8 assay kit.

[0455] The results are as follows Figure 24 , Figure 25 As shown in the figure. The results indicate that the killing activity trends of each ADC are the same in MDA-MB-231 and SKOV3 cells.

[0456] Killing activity assay of ADCs prepared with different hz5H6 mutants: A2780 cells were plated and adhered overnight. ADCs prepared with each hz5H6 mutant and / or Mab-A were diluted to 10 μg / ml, then serially diluted 3-fold and added to the cells. The cells were incubated at 37°C for 96 h. The killing activity of each mutant was then detected using the CCK8 assay kit.

[0457] The results are as follows Figure 26 , Figure 27 As shown in the figure. The results indicate that the cytotoxic activities of ADCs made from different humanized antibody hz5H6 mutants are similar, and their cytotoxic activities are similar to those of ADCs made from Mab-A.

[0458] Example 17: Pharmacodynamic evaluation of ADCs obtained by conjugating humanized antibodies with small molecule cytotoxic compounds in NCG mouse model of human ovarian cancer A2780

[0459] To evaluate the antitumor therapeutic effect of ADC in an NCG mouse model of human ovarian cancer A2780.

[0460] Human ovarian cancer A2780 cells were subcutaneously in the right anterior flank of female NCG mice. The tumor was allowed to grow to 100 mm. 3 The animals were divided into groups of 6 each, and the dosage was 3 mg / kg, administered intraperitoneally (IP).

[0461] The tumor growth inhibition status is shown in Table 8. Figure 28 and Figure 29 As shown. The results indicated that, compared with the NC group, all groups had a clear anti-tumor effect, and the efficacy of hz5H6Lm8-MMAE(BL20E) (TGI) was significantly higher. TV and TGI Tw The rates of 86% and 84% respectively were significantly better than those of the control antibody Mab-A-MMAE(BL20E)(TGI). TV and TGI Tw (55% and 53% respectively).

[0462] Table 8. Tumor growth inhibition status in each group

[0463]

[0464]

[0465] Example 18: Pharmacodynamic evaluation of ADCs conjugated with humanized antibodies in M-NSG mouse model of human breast cancer MDA-MB-231

[0466] In addition, the antitumor therapeutic effect of ADC (mutant-MMAE(BL20E)) was evaluated in the M-NSG mouse model of human breast cancer MDA-MB-231.

[0467] Human breast cancer MDA-MB-231 cells were subcutaneously in the right anterior flank of NSG mice. When the tumor grew to 180 mm... 3 The animals were divided into groups of 6 each, and the dosage was 5 mg / kg, administered once via intraperitoneal injection.

[0468] The tumor growth inhibition status is shown in Table 9. Figure 30 and Figure 31 As shown in the figure. The results indicated that all groups had a clear anti-tumor effect compared with the NC control group. Tumors reached the euthanasia criteria on day 28 and were euthanized, with tumor weight measured. The drug-treated groups completed the experiment on day 45, with tumor volume and weight compared. The results showed that hz5H6Lm8-MMAE(BL20E), hz5H6Hm6Lm14-MMAE(BL20E), and hz5H6Hm8Lm14-MMAE(BL20E) all had better tumor-suppressing effects than the control antibody Mab-A-MMAE(BL20E); among them, hz5H6Hm6Lm14-MMAE(BL20E) showed the best tumor-suppressing effect.

[0469] Table 9. Tumor growth inhibition status in each group

[0470]

[0471] Example 19: Pharmacodynamic evaluation of ADCs conjugated with humanized antibodies in a Balb / C-nude mouse model of human colorectal cancer RKO

[0472] Furthermore, the antitumor therapeutic effects of the ADAM9-targeting ADC of the present invention and the control ADC IMGC936 (Mab-A-DM21, DAR value of 2; wherein the compound is DM21-C, from patent application publication WO2022192134A1) were evaluated in a Balb / C-nude mouse RKO model of human colorectal cancer.

[0473] Human colorectal cancer RKO cells were subcutaneously in the right anterior flank of Balb / C-nude mice. The cells were allowed to grow to 300 mm. 3 The animals were divided into groups of 6 each, and the dosage was 10 mg / kg, administered once via intraperitoneal injection.

[0474] Tumor growth inhibition status, such as Figure 32 , Figure 33 As shown. The results indicate that the hz5H6Hm6Lm14-ADC is more effective, with hz5H6Hm6Lm14-MF-L6 showing better tumor-suppressing effect than the similar toxin Mab-A-MF-L6, and significantly better than the control molecule IMGC936. MF-L6 is the same as MWF-L6, and can be used interchangeably in this application; its structure is shown in the examples below.

[0475] Example 20: Anti-ADAM9 antibodies bind to normal human tissues or cells with ADAM9+.

[0476] ADAM9 is expressed in various normal tissues, including the kidney and gastrointestinal tract. Using frozen sections (three donors each) of human kidney, intestinal, and gastric tissues, immunohistochemical staining was performed using hz5H6Hm6Lm14, the positive control antibody Mab-A, and the isotype control antibody NC-IgG1 at a concentration of 10 μg / mL as primary antibodies. The binding of each antibody molecule to normal ADAM9+ tissues was evaluated.

[0477] In addition, ADAM9 is expressed on the surface of monocytes. The binding characteristics of hz5H6Hm6Lm14, hz5H6Hm8Lm14, and Mab-A to ADAM9+ human PBMCs were analyzed using FACS. After blocking PBMCs (derived from healthy volunteers) with 20 μg / ml NC-hIgG1 at room temperature for 30 min, CF488 (Cat.MX488AS100-1KT, Sigma) labeled hz5H6Hm6Lm14, hz5H6Hm8Lm14, control antibody Mab-A, and isotype control antibody NC-hIgG1 (starting concentration 20 μg / mL, serially diluted 2-fold to 6 concentrations) were added to each well. At the same time, APC Anti-Human CD14 Antibody (Cat.E-AB-F1209E, Elabscience) was added to each well. The cells were incubated at 4°C in the dark for 30 min. After thorough washing and resuspending with 4°C pre-cooled PBS, the FITC and APC signal values ​​were detected by flow cytometry. The CD14+ subset (APC) was circled from the total cells, and the FITC signal values ​​of each antibody were analyzed and compared in the CD14+ subset.

[0478] The results are as follows Figure 34 , Figure 35 , Figure 36 , Figure 37 As shown in Table 10, the results indicate that hz5H6Hm6Lm14 binds significantly weakly to positive tissues of normal individuals compared to the control antibody Mab-A. Furthermore, hz5H6Hm6Lm14 and hz5H6Hm8Lm14 also bind significantly weakly to monocytes (CD14+) derived from human PBMCs compared to Mab-A, suggesting that hz5H6-based ADC drugs have lower toxicity risks to normal tissues and better safety.

[0479] Table 10. Summary of immunohistochemical staining results of frozen sections

[0480]

[0481]

[0482] Note: -, negative; +, weak positive; ++, positive.

[0483] Example 21: Effect of Fc variants on anti-ADAM9 antibody activity

[0484] Human IgG1 variants L234A / L235A (“LALA”; Xu et al., Cell Immunol 2000 Feb 25; 200(1):16-26) can eliminate the binding of FcγRs without affecting the binding of FcRn, thereby eliminating the Fc-mediated effector function. To further confirm the effect of FcγRs on the activity of the anti-ADAM9 antibody of the present invention, the gene sequence encoding the heavy chain variable region of hz5H6Hm6Lm14 was cloned into a eukaryotic transient expression vector containing the gene sequence encoding the heavy chain constant region (SEQ ID NO. 57) of human IgG1-LALA (human IgG1 variants L234A / L235A) to obtain a heavy chain expression plasmid; the gene sequence encoding the light chain variable region of hz5H6Hm6Lm14 was cloned into a eukaryotic transient expression plasmid containing the light chain constant region (SEQ ID NO. 4) of human kappa to obtain a light chain expression vector. The two expression vectors were recombinantly expressed in HEK293 cells to obtain the recombinant human IgG1-LALA subtype protein, named "hz5H6Hm6Lm14-LALA".

[0485] SEQ ID NO.57: Amino acid sequence of the constant region of the IgG1-LALA heavy chain

[0486] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

[0487] SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAA

[0488] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

[0489] EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL

[0490] PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

[0491] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0492] 2E5 human large cell lung cancer NCI-H460 and human colorectal cancer DLD-1 cells were collected and, following the same procedure as in Examples 14 and 15, the binding and internalization of hz5H6Hm6Lm14-LALA with the cells were detected by flow cytometry. The results are as follows: Figures 38 to 41 As shown, hz5H6Hm6Lm14-LALA exhibits the same binding and internalization activity as hz5H6Hm6Lm14.

[0493] Furthermore, the antitumor therapeutic effect of ADC (IgG1 variant) was evaluated in the Balb / C-nude mouse model of human large cell lung cancer NCI-H460.

[0494] Human large cell lung cancer NCI-H460 cells were subcutaneously in the right anterior flank of Balb / C-nude mice. The tumor was allowed to grow to 200 mm. 3 The animals were divided into groups of 6 each, and the dosage was 10 mg / kg, administered once via intraperitoneal injection.

[0495] Tumor growth inhibition status, such as Figure 42 As shown in the figure. The results indicate that hz5H6Hm6Lm14-LALA-MF-L6 has comparable tumor-suppressing effects to hz5H6Hm6Lm14-MF-L6, and the use of human IgG1-LALA subtype does not affect the antitumor activity of ADAM9.

[0496] Example 22 Distribution of anti-ADAM9 antibody in the tumor microenvironment of an AsPC-1 cell CDX subcutaneous tumor mouse model

[0497] ADAM9-positive tumor AsPC-1 cells were inoculated into nude mice at a concentration of 5 × 10⁶ cells / mL. 6 Each animal was divided into 3 groups of 3, and the tumors were allowed to grow to 400-600 mm. 3 Mice were intraperitoneally administered CY7(MCE, HY-D0824A) labeled antibodies: hz5H6Hm6Lm14, Mab-A, and isotype control antibody NC, at a dose of 10 mg / kg. Five days later, the mice underwent cardiac perfusion. The whole mouse and the heart, liver, spleen, lung, kidney, and tumor sites were then imaged using a PerkinElmerIVIS Lumina III instrument to obtain the fluorescence signal values ​​of each tissue. The ratio of the signal of each tissue to the corresponding whole mouse signal was then calculated to confirm the distribution of the antibodies in the tissues, and the statistical differences were analyzed using a t-test.

[0498] The tissue distribution of antibodies is as follows: Figure 43 As shown in the figure. The results indicate that the detection amount of hz5H6Hm6Lm14 in tumors was higher than that of Mab-A, and there was a statistically significant difference in the detection amount of hz5H6Hm6Lm14 in tumors compared with the NC control group (P<0.05), suggesting that hz5H6Hm6Lm14 has better tumor accumulation.

[0499] Example 23: Preparation of drug-containing linkers

[0500] 1. Synthesis of compound A

[0501] According to the method described in patent application publication WO2023109965A1, compound A of formula shown in Table 11 was synthesized.

[0502] Table 11. Compounds of Formula A

[0503]

[0504]

[0505]

[0506]

[0507]

[0508] 2. Synthesis of Camptothecin-like Compounds

[0509] According to the method described in patent application publication WO2023109965A1, camptothecin-like compounds as shown in Table 12 were synthesized.

[0510] Table 12. Camptothecin-like compounds

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518] 3. Synthesis of drug-containing linkers

[0519] The drug-containing linkers shown in Table 13 were synthesized according to the method described in patent application publications WO2023109965A1 or WO2018095422A1.

[0520] Table 13. Drug-containing connectors

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528] Example 24 Preparation and Characterization of Antibody-Drug Conjugates Targeting ADAM9 1. General Preparation Methods for Antibody-Drug Conjugates

[0529] 1.1 General preparation method for site-directed coupling

[0530]

[0531] Antibody reduction: 120 mg of antibody sample was used with a Sephadex G25-supported NAP-25 column, purged to pH 7.0 in a buffer solution containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate, and the antibody concentration was diluted to 10 mg / ml. 10 ml of a total of 100 mg of antibody sample was taken, and 2.1 ml of 10 mg / ml TCEP (Sigma-Aldrich) aqueous solution was added at an antibody-TCEP molar ratio of 1:10. After incubation for 2 hours, the sample was purged using Sephadex G25 column reaction solution to a buffer solution at pH 6.5 containing 50 mM sodium chloride and 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate.

[0532] Antibody-drug-linker coupling and hydrolysis: The reduced antibody was diluted to 5 mg / mL, and 0.38 mL of N,N-dimethylacetamide (DMA) (2% of the total reaction volume) was added as a pre-solvent. A DMA-drug-linker mixture containing 10 mg / mL of drug-linker was added at an antibody-small molecule drug molar ratio of 1:5.5 as the reaction solution. The mixture was stirred at room temperature for 30 minutes. The reaction solution was then replaced with a pH 8.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer using a Sephadex G25 support NAP-25 column to remove excess drug-linker. The mixture was then heated in a 37°C water bath for 3 hours.

[0533] Purification of the antibody-drug conjugate: The above sample was concentrated using an AMICOM ultrafiltration centrifuge tube to approximately 15 mg / mL. A 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 3 M ammonium phosphate buffer solution was added until the conductivity reached 100 mS / cm. The sample was loaded onto a TOYOPEAL Butyl-650M hydrophobic column (purchased from TOSOH). Phase A consisted of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate + 0.6 M ammonium sulfate, and Phase B consisted of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution. Elution was performed using an 8-fold column volume gradient from 0-100% in Phase B, and the main peak was collected.

[0534] The final sample was replaced with a 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 using an AMICOM ultrafiltration centrifuge tube and filtered using a 0.22 μm filter membrane (Sartorius stedim Ministart).

[0535] 1.2 General preparation method for random coupling

[0536]

[0537] It was prepared according to the preparation method disclosed in patent CN105849126A.

[0538] 1.3 Preparation method of control ADC IMGC936

[0539] The ADC IMGC936 was prepared according to the method described in patent application publication CN112543770A (CN201980049672.2).

[0540] 2. General analytical methods for antibody-drug conjugates

[0541] 2.1 Determination of Antibody-Drug Conjugate Ratio and Concentration by Ultraviolet Spectrophotometry (UV-DAR Method)

[0542] According to the method described in patent application publication WO2023109965A1, the antibody-drug conjugation ratio (DAR value) and concentration were determined by ultraviolet spectrophotometry. The molar absorption coefficients of each drug-containing linker at 280 nm and its characteristic wavelength are shown below, where... The molar absorption coefficient of the drug-containing linker at its characteristic absorption wavelength Z nm is given. The molar absorption coefficient of the drug-containing linker at 280 nm is given.

[0543]

[0544] 2.2 Hydrophobic Chromatography

[0545] a. Determination of DAR value of antibody-drug conjugates by hydrophobic chromatography-HIC-HPLC

[0546] 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.

[0547] Chromatographic column: Sepax Proteomix HIC Butyl-NP5, 5μm, 4.6mm*35mm;

[0548] Mobile phase A: 1.5M (NH4)2SO4 + 25mM PB, pH 7.0

[0549] Mobile phase B: 25 mM PB + 20% IPA, pH 7.0

[0550] Flow rate: 0.6 mL / min;

[0551] Detection wavelength: 280nm;

[0552] Column temperature: 30℃;

[0553] Sample loading volume: 10 μL;

[0554] HIC chromatographic gradient:

[0555] Time (min) ％A ％B 0 100 0 10 80 20 20 40 60 25 0 100 25.1 100 0 35 100 0

[0556] DAR calculation formula:

[0557] DAR = ∑(weighted peak area) / 100, that is, 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.

[0558] b. Determination of DAR value of antibody-drug conjugates by hydrophobic chromatography-HIC-HPLC

[0559] 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.

[0560] Chromatographic column: Sepax Proteomix HIC Butyl-NP5, 5μm, 4.6mm*35mm;

[0561] Mobile phase A: 1.2M (NH4)2SO4 + 25mM PB, pH 7.0

[0562] Mobile phase B: 25 mM PB + 20% IPA, pH 7.0

[0563] Flow rate: 0.6 mL / min;

[0564] Detection wavelength: 280nm;

[0565] Column temperature: 30℃;

[0566] Sample loading volume: 10 μL;

[0567] HIC chromatographic gradient

[0568] Time (min) ％A ％B 0 100 0 1 100 0 20 0 100 20.1 100 0 30 100 0

[0569] DAR calculation formula: same as a.

[0570] 2.3 Size Exclusion Chromatography-HPLC for Determination of Molecular Size Heterogeneity

[0571] Sample preparation: The sample was diluted with the mobile phase to about 1.0 mg / ml, centrifuged at 12000 rpm for 10 min, and the supernatant was injected for analysis.

[0572] Chromatographic column: TOSOH, TSKgel G3000SWXL, 5μm, 7.8mm*300mm;

[0573] Mobile phase: 100 mM PB + 200 mM arginine hydrochloride, 5% isopropanol (pH 6.8);

[0574] Flow rate: 0.6 mL / min;

[0575] Detection wavelength: 280nm;

[0576] Column temperature: 30℃;

[0577] Sample loading volume: 20 μL;

[0578] Washing time: 20 min;

[0579] Elution gradient: isocratic elution.

[0580] 3. Preparation and characterization of antibody-drug conjugates targeting ADAM9

[0581] According to the general preparation method described above in this embodiment, anti-ADAM9 antibodies hz5H6Lm1, hz5H6Lm3, Mab-A, hz5H6, hz5H6Hm6Lm14, hz5H6Hm6Lm14-LALA, and hz5H6Lm8 were taken and coupled with drug-containing linkers to prepare ADCs.

[0582] According to the preparation method of the control ADC IMGC936 described above in this embodiment, anti-ADAM9 antibody Mab-A was conjugated with DM21-C to prepare control ADC IMGC936 (i.e., ADAM9-ADC-14 in Table 14).

[0583] The DAR value, concentration, and purity of the site-coupled ADC were determined using the ultraviolet spectrophotometry, hydrophobic chromatography, and size exclusion chromatography methods described in this embodiment.

[0584] The DAR and purity of the control ADC IMGC936 were determined using size exclusion chromatography (SEC-HPLC) in this embodiment; 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 General Chapter 0731 "Protein Content Determination" of the Chinese Pharmacopoeia 2015 edition.

[0585] The results are shown in Table 14.

[0586] Table 14. Characterization results of antibody-drug conjugates

[0587]

[0588]

[0589] Example 25 Activity evaluation of antibody-drug conjugates targeting ADAM9

[0590] The cell density of the ADAM9-overexpressing cell line HCT15 (purchased from ATCC) was adjusted to 1.5 × 10⁻⁶ cells using complete culture medium. 4 Add 100 μl / well to a 96-well cell culture plate and incubate overnight.

[0591] The ADAM9-targeting ADC was diluted to 20 μg / ml using complete culture medium, followed by 4-fold serial dilutions, for a total of 8 serial dilutions. 50 μL of the diluted ADC was added to each well of a 96-well cell culture plate containing HCT15 cells, followed by 150 μL of complete culture medium. All samples were prepared in duplicate. Negative control wells (cells + culture medium) and blank control wells (cell-free, culture medium only) were also included in the cell culture plate. After incubation for 168 hours, the cell culture plate was removed, the supernatant was discarded, and 100 μL of CCK-8 was added to each well. The plate was incubated at 37°C for 1–4 hours. The cell culture plate was then removed, and the OD value was read at 450 nm.

[0592] The results are shown in Table 15 and... Figure 44 The results showed that the ADAM9-ADC-8, which targets ADAM9, was significantly more effective at killing ADAM9-expressing HCT15 cells than the control ADC IMGC936 (ADAM9-ADC-14).

[0593] Table 15. In vitro cytotoxic activity of ADAM9-targeting ADCs

[0594]

[0595] Example 26 Evaluation of drug resistance characteristics of antibody-drug conjugates targeting ADAM9

[0596] Multidrug residence (MDR) was first discovered in tumor cells. This phenomenon refers to the phenomenon where drug-sensitive tumor cells, after long-term treatment with a single antitumor drug, develop resistance to that drug and also become less sensitive to other types of antitumor drugs. Assessing whether tumor cells exhibit drug resistance is crucial for evaluating the efficacy of that drug.

[0597] P-glycoprotein (p-gp), also known as ABCB1, mediates drug resistance (MDR), a classic MDR pathway known in the art. It is generally believed that the drug efflux mediated by this protein leads to drug resistance in the body. In this embodiment, the p-gp inhibitor Tariquidar was used to detect whether the ADC of the present invention is easily effluxed through p-gp, thereby understanding its drug resistance characteristics.

[0598] The cell density of the ADAM9-overexpressing HCT15 cell line was adjusted to 1.5 × 10⁻⁶ cells using complete culture medium. 4 Add 100 μl / well to each of the 100 cells / ml of the ADAM9-targeted ADC to a 96-well cell culture plate and incubate overnight. Dilute the ADAM9-targeted ADC to 20 μg / ml using complete culture medium, then perform 4-fold serial dilutions, for a total of 8 serial dilutions. Set up the following experimental groups:

[0599] No inhibitor group: 50 μl of complete culture medium was added to the above 96-well cell culture plate containing HCT15 cells and incubated at 37°C for 1 hour. Then, 50 μl of diluted ADC was added to the 96-well cell culture plate and mixed. All samples were in duplicate.

[0600] Inhibitor group: The dilution of the drug-resistant protein p-gp inhibitor Tariquidar in complete culture medium was added at 50 μl / well to the above-mentioned 96-well cell culture plate containing HCT15 cells, so that the concentration in the final volume of 200 μl was 200 nM. The plate was incubated at 37°C for 1 hour. Then, the diluted ADC was added at 50 μl / well to the 96-well cell culture plate and mixed. All samples were in duplicate.

[0601] Simultaneously, negative control wells (cells + culture medium) and blank control wells (cells-free, culture medium only) were set up in the cell culture plate. After incubation for 168 hours, the cell culture plate was removed, the supernatant was discarded, and 100 μl of CCK-8 was added to each well. The reaction was carried out at 37°C for 1-4 hours. The cell culture plate was then removed, and the OD value was read at 450 nm.

[0602] The results are shown in Table 16 and... Figure 45 The results showed that in the p-gp-highly expressed cell line HCT15, the activity of the control ADC ADAM9-ADC-14 was affected by p-gp expression: without p-gp inhibitors, the control ADC had no cytotoxic activity, while with the addition of p-gp inhibitors, the cytotoxic activity of the control ADC increased significantly, with an EC50 of 184.3 ng / mL. In contrast, the cytotoxic activity of the ADC ADAM9-ADC-8 of this invention was not affected by p-gp protein expression. Therefore, it can be concluded that ADAM9-ADC-8 is not a p-gp substrate, while ADAM9-ADC-14 is a p-gp substrate and is more likely to induce drug resistance.

[0603] Table 16. Drug resistance characteristics of ADAM9-targeting ADCs

[0604]

[0605] Example 27 Pharmacodynamic evaluation of antibody-drug conjugates targeting ADAM9 in a Balb / C-nude mouse model of human colorectal cancer

[0606] The small molecule cytotoxic compound DXD was coupled with hz5H6Hm6Lm14 via a drug-containing linker to prepare an ADC molecule, named "hz5H6Hm6Lm14-GGFG-DXD(ADAM9-ADC-17)". The efficacy of hz5H6Hm6Lm14-GGFG-DXD(ADAM9-ADC-17) and hz5H6Hm6Lm14-MF-L6(ADAM9-ADC-16) was evaluated.

[0607] Human colorectal cancer RKO cells were subcutaneously in the right anterior flank of Balb / C-nude mice. The cells were allowed to grow to 200 mm. 3 The animals were divided into groups of 6 for each group. Two doses (5 and 2.5 mg / kg) were set for each antibody-drug conjugate, and the drugs were administered once per intraperitoneal (IP).

[0608] Tumor growth inhibition status, such as Figure 46 As shown in the figure. The results indicate that different toxin ADCs all have clear antitumor effects in the RKO colorectal cancer model. hz5H6Hm6Lm14-MF-L6 showed superior antitumor effects.

[0609] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

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 sequence; and L-CDRs of the amino acid sequences set forth in SEQ ID NO. 51, SEQ ID NO. 49, SEQ ID NO. 47, in sequence; (20) H-CDRs of the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 44, in sequence; and L-CDRs of the amino acid sequences set forth in SEQ ID NO. 51, SEQ ID NO. 49, SEQ ID NO. 47, in sequence; (21) H-CDRs of the amino acid sequences set forth in SEQ ID NO. 36, SEQ ID NO. 41, SEQ ID NO. 38, in sequence; and L-CDRs of the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 53, in sequence; 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 sequence; and L-CDRs of the amino acid sequences set forth in SEQ ID NO. 45, SEQ ID NO. 49, SEQ ID NO. 53, in sequence.

2. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of claim 1.

3. A vector comprising the nucleic acid molecule of claim 2.

4. A host cell comprising the nucleic acid molecule of claim 2 or the vector of claim 3.

5. An antibody drug conjugate targeting ADAM9 or a salt thereof, comprising the anti-ADAM9 antibody or antigen-binding fragment thereof of claim 1; Preferably, the antibody drug conjugate or a salt thereof is formed by conjugating the antibody or antigen-binding fragment thereof of claim 1 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 a salt thereof is formed by conjugating the antibody or antigen-binding fragment thereof of claim 1 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; Further preferably, the antibody drug conjugate or salt thereof has the structure as shown, wherein: Ab represents an anti-ADAM9 antibody or antigen-binding fragment thereof according to claim 1 ; E L selected from the group consisting of represents the attachment to Ab via the thiol group 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 chemical 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 of the amino terminus of the short peptide structure of the group A; m is 1-10, preferably 1-8 (e.g. 1-5), more preferably 3-8; and m can be an integer or a non-integer; and D represents the small molecule cytotoxic compound.

6. A composition comprising the antibody or antigen-binding fragment thereof of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, the host cell of claim 4, or the antibody drug conjugate of claim 5, or a salt thereof, and optionally a pharmaceutically acceptable excipient.

7. Use of the antibody or antigen-binding fragment thereof of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, the host cell of claim 4, the antibody drug conjugate of claim 5, or a salt thereof, and / or the composition of claim 6 in the manufacture of a medicament for preventing, treating and / or ameliorating a disease or a disorder.

8. Use of the antibody or antigen-binding fragment thereof of claim 1 in the manufacture of an antibody drug conjugate for preventing, treating and / or ameliorating a disease or a disorder.

9. Use of the antibody or antigen-binding fragment thereof of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, the host cell of claim 4, the antibody drug conjugate of claim 5, or a salt thereof, and / or the composition of claim 6 in the manufacture of a reagent for diagnosing a disease or a disorder. 10.A kit comprising the antibody or antigen binding fragment thereof of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, the host cell of claim 4, the antibody drug conjugate of claim 5 or a salt thereof, and / or the composition of claim 6.

Images