Antibody-drug conjugates, their production methods, and uses

Abstract

The present application relates to an antibody-drug conjugate, a method for producing the same, and uses thereof. Specifically, the present application relates to an antibody-drug conjugate for use in treating B7-H3-positive tumors. The antibody-drug conjugate of the present application comprises a B7-H3 antibody, which has excellent binding activity to B7-H3-positive cells and can efficiently transport drugs to B7-H3-positive cells. The antibody-drug conjugate of the present application has an excellent drug-antibody binding ratio and exhibits excellent targeted killing effects against tumors such as lung cancer, breast cancer, gastric cancer, and colon cancer. Therefore, the present application further provides a method for producing the antibody-drug conjugate and its use in treating B7-H3-positive tumors.

Description

[Technical Field]

[0001] This application relates to the field of targeted therapy, specifically to antibody-drug conjugates for use in treating B7-H3-positive tumors. Specifically, this application provides antibody-drug conjugates of B7-H3 antibodies that have excellent binding activity to B7-H3-positive cells and can efficiently deliver drugs to B7-H3-positive cells. The resulting antibody-drug conjugates have excellent drug-antibody binding ratios and excellent targeted killing effects against tumors such as breast cancer, colon cancer, and gastric cancer. Therefore, this application also provides methods for producing the antibody-drug conjugates and their use in treating B7-H3-positive tumors. [Background technology]

[0002] Oncology is a serious disease field, and research and development of anti-cancer drugs has attracted worldwide attention and posed a challenging challenge. Potential indications for B7-H3 include solid tumors with high incidence rates, such as colorectal cancer, gastric cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, melanoma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), kidney cancer, bladder cancer, thyroid cancer, mesothelioma, pancreatic cancer, ovarian cancer, endometrial cancer, esophageal cancer, liver cancer, salivary gland cancer, bile duct cancer, and meningioma. This disease field is experiencing significant clinical demand and is a focus of major pharmaceutical companies.

[0003] B7-H3 (CD276) is a type I transmembrane protein (45-66 kD) located on human chromosome 15. It is a costimulatory molecule of the B7 family and shares 20-27% amino acid sequence identity with other family members. Structurally, B7-H3 contains an extracellular IgV / IgC tandem repeat sequence, a transmembrane domain, and an intracellular domain (similar to PD-L1). Based on the number of extracellular tandem repeat units, two forms of B7-H3 have been identified: 2Ig-B7-H3 and 4Ig-B7-H3 (with one extra IgV / C repeat unit), although 4Ig-B7-H3 is considered the more common form. Studies have shown that matrix metalloproteinases can lyse 2Ig-B7-H3 to form a serum-free form. Although B7-H3 mRNA is distributed relatively widely, no positive expression has been detected in lymphoid organs such as the spleen, lymph nodes, bone marrow, or thymus. However, B7-H3 protein is constitutively expressed only in non-immune resting fibrocytes, endothelial cells, osteoblasts, and amniotic fluid stem cells, and is inducibly expressed in activated T, NK, DC, and macrophages. In normal tissues, IHC shows negative expression in many tissues, but low to moderate antigen expression is detected in tissues such as the pancreas, liver, colon, stomach, placenta, skin, and adrenal gland.

[0004] Studies have shown that B7-H3 mRNA is overexpressed in a variety of tumors, including breast cancer, colorectal cancer, head and neck cancer, clear cell renal cell carcinoma, papillary renal cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, and thyroid cancer. Numerous studies have demonstrated that B7-H3 plays an important role in tumor progression, including promoting tumor growth / metastasis, mediating tumor cell metastasis (EMT) transformation, and influencing tumor cell metabolism. B7-H3 expression is regulated by oncogenes, and elevated B7-H3 promotes tumor growth and metastasis through various signaling pathways. Currently, companies focusing on this target are relatively focused: Macrogenics focuses on monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates (ADCs); Daiichi Sankyo focuses on monoclonal antibodies and ADCs; Y-mAbs focuses on monoclonal antibodies, bispecific antibodies, and ADCs; and AbbVie and GT Biopharma focus on ADCs and carrier-linked bispecific antibodies, respectively. Preclinical studies have shown that B7-H3 expression is elevated in tumors, macrophages, and dendritic cells of cancer patients compared to normal tissues, and tumor growth was significantly suppressed (approximately 50%) when tumor cells were inoculated into B7-H3 knockout mice. DS7300, an antibody-drug conjugate targeting B7-H3, developed by Daiichi Sankyo, has demonstrated good safety and initial efficacy in Phase I clinical trials. Summary of the Invention [Problem to be solved by the invention]

[0005] Macromolecular biopharmaceuticals have already become an important component of anticancer drugs and are showing a rapid growth trend. Antibody-drug conjugates are powerful anticancer drugs that pinpoint cancer cells and are composed of a monoclonal antibody and a highly toxic drug linked via a biologically active linker. The ability to accurately identify the target significantly improves efficacy and reduces toxicity and side effects, making them one of the main research directions for future anticancer drugs. [Means for solving the problem]

[0006] This application relates to antibody-drug conjugates for the treatment of B7-H3 positive tumors, with the targeting moiety being fully humanized antibody 2#8890, and having the general formula Ab-[MLED] x The present application exemplarily discloses an antibody-drug conjugate having the structure shown in Figure 1. The results showed that the conjugate has an excellent drug-antibody binding ratio, excellent binding activity to B7-H3-positive cells, and excellent targeted killing effect against B7-H3-positive tumors, such as non-small cell lung cancer, glioma, breast cancer, gastric cancer, and colon cancer. Therefore, the present application provides an antibody-drug conjugate for use in treating B7-H3-positive tumors, a pharmaceutical composition containing the antibody-drug conjugate, and their use in treating B7-H3-positive tumors.

[0007] antibody-drug conjugates In one aspect, the present application provides a compound of formula Ab-[MLED] x The invention provides an antibody-drug conjugate having the structure: An Ab is an antibody or antigen-binding fragment thereof that specifically binds to B7-H3. M is the attachment site to the antibody or antigen-binding fragment thereof. L is a linker connecting the connecting sites M and E. E is a structural fragment that connects L and D. D is the payload fragment. x is selected from 1 to 10.

[0008] In this application, the inventors have developed a high-affinity fully human antibody with excellent properties that can specifically identify and bind to B7-H3, does not bind to or essentially does not bind to B7-1, B7-2, B7-H1, B7-H2 and / or B7-H4, and has no ADCC activity, thereby effectively avoiding side effects caused by ADCC function.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): (a) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1, and / or CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO: 2; (b) CDR-H1, CDR-H2, CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 3, and / or CDR-L1, CDR-L2, CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO: 4; or (c) CDR-H1, CDR-H2, CDR-H3 contained in the heavy chain variable region (VH), and / or CDR-L1, CDR-L2, CDR-L3 contained in the light chain variable region (VL), which contain a mutation in at least one CDR that is a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of one, two, or three amino acids) compared to the heavy chain variable region and / or light chain variable region described in either (a) or (b).

[0010] In some embodiments, the substitution is a conservative substitution.

[0011] In some embodiments, the CDRs are defined according to the IMGT, Kabat, Chothia, or AbM numbering scheme.

[0012] In some embodiments, the B7-H3 comprises human B7-H3 and / or monkey B7-H3. In some embodiments, the monkey is a rhesus monkey (Macaca mulatta).

[0013] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and / or a light chain variable region (VL) whose CDRs are defined according to the IMGT numbering scheme as follows: (1a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 5 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 6 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 8 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 9 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; or (1b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 18 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 19 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 20 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 8 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 9 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; Here, the variant described in either (1a) or (1b) has one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, and preferably, the substitutions are conservative substitutions.

[0014] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and / or a light chain variable region (VL) whose CDRs are defined according to the Chothia numbering scheme as follows: (2a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 11 or a variant thereof; CDR-H2 whose sequence is SEQ ID NO: 12 or a variant thereof; and CDR-H3 whose sequence is SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14 or a variant thereof; CDR-L2 whose sequence is SEQ ID NO: 15 or a variant thereof; and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; or (2b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 21 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 22 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 15 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; Here, the variant described in either (2a) or (2b) has one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, and preferably, the substitutions are conservative substitutions.

[0015] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the Kabat numbering scheme as follows: (3a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 16 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 17 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 15 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; or (3b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 24 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 25 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 15 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; Here, the variant described in either (3a) or (3b) has one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, and preferably, the substitutions are conservative substitutions.

[0016] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and / or a light chain variable region (VL) whose CDRs are defined according to the AbM numbering scheme as follows: (4a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 26 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 27 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 15 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; or (4b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence of SEQ ID NO: 28 or a variant thereof, CDR-H2 having the sequence of SEQ ID NO: 29 or a variant thereof, and CDR-H3 having the sequence of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence of SEQ ID NO: 14 or a variant thereof, CDR-L2 having the sequence of SEQ ID NO: 15 or a variant thereof, and CDR-L3 having the sequence of SEQ ID NO: 10 or a variant thereof; Here, the variant described in either (4a) or (4b) has one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, and preferably, the substitutions are conservative substitutions.

[0017] In some embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain variable region (VH) and / or a light chain variable region (VL): (1a) a heavy chain variable region (VH) comprising three CDRs, CDR-H1 having the sequence of SEQ ID NO: 5, CDR-H2 having the sequence of SEQ ID NO: 6, and CDR-H3 having the sequence of SEQ ID NO: 7, and / or a light chain variable region (VL) comprising three CDRs, CDR-L1 having the sequence of SEQ ID NO: 8, CDR-L2 having the sequence of SEQ ID NO: 9, and CDR-L3 having the sequence of SEQ ID NO: 10; or (1b) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 18, CDR-H2 whose sequence is SEQ ID NO: 19, and CDR-H3 whose sequence is SEQ ID NO: 20; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 8, CDR-L2 whose sequence is SEQ ID NO: 9, and CDR-L3 whose sequence is SEQ ID NO: 10.

[0018] In some embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain variable region (VH) and / or a light chain variable region (VL): (2a) a heavy chain variable region (VH) comprising three CDRs, namely, CDR-H1 having the sequence of SEQ ID NO: 11, CDR-H2 having the sequence of SEQ ID NO: 12, and CDR-H3 having the sequence of SEQ ID NO: 13, and / or a light chain variable region (VL) comprising three CDRs, namely, CDR-L1 having the sequence of SEQ ID NO: 14, CDR-L2 having the sequence of SEQ ID NO: 15, and CDR-L3 having the sequence of SEQ ID NO: 10; or (2b) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 21, CDR-H2 whose sequence is SEQ ID NO: 22, and CDR-H3 whose sequence is SEQ ID NO: 23; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14, CDR-L2 whose sequence is SEQ ID NO: 15, and CDR-L3 whose sequence is SEQ ID NO: 10.

[0019] In some embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain variable region (VH) and / or a light chain variable region (VL): (3a) a heavy chain variable region (VH) comprising three CDRs, CDR-H1 having the sequence of SEQ ID NO: 16, CDR-H2 having the sequence of SEQ ID NO: 17, and CDR-H3 having the sequence of SEQ ID NO: 13, and / or a light chain variable region (VL) comprising three CDRs, CDR-L1 having the sequence of SEQ ID NO: 14, CDR-L2 having the sequence of SEQ ID NO: 15, and CDR-L3 having the sequence of SEQ ID NO: 10; or (3b) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 24, CDR-H2 whose sequence is SEQ ID NO: 25, and CDR-H3 whose sequence is SEQ ID NO: 23; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14, CDR-L2 whose sequence is SEQ ID NO: 15, and CDR-L3 whose sequence is SEQ ID NO: 10.

[0020] In some embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain variable region (VH) and / or a light chain variable region (VL): (4a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 having the sequence of SEQ ID NO: 26, CDR-H2 having the sequence of SEQ ID NO: 27, and CDR-H3 having the sequence of SEQ ID NO: 13; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 having the sequence of SEQ ID NO: 14, CDR-L2 having the sequence of SEQ ID NO: 15, and CDR-L3 having the sequence of SEQ ID NO: 10; or (4b) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 28, CDR-H2 whose sequence is SEQ ID NO: 29, and CDR-H3 whose sequence is SEQ ID NO: 23; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14, CDR-L2 whose sequence is SEQ ID NO: 15, and CDR-L3 whose sequence is SEQ ID NO: 10.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 5, CDR-H2 whose sequence is SEQ ID NO: 6, and CDR-H3 whose sequence is SEQ ID NO: 7; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 8, CDR-L2 whose sequence is SEQ ID NO: 9, and CDR-L3 whose sequence is SEQ ID NO: 10.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 18, CDR-H2 whose sequence is SEQ ID NO: 19, and CDR-H3 whose sequence is SEQ ID NO: 20; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 8, CDR-L2 whose sequence is SEQ ID NO: 9, and CDR-L3 whose sequence is SEQ ID NO: 10.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 11, CDR-H2 whose sequence is SEQ ID NO: 12, and CDR-H3 whose sequence is SEQ ID NO: 13; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14, CDR-L2 whose sequence is SEQ ID NO: 15, and CDR-L3 whose sequence is SEQ ID NO: 10.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 21, CDR-H2 whose sequence is SEQ ID NO: 22, and CDR-H3 whose sequence is SEQ ID NO: 23; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14, CDR-L2 whose sequence is SEQ ID NO: 15, and CDR-L3 whose sequence is SEQ ID NO: 10.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 16, CDR-H2 whose sequence is SEQ ID NO: 17, and CDR-H3 whose sequence is SEQ ID NO: 13; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14, CDR-L2 whose sequence is SEQ ID NO: 15, and CDR-L3 whose sequence is SEQ ID NO: 10.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 24, CDR-H2 whose sequence is SEQ ID NO: 25, and CDR-H3 whose sequence is SEQ ID NO: 23; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14, CDR-L2 whose sequence is SEQ ID NO: 15, and CDR-L3 whose sequence is SEQ ID NO: 10.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 26, CDR-H2 whose sequence is SEQ ID NO: 27, and CDR-H3 whose sequence is SEQ ID NO: 13; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14, CDR-L2 whose sequence is SEQ ID NO: 15, and CDR-L3 whose sequence is SEQ ID NO: 10.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 28, CDR-H2 whose sequence is SEQ ID NO: 29, and CDR-H3 whose sequence is SEQ ID NO: 23; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14, CDR-L2 whose sequence is SEQ ID NO: 15, and CDR-L3 whose sequence is SEQ ID NO: 10.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: (a) a VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; or (b) a VH comprising the sequence shown in SEQ ID NO: 3 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 4 or a variant thereof; Here, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) with respect to the sequence from which it is derived, and preferably, the substitutions are conservative substitutions.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof according to any of the above embodiments may comprise a constant region originating from or derived from a human immunoglobulin.

[0031] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region originating from or derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a wild-type Fc region or a mutated or chemically modified Fc region having altered effector function (e.g., reduced ADCC activity) compared to the wild-type Fc region. In some exemplary embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a mutant of the human IgG1 heavy chain constant region, which has substitutions of Leu234Ala, Leu235Ala, and Gly237Ala (positions according to the EU numbering scheme) compared to the wild-type sequence from which it is derived. In such embodiments, the antibody or antigen-binding fragment thereof of the present invention has reduced ADCC activity. In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 30 or a variant thereof, wherein the variant has 20 or fewer conservative amino acid substitutions (e.g., 15 or fewer, 10 or fewer, or 5 or fewer conservative amino acid substitutions, e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared to the sequence set forth in SEQ ID NO: 31 or a variant thereof, wherein the variant has 20 or fewer conservative amino acid substitutions (e.g., 15 or fewer, 10 or fewer, or 5 or fewer conservative amino acid substitutions, e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared to the sequence set forth in SEQ ID NO: 31.

[0032] In some embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region originating from or derived from a human immunoglobulin (e.g., kappa or lambda). In some embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 32 or a variant thereof, wherein the variant has 20 or fewer conservative amino acid substitutions (e.g., 15 or fewer, 10 or fewer, or 5 or fewer conservative amino acid substitutions, e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared thereto.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: (1) a heavy chain comprising a VH sequence set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) set forth in SEQ ID NO: 30, and a light chain comprising a VL sequence set forth in SEQ ID NO: 2 and a light chain constant region (CL) set forth in SEQ ID NO: 32; (2) a heavy chain comprising a VH of SEQ ID NO: 3 and a heavy chain constant region (CH) of SEQ ID NO: 30, and a light chain comprising a VL of the sequence of SEQ ID NO: 4 and a light chain constant region (CL) of SEQ ID NO: 32; (3) a heavy chain comprising a VH of SEQ ID NO: 1 and a heavy chain constant region (CH) of SEQ ID NO: 31, and a light chain comprising a VL of the sequence of SEQ ID NO: 2 and a light chain constant region (CL) of SEQ ID NO: 32; Or, (4) A heavy chain comprising a VH of sequence number 3 and a heavy chain constant region (CH) of sequence number 31, and a light chain comprising a VL of sequence number 4 and a light chain constant region (CL) of sequence number 32.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises: (1) a heavy chain having the sequence set forth in SEQ ID NO: 40 and a light chain having the sequence set forth in SEQ ID NO: 41; or (2) A heavy chain having the sequence shown in SEQ ID NO: 42 and a light chain having the sequence shown in SEQ ID NO: 43.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof of any of the above embodiments is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0036] In some embodiments, the variable region of the antibody or antigen-binding fragment thereof of any of the above embodiments is of human origin.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof of any of the above embodiments is selected from an ScFv, a Fab, a Fab', a (Fab')2, a Fab'-SH, an Fv fragment, a disulfide-linked Fv (dsFv), a diabody, a bispecific antibody, and a multispecific antibody.

[0038] In specific embodiments of the antibodies or antibody-drug conjugates disclosed herein, the heavy chain constant region may include a C-terminal lysine, or may lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

[0039] In some embodiments, the N-terminal amino acid of the variable region of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid.

[0040] Thus, a composition may comprise a group of antibody-drug conjugates in which the antibody or antigen-binding fragment thereof of each antibody-drug conjugate independently comprises a C-terminal lysine, lacks a C-terminal lysine, lacks a C-terminal glycine-lysine, and / or comprises an N-terminal glutamine or glutamic acid, or the N-terminal amino acid may be cyclized to pyroglutamic acid.

[0041] Therefore, in specific embodiments, the present invention further provides compositions comprising the antibody-drug conjugates disclosed herein, wherein the main antibody-drug conjugate comprises: (i) an antibody lacking a lysine residue at the C-terminus of its heavy chain; (ii) an antibody whose heavy chain N-terminus is glutamine, glutamic acid, or pyroglutamic acid; (iii) an antibody whose heavy chain C-terminus is lacking a lysine residue and whose heavy chain N-terminus is glutamine, glutamic acid, or pyroglutamic acid; (iv) an antibody whose heavy chain C-terminus is lacking a lysine residue and whose heavy chain N-terminus is a pyroglutamic acid residue; or (v) an antibody whose heavy chain C-terminus is lacking a lysine residue and whose heavy chain N-terminus is glutamine or glutamic acid residue.

[0042] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution. Therefore, the present application further provides a composition comprising one or more antibody-drug conjugates described in any one of the above.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region encoded by a nucleic acid molecule having (i) the nucleotide sequence set forth in SEQ ID NO: 33, (ii) a sequence essentially the same as SEQ ID NO: 33 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 33, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above; and / or the nucleic acid molecule encoding the antibody light chain variable region comprises (iv) the nucleotide sequence set forth in SEQ ID NO: 34, (v) a sequence essentially the same as SEQ ID NO: 34 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 34, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v) above.

[0044] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof, of the present invention. Due to codon degeneracy in the art, in some embodiments, the nucleotide sequence can be substituted by codon degeneracy. In some embodiments, the nucleotide sequence is codon-optimized.

[0045] In some embodiments, the isolated nucleic acid molecule comprises (i) a nucleic acid molecule encoding a heavy chain variable region of an antibody, comprising: (i) the nucleotide sequence set forth in SEQ ID NO: 33; (ii) a sequence essentially the same as SEQ ID NO: 33 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 33, or a sequence having one or more nucleotide substitutions); or (iii) a degenerate sequence of (i) or (ii) above; and / or (iv) a nucleic acid molecule encoding a light chain variable region of an antibody, comprising: (v) a sequence essentially the same as SEQ ID NO: 34 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 34, or a sequence having one or more nucleotide substitutions); or (vi) a nucleic acid molecule encoding a light chain variable region of an antibody, comprising: (i) the nucleotide sequence set forth in SEQ ID NO: 34; (v) a sequence essentially the same as SEQ ID NO: 34 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 34, or a sequence having one or more nucleotide substitutions); or (vi) a degenerate sequence of (iv) or (v) above.

[0046] In some embodiments, the isolated nucleic acid molecule comprises (i) a nucleic acid molecule encoding a heavy chain variable region of an antibody, comprising the nucleotide sequence set forth in SEQ ID NO: 35, (ii) a sequence essentially the same as SEQ ID NO: 35 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 35, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above, and / or (iv) a nucleic acid molecule encoding a light chain variable region of an antibody, comprising the nucleotide sequence set forth in SEQ ID NO: 36, (v) a sequence essentially the same as SEQ ID NO: 36 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 36, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v) above. In some embodiments, the antibody or antigen-binding fragment thereof comprising any of the characteristics (1a), (2a), (3a), (4a), or (a) in the above embodiments further has a characteristic selected from the following: (1) binds to B7-H3 (e.g., human or monkey B7-H3) with an EC50 of less than about 100 ng / mL, e.g., less than about 80 ng / mL, 50 ng / mL, 20 ng / mL, 15 ng / mL, 14 ng / mL, 13 ng / mL, 12 ng / mL, 11 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL, or less, preferably wherein the EC50 is measured by ELISA; (2) binds to B7-H3 (e.g., human or monkey B7-H3) with a KD of less than about 100 nM, e.g., less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 5 nM, or less, preferably as measured by biolayer interferometry (BLI) (e.g., ForteBio Octet®); (3) no or essentially no binding to B7-1, B7-2, B7-H1, B7-H2, and / or B7-H4, e.g., as measured by ELISA; (4) have CDC activity, e.g., induce killing of cells expressing B7-H3 (e.g., tumor cells) by CDC; (5) No ADCC activity; (6) inducing internalization of B7-H3, as measured, for example, by flow cytometry; (7) inhibiting cell (e.g., tumor cell) proliferation; and / or (8) Inhibit tumor growth.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof comprising any of the features (1b), (2b), (3b), (4b), or (b) in the above embodiments further comprises a feature selected from the following: (1) binds to B7-H3 (e.g., human or monkey B7-H3) with an EC50 of less than about 100 ng / mL, e.g., less than about 80 ng / mL, 50 ng / mL, 20 ng / mL, 15 ng / mL, 14 ng / mL, 13 ng / mL, 12 ng / mL, 11 ng / mL, 10 ng / mL, 9 ng / mL, 8 ng / mL, 7 ng / mL, 6 ng / mL, 5 ng / mL, 4 ng / mL, or less, preferably wherein the EC50 is measured by ELISA; (2) binds to B7-H3 (e.g., human or monkey B7-H3) with a KD of less than about 100 nM, e.g., less than about 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 5 nM, or less, preferably as measured by biolayer interferometry (BLI) (e.g., ForteBio Octet®); (3) no or essentially no binding to B7-1, B7-2, B7-H1, B7-H2, and / or B7-H4, e.g., as measured by ELISA; (4) have CDC activity, e.g., induce killing of cells expressing B7-H3 (e.g., tumor cells) by CDC; (5) No ADCC activity; (6) inducing internalization of B7-H3, as measured, for example, by flow cytometry; (7) inhibiting cell (e.g., tumor cell) proliferation; and / or (8) Inhibit tumor growth.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof of any of the above embodiments is labeled, ie, labeled with a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent material (such as a chemiluminescent material), or biotin.

[0049] Induced antibodies The antibodies or antigen-binding fragments thereof of the invention can be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to B7-H3 (particularly human B7-H3). Thus, the antibodies or antigen-binding fragments thereof of the invention are further intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof of the invention can be functionally linked (by chemical bond, genetic fusion, noncovalent bonding, or otherwise) to one or more other molecular groups, such as, for example, another antibody (e.g., formed bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or can mediate the attachment of the antibody or antigen-binding fragment to a protein or polypeptide (e.g., avidin or polyhistidine tag) to another molecule.

[0050] As derivatives of antibodies, the present invention provides conjugates comprising the antibodies of the invention or antigen-binding fragments and binding portions thereof.

[0051] In some embodiments, the binding moiety is selected from a detectable label. A detectable label according to the present invention can be any substance detectable by fluorescent, spectral, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, and examples include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., H, I, S, C, or P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine ester compounds, magnetic beads (e.g., Dynabeads®), calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such labels are applicable to immunological detection (e.g., ELISA, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In some embodiments, the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. In some embodiments, the detectable label may be linked to the antibody or antigen-binding fragment thereof of the present invention via a linker of different lengths to reduce potential steric hindrance.

[0052] In some embodiments, the binding moiety is selected from a therapeutic agent, which in some embodiments is preferably an anti-tumor agent, such as, for example, a cytotoxic drug, cytokine, toxin, or radionuclide.

[0053] In some embodiments, the binding moiety is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), and may be, for example, a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a glycosyl group.

[0054] As one type of antibody derivative, the present invention provides a multispecific antibody comprising an antibody of the present invention or an antigen-binding fragment thereof.

[0055] In some embodiments, the multispecific antibody comprises an antibody of the invention or an antigen-binding fragment thereof as a first antigen-binding domain, and further comprises at least one second antigen-binding domain against another target.

[0056] In some embodiments, each antigen-binding domain of the multispecific antibody retains its original binding specificity.

[0057] In some embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0058] As one of the antibody derivatives, the present invention provides a chimeric antigen receptor comprising the antibody of the present invention or an antigen-binding fragment thereof. In some embodiments, the chimeric antigen receptor comprises an antibody of the present invention or an antigen-binding fragment thereof (e.g., an ScFv) that is an extracellular antigen-binding domain that specifically binds to B7-H3, a transmembrane domain, and one or more intracellular T cell signaling domains. The present invention also provides host cells (e.g., immune cells such as T lymphocytes, NK cells, dendritic cells, and macrophages) that contain or express the chimeric antigen receptor.

[0059] The antibodies of the present invention can be prepared by various methods known in the art, for example, by recombinant genetic engineering techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0060] Antigen-binding fragments of the present invention can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). These antigen-binding fragments can also be produced directly by recombinant host cells (see Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). Thus, Fab' fragments can be obtained directly from host cells, and Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). Fv, Fab or F(ab')2 fragments can also be obtained directly by isolation from recombinant host cell culture. Those skilled in the art will know of other techniques for preparing these antigen-binding fragments.

[0061] In the antibody-drug conjugate, the payload can be linked to the antibody or antigen-binding fragment thereof by a linker (referred to herein as an "MLE" fragment).

[0062] In some embodiments, M is [ka] wherein ring A is a 5- to 6-membered aliphatic heterocycle or a 5- to 20-membered aromatic ring system, and the aliphatic heterocycle and aromatic ring system may optionally contain an oxygen group (=O), a halogen, a cyano group, an amino group, a carboxyl group, a thiol group, and C 1~6 alkyl group, and M is a single bond and C 1~20 Alkylene group, C 2~20 Alkenylene group, C 2~20 It is selected from an alkynylene group or an amine group.

[0063] In some embodiments, M is [ka] In the formula, ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycycle formed by connecting one or more (for example, two) 6-membered aromatic heterocycles with a benzene ring or a 6-membered heteroaromatic ring via a single bond, and the aliphatic heterocycle may optionally contain an oxygen group (═O), a halogen, and C 1~4 alkyl group, and M1 is a single bond, C 1~20 Alkylene group, C 2~20 Alkenylene group or C 2~20 It is selected from an alkynylene group or an amine group.

[0064] In some embodiments, M is [ka] wherein ring A is [ka] M is selected from a single bond and C 1~6 Alkylene group, C 2~6 Alkenylene group, C 2~6 It is selected from an alkynylene group or an amine group.

[0065] In some embodiments, M is [ka] is selected from.

[0066] In some embodiments, M is [ka] is selected from.

[0067] In some embodiments, M is [ka] is selected from.

[0068] In some embodiments, M is [ka] is selected from.

[0069] In some embodiments, L is C 1~6 Alkylene group, -N(R')-, carbonyl group, -O-, natural amino acids or unnatural amino acids and their analogs (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2) rOCH3), as well as short peptides consisting of amino acids (e.g., Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly- Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Va l-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), [ka] and wherein the structure is selected from one or more of: In the formula, R' is hydrogen, C 1~6 Alkyl groups or polyethylene glycol fragments having 1 to 10 EO units (i.e., -(CH2CH2O) r -an alkyl group, wherein r is selected from an integer of 1 to 10, and the alkyl group is a methyl group or an ethyl group; and s is selected from an integer of 1 to 20.

[0070] In some embodiments, L is C 1~6Alkylene group, carbonyl group, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gl y-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, G ly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, [ka] In the formula, s is selected from an integer of 1 to 20.

[0071] In some embodiments, L is [ka] [ka] The structure is selected from one or more of:

[0072] In some embodiments, L is [ka] The structure is selected from:

[0073] In some embodiments, L is [ka] The structure is selected from:

[0074] In some embodiments, L is [ka] The structure is selected from:

[0075] In some embodiments, L is [ka] The structure is selected from:

[0076] In some embodiments, E is a single bond, —NHCH 2 —, or [ka] The structure is selected from:

[0077] In some embodiments, E is a single bond, -NHCH2-, [ka] In some embodiments, E is -NHCH2- or [ka] is.

[0078] In some embodiments, E is —NHCH 2 —.

[0079] In some embodiments, E is a single bond. In some embodiments, E is [ka] In some embodiments, [ka] is selected from the following structures: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0080] In some embodiments, [ka] is selected from the following structures: [ka] [ka] [ka]

[0081] In some embodiments, the payload is selected from a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor. In some embodiments, the tubulin inhibitor is an auristatin compound or a maytansine compound. In some embodiments, the DNA intercalator is a pyrrolobenzodiazepine (PBD). In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, nogitecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., adriamycin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analog thereof.

[0082] The payloads disclosed herein typically include, for example, a hydroxyl group (-OH), a carboxyl group (-COOH), a thiol group (-SH), a primary amino group (-NH), a secondary amine group (-NR A H) or tertiary amine groups (-NR B R C )(wherein, R A , R B , R C represents a non-hydrogen substituent on N), and the payload can be linked to a linker in the conjugate by these functional groups.

[0083] In some embodiments, the payload is linked to E in the antibody-drug conjugate through an -OH, -SH, primary amino group, secondary amine group, or tertiary amine group thereon.

[0084] In some embodiments, the payload is selected from a compound of Formula I or Formula II, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, or prodrug of a compound of Formula I or Formula II. [ka] In the formula, R1 and R2 each independently represent C 1~6 It is selected from alkyl groups and halogens. R3 is selected from H and -CO-CH2OH. R4 and R5 are each independently selected from H, halogen and hydroxyl groups, or R4 and R5 combine with the carbon atoms to which they are attached to form a 5- to 6-membered oxygen-containing heterocycle. R6 is hydrogen or -C 1~4 Alkylene group -NR a R b is selected from. R7 is C 1~6 Alkyl groups and -C 1~4 Alkylene group -NR a R b is selected from. In the formula, R a , R b are independently H, C for each occurrence. 1~6 Alkyl group, -SO2-C 1~6 Alkyl groups and -CO-C 1~6 The alkyl group is selected from the group consisting of:

[0085] In some embodiments, the payload is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: [ka] [ka]

[0086] The fragment corresponding to the payload obtained after the payload is linked to a linker is D in the general formula, and preferably, D is a monovalent structure obtained by losing one H from an -OH, -NH2, or secondary amine group on the payload.

[0087] In some embodiments, the payload is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: [ka]

[0088] In some embodiments, the payload is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: [ka]

[0089] In some embodiments, D is selected from the following structures: [ka]

[0090] In some embodiments, the antibody-drug conjugate is selected from ADC A-01 to ADC A-34, ADC B-01 to ADC B-07, and ADC C-01 to ADC C-28 shown below. The Abs shown in the diagram below are defined as above, and the thiol group on the antibody forms a sulfide bond with the linker compound of the drug through an addition or substitution reaction to obtain a complete antibody-drug conjugate. x represents the drug-antibody ratio. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] wherein Ab in the antibody-drug conjugate represents any one of the antibodies or antigen-binding fragments described above. During the ceremony, [ka] represents a specific linking scheme between a thiol group in an antibody or antigen-binding fragment thereof and a linker.

[0091] In some embodiments, the Ab in each antibody-drug conjugate represents an antibody or antigen-binding fragment thereof comprising a VH set forth in SEQ ID NO: 3 and a VL set forth in SEQ ID NO: 4, e.g., an antibody or antigen-binding fragment thereof comprising a VH set forth in SEQ ID NO: 3 and a CH set forth in SEQ ID NO: 31, and a VL set forth in SEQ ID NO: 4 and a CL set forth in SEQ ID NO: 32.

[0092] In some embodiments, each antibody drug conjugate comprises a heavy chain set forth in SEQ ID NO:42 and a light chain set forth in SEQ ID NO:43.

[0093] In some embodiments, the antibody-drug conjugate is: [ka] In the formula, Ab in the antibody-drug conjugate represents an antibody or antigen-binding fragment thereof comprising VH shown in SEQ ID NO: 3 and VL shown in SEQ ID NO: 4, and x is an integer of 1 to 10. During the ceremony, [ka] represents a specific linking scheme between a thiol group in an antibody or antigen-binding fragment thereof and a linker.

[0094] In some embodiments, the antibody-drug conjugate is: [ka] In the formula, Ab in the antibody-drug conjugate represents an antibody comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising three CDRs, CDR-H1 having the sequence of SEQ ID NO: 18, CDR-H2 having the sequence of SEQ ID NO: 19, and CDR-H3 having the sequence of SEQ ID NO: 20, and / or a light chain variable region comprising three CDRs, CDR-L1 having the sequence of SEQ ID NO: 8, CDR-L2 having the sequence of SEQ ID NO: 9, and CDR-L3 having the sequence of SEQ ID NO: 10; or (ii) a heavy chain variable region comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 21, CDR-H2 whose sequence is SEQ ID NO: 22, and CDR-H3 whose sequence is SEQ ID NO: 23; and / or a light chain variable region comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14, CDR-L2 whose sequence is SEQ ID NO: 15, and CDR-L3 whose sequence is SEQ ID NO: 10; or (iii) a heavy chain variable region comprising three CDRs, namely, CDR-H1 having the sequence of SEQ ID NO: 24, CDR-H2 having the sequence of SEQ ID NO: 25, and CDR-H3 having the sequence of SEQ ID NO: 23, and / or a light chain variable region comprising three CDRs, namely, CDR-L1 having the sequence of SEQ ID NO: 14, CDR-L2 having the sequence of SEQ ID NO: 15, and CDR-L3 having the sequence of SEQ ID NO: 10; (iv) A heavy chain variable region comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 28, CDR-H2 whose sequence is SEQ ID NO: 29, and CDR-H3 whose sequence is SEQ ID NO: 23; and / or a light chain variable region comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14, CDR-L2 whose sequence is SEQ ID NO: 15, and CDR-L3 whose sequence is SEQ ID NO: 10. During the ceremony, [ka] represents a specific linking scheme between a thiol group in an antibody or antigen-binding fragment thereof and a linker.

[0095] In some embodiments, the antibody-drug conjugate is: [ka] In the formula, Ab in the antibody-drug conjugate represents an antibody or antigen-binding fragment thereof having a heavy chain having the sequence shown in SEQ ID NO: 42 and a light chain having the sequence shown in SEQ ID NO: 43, x is an integer of 1 to 10, and Ab forms a conjugate via one or more thiol bonds.

[0096] In some embodiments, x is an integer from 1 to 10, for example, x is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10.

[0097] In some embodiments, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0098] In some embodiments, the antibodies disclosed herein have been genetically engineered to substitute one or more cysteine ​​residues or non-standard amino acids, including amino acids, at specific sites within the antibody. Furthermore, these cysteine ​​residues or non-standard amino acid residues can be conjugated to a drug-linker via the thiol group of the cysteine ​​residue or the reactive group of the non-standard amino acid. Therefore, the antibody-drug conjugates of the present invention may comprise substituting one or more amino acids in the heavy or light chain of an antibody with a cysteine ​​residue or a non-standard amino acid residue, followed by conjugation with a drug-linker disclosed herein. In specific embodiments, the substitutable amino acid positions are selected from positions 152, 153, 171, 172, 173, and 375 (numbered according to the EU numbering scheme) in the heavy chain constant region and positions 165 and 168 in the light chain constant region (numbering begins with amino acid 1 at the N-terminus). In a specific embodiment, cysteine ​​may replace one or more amino acids at positions 152, 153, 171, 172, 173, and 375 in the heavy chain constant region (numbered according to the EU numbering scheme) and positions 165 and 168 in the light chain constant region (numbering starting from amino acid 1 at the N-terminus). In a specific embodiment, the antibody-drug conjugate comprises an S375C amino acid substitution conjugated to a drug linker disclosed herein. In a specific embodiment, the antibody comprises an S375C amino acid substitution and an E152C amino acid substitution, each conjugated to a drug linker disclosed herein. In a specific embodiment, the antibody comprises an S375C amino acid substitution and an S168C amino acid substitution, each conjugated to a drug linker disclosed herein.

[0099] In some embodiments, the DAR value (drug-antibody conjugate ratio) of the antibody-drug conjugate is 1 to 10, for example, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10, preferably 3 to 9, for example, 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.0 to 5.5, 3.0 to 6.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 3.5 ~5.5, 3.5~6.0, 3.5~6.5, 3.5~7.0, 3.5~7.5, 3.5~8.0, 4.0~4.5, 4.0~5.0, 4.0~5.5, 4.0~6.0, 4.0~6.5, 4.0~7.0, 4.0~7.5, 4.0~8.0, 4.5~5.0, 4.5~5.5, 4.5~6.0, 4.5~6.5, 4.5~7.0, 4.5~7.5, 4.5~8.0, 5.0 In some embodiments, the DAR value of the antibody-drug conjugate is 4 to 8.

[0100] Those skilled in the art will appreciate that the antibody-drug conjugates described herein may be prepared by modularizing the drug-linker. For example, first prepare the free form of the "drug-linker" (GM-[LED] xwhere GM is in a structural form prior to being covalently bound to an antibody or antigen-binding fragment thereof), which is then covalently bound to an antibody or antigen-binding fragment thereof to obtain the antibody-drug conjugate described herein. Accordingly, GM in the free "drug-linker" is linked to one or more thiol groups (-SH), amino groups (-NH), or carboxyl groups (-COOH) on the antibody or antigen-binding fragment thereof by a method such as a substitution reaction (e.g., by removing a structure such as -SOMe or -Br thereon) or an addition reaction.

[0101] In another aspect, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the invention. In some embodiments, a vector of the invention is, for example, a plasmid, cosmid, bacteriophage, lentivirus, etc. In some embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof of the invention in a subject (e.g., a mammal such as a human).

[0102] In some embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding the light chain or light chain variable region thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector described in the present invention comprises a first vector containing the first nucleotide sequence and a second vector containing the second nucleotide sequence.

[0103] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can be used to construct chimeric antigen receptors (CARs) comprising an extracellular antigen-binding domain (e.g., ScFv) that specifically binds to B7-H3, a transmembrane domain, and one or more intracellular T cell signaling domains. In such embodiments, the isolated nucleic acid molecules of the present invention may comprise a nucleotide sequence encoding a chimeric antigen receptor, wherein the nucleotide sequence encoding the chimeric antigen receptor further comprises a nucleotide sequence encoding an antibody or antigen-binding fragment thereof (e.g., ScFv) of the present invention. In some embodiments, the isolated nucleic acid molecules of the present invention encode a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention.

[0104] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can be used to construct chimeric antigen receptor-modified immune cells, including chimeric antigen receptors (CARs) and immune cells (e.g., T lymphocytes, NK cells, dendritic cells, macrophages).

[0105] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention or the vector of the present invention. The host cell may be a eukaryotic cell (e.g., a mammalian cell, an insect cell, or a yeast cell) or a prokaryotic cell (e.g., an E. coli cell). Suitable eukaryotic cells include NSO cells, Vero cells, HeLa cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include Sf9 cells. In some embodiments, the host cell of the present invention is a mammalian cell, such as a CHO cell (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44, or CHO-EBNA).

[0106] In some embodiments, the host cell of the invention may be a chimeric antigen receptor T cell (CAR-T). In such embodiments, the isolated nucleic acid molecule contained in the host cell may comprise a nucleotide sequence encoding a chimeric antigen receptor, wherein the nucleotide sequence encoding the chimeric antigen receptor further comprises a nucleotide sequence encoding an antibody of the invention or an antigen-binding fragment thereof (e.g., an ScFv). In some embodiments, the isolated nucleic acid molecule contained in the host cell encodes a chimeric antigen receptor comprising an antigen-binding fragment of an antibody of the invention (e.g., an ScFv).

[0107] In another aspect of the invention, there is provided a method for preparing an antibody or antigen-binding fragment thereof of the invention, comprising culturing a host cell of the invention under conditions permissive for expression of said antibody or antigen-binding fragment thereof, and recovering said antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0108] composition In another aspect, the present application provides a composition of an antibody-drug conjugate (ADC) described herein. The composition may comprise a plurality of ADCs described herein, each ADC comprising a drug-linker described herein, where x is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other words, each antibody molecule in the composition can be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug-linkers. Thus, the composition is characterized by a "drug-antibody" ratio (DAR) ranging from about 1 to about 10. Methods for measuring the DAR are well known to those skilled in the art and include methods using reverse-phase chromatography or HPLC-MS.

[0109] For example, in any embodiment, the ADC compositions described herein may be, for example, about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to and about 1 to about 10, or any subrange therebetween, such as about 1 to about 9, about 3 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10, or about 9 to 10.

[0110] In some embodiments, the DAR of the ADC compositions described herein is about 3 to 9, e.g., about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, About 4.0 to 8.0, about 4.5 to 5.0, about 4.5 to 5.5, about 4.5 to 6.0, about 4.5 to 6.5, about 4.5 to 7.0, about 4.5 to 7.5, about 5.0 to 8.0, about 5.5 to 6.0, about 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 7.5, about 6.5 to 8.5, about 7.0 to 7.5.

[0111] Pharmaceutical Composition 1. Antibody-drug conjugate pharmaceutical composition In another aspect, the present application provides a pharmaceutical composition comprising any one of the antibody-drug conjugates described above and one or more pharmaceutical excipients.

[0112] The antibody-drug conjugates described herein are typically prepared in a unit injectable form together with a pharmaceutically acceptable parenteral vehicle and used parenterally, for example, by bolus administration, intravenous injection, intratumoral injection, etc. Optionally, the antibody-drug conjugates having the desired purity, in the form of a lyophilized or solution, are mixed with a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1980) 16). th (Edition, Osol, A. Ed.) The antibody-drug conjugates described herein or pharmaceutical compositions containing said antibody-drug conjugates may be administered to an individual in need of treatment by any suitable route.

[0113] 2. Pharmaceutical compositions of antibodies or antigen-binding fragments thereof In another aspect of the present invention, there is provided a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or chimeric antigen receptor of the present invention or a host cell expressing the chimeric antigen receptor, and a pharmaceutically acceptable carrier and / or excipient.

[0114] In some embodiments, pharmaceutical compositions of the invention comprise an antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier and / or excipient.

[0115] In some embodiments, the pharmaceutical composition of the invention comprises a vector or host cell of the invention and a pharmaceutically acceptable carrier and / or excipient. In such embodiments, the isolated nucleic acid molecule contained in the vector comprises a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further comprises a nucleotide sequence encoding an antibody of the invention or an antigen-binding fragment thereof (e.g., an ScFv), and the host cell comprises the isolated nucleic acid molecule or vector. In some embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment of an antibody of the invention (e.g., an ScFv). In some embodiments, the host cell is an immune cell, such as a T cell. In some embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0116] In some embodiments, the pharmaceutical composition may further comprise another pharmacologically active agent. In some embodiments, the other pharmacologically active agent is a drug with anti-tumor activity. In some embodiments, the other pharmacologically active agent is selected from a B7-H3 inhibitor, an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-met or VEGF inhibitor, a chemotherapeutic agent, or any combination thereof. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention and the other pharmacologically active agent are provided as separate components or as a mixed component. Therefore, the antibody or antigen-binding fragment thereof and the other pharmacologically active agent can be administered simultaneously, separately, or sequentially.

[0117] In some embodiments, the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or chimeric antigen receptor or host cell expressing said chimeric antigen receptor in the pharmaceutical composition of the invention is sufficient to do the following (e.g., in a subject): (a) inhibiting cell (e.g., tumor cell) proliferation; (b) inhibiting tumor growth; (c) induce and / or increase antibody-dependent cellular cytotoxicity; (d) inhibiting B7-H3-mediated signaling; (e) preventing and / or treating a disease / condition mediated by B7-H3; or (f) Any combination of (a) to (e).

[0118] In some embodiments, the B7-H3-mediated disease / condition is a tumor, e.g., a B7-H3-expressing tumor, hi some embodiments, the tumor is selected from breast cancer, colorectal cancer, head and neck cancer, clear cell renal cell carcinoma, papillary renal cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, thyroid cancer, or any combination thereof.

[0119] The antibody or antigen-binding fragment thereof of the present invention, and the pharmaceutical composition of the present invention can be prepared into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, tablets, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, and aerosols.

[0120] Purpose 1. Applications of antibody-drug conjugates The antibody drug conjugates or pharmaceutical compositions described herein can be used to treat a number of diseases or conditions, such as, for example, B7-H3 positive tumors.

[0121] Therefore, the present application provides a use of any one of the antibody-drug conjugates described above or a pharmaceutical composition containing the same in the manufacture of a drug for the prevention and / or treatment and / or adjuvant treatment of B7-H3-positive tumors.

[0122] The present application also provides a method for preventing and / or treating and / or supporting the treatment of B7-H3-positive tumors, comprising administering to a subject in need thereof any one of the antibody-drug conjugates described above or a pharmaceutical composition containing the same.

[0123] The present application further provides a use of any one of the antibody-drug conjugates or pharmaceutical compositions described above in inhibiting the growth of B7-H3-positive tumor cells. In some embodiments, the antibody-drug conjugates or pharmaceutical compositions are administered to cells in vitro or to cells in vivo in a subject, e.g., used in a subject to inhibit the growth of tumor cells in the subject, or used in vitro on tumor cells (e.g., a cell line or cells derived from a subject) to inhibit the growth of tumor cells in vitro.

[0124] In the present application, B7-H3-positive tumors include solid tumors or blood cancers such as colorectal cancer, gastric cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, melanoma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, etc.), kidney cancer, bladder cancer, thyroid cancer, mesothelioma, pancreatic cancer, ovarian cancer, endometrial cancer, esophageal cancer, liver cancer, salivary gland cancer, bile duct cancer, and meningioma.

[0125] In the present application, the subject is preferably a mammal, such as, for example, a bovine, equine, porcine, canine, feline, rodent, or primate, eg, a human.

[0126] 2. Uses of antibodies or antigen-binding fragments thereof In another aspect of the present invention, there is provided use of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, complex, multispecific antibody, chimeric antigen receptor or host cell expressing said chimeric antigen receptor, or pharmaceutical composition of the present invention in the preparation of a medicament for use in inhibiting cell proliferation, or for the prevention and / or treatment and / or adjuvant treatment of a tumor.

[0127] In some embodiments, the drug is used to inhibit the proliferation of cells that express B7-H3 (eg, tumor cells).

[0128] In another aspect of the present invention, there is provided a method for inhibiting cell proliferation, comprising contacting a cell with an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor, or host cell expressing the chimeric antigen receptor of the present invention. In some embodiments, the cell is a cell that expresses B7-H3, such as a tumor cell.

[0129] In another aspect of the present invention, there is provided a method for preventing and / or treating and / or adjunctively treating a tumor in a subject in need thereof, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention.

[0130] In some embodiments, the method further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjunctive therapy, and any combination thereof. In some embodiments, the second therapy can be administered simultaneously, separately, or sequentially with the method.

[0131] In any of the above embodiments, the tumor affected by the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor, or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention may be of any tumor type. In some embodiments, the tumor affected by the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor, or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention is a B7-H3-positive tumor. In some embodiments, the tumor affected by the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor, or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention is selected from breast cancer, colorectal cancer, head and neck cancer, clear cell renal cell carcinoma, papillary renal cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, thyroid cancer, or any combination thereof.

[0132] Use for detecting antibodies or antigen-binding fragments thereof The antibodies or antigen-binding fragments thereof of the present invention can specifically bind to B7-H3 and can therefore be used to detect the presence or level of B7-H3 in a sample.

[0133] Therefore, in another aspect, the present invention provides a reagent kit comprising an antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention is labeled with a detectable label. In a preferred embodiment, the reagent kit further comprises a second antibody that specifically identifies the antibody or antigen-binding fragment thereof of the present invention. Preferably, the second antibody further comprises a detectable label.

[0134] In the present invention, the detectable label may be any substance that can be detected by fluorescent, spectral, photochemical, biochemical, immunological, electrical, optical or chemical means, and it is particularly preferred that such labels are applicable to immunological detection (e.g., ELISA, radioimmunoassay, fluoroimmunoassay, chemiluminescent immunoassay, etc.). Such labels are well known in the art and include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., H, I, S, C, or P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine ester compounds, magnetic beads (e.g., Dynabeads®), calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above markers. In some embodiments, the detectable labels described above may be linked to the antibodies of the invention via linkers of different lengths to reduce potential steric hindrance.

[0135] In another aspect, the present invention provides a method for detecting the presence or level of B7-H3 in a sample, comprising using an antibody or antigen-binding fragment thereof of the present invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof is labeled with a detectable label. In another preferred embodiment, the method further comprises detecting the antibody or antigen-binding fragment thereof of the present invention using a reagent labeled with a detectable label. The method can be used for diagnostic or non-diagnostic purposes (e.g., the sample is a cell sample rather than a patient-derived sample).

[0136] In some embodiments, the method comprises contacting the sample with an antibody or antigen-binding fragment thereof described in the invention under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and B7-H3, and detecting the formation of the complex.

[0137] Given that B7-H3 is expressed at low or no levels in normal tissues and at high or low levels in cancers, tumors can be diagnosed by detecting the presence or level of B7-H3 in a sample. Thus, in some embodiments, the methods are used to diagnose tumors, such as B7-H3-positive tumors, including breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer), colorectal cancer, pancreatic cancer, head and neck squamous cell carcinoma, melanoma, ovarian cancer, prostate cancer, liver cancer, kidney cancer, bladder cancer, or any combination thereof.

[0138] In some embodiments, the method includes detecting the expression level of B7-H3 in a measurement sample from a subject, comparing the expression level with a reference value (e.g., a healthy control), and determining that an elevated expression level compared to the reference value is indicative of a tumor.

[0139] In another aspect, there is provided a use of the antibody or antigen-binding fragment thereof of the present invention in the preparation of a reagent kit for use in detecting the presence or level of B7-H3 in a sample and / or diagnosing tumors.

[0140] In another aspect, the present invention provides a diagnostic or therapeutic reagent kit comprising an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, or multispecific antibody according to the present invention, and instructions for use. The reagent kit may further comprise an administration device for topical administration. The administration device may comprise a pre-filled syringe or a needleless syringe.

[0141] definition Unless otherwise defined below, the meanings of all technical and scientific terms used herein are intended to be the same as those commonly understood by those skilled in the art. The technical terms used herein refer to techniques commonly understood in the art and include modifications of techniques or equivalent technical substitutions that are obvious to those skilled in the art. Furthermore, all laboratory procedures used herein, such as genomics, nucleic acid chemistry, and molecular biology, are conventional procedures widely used in the corresponding fields. While the following terms are believed to be well understood by those skilled in the art, the following definitions are provided to better explain the present invention.

[0142] The term "antibody" generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Antibody light chains are classified as κ (kappa) light chains and λ (lambda) light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable and constant regions are connected via a "J" region of about 12 or more amino acids, and heavy chains further contain a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions are not directly involved in antibody-antigen binding but perform various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can be subdivided into hypervariable regions (called complementarity-determining regions (CDRs)), interspersed with relatively invariant regions called framework regions (FRs). Each VH and VL region consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of each heavy / light chain pair (VH and VL) form the respective antigen-binding sites. The amino acid sequences of each region or domain can be numbered according to various schemes known in the art.

[0143] The term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. The heavy and light chain variable regions each contain three CDRs, designated CDR1, CDR2, and CDR3. The exact boundaries of these CDRs can be determined, for example, using the Kabat numbering scheme (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering scheme (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering scheme (Lefranc et al., Dev. Compare. Immunol. 27:55-77, 2003) or the AbM numbering scheme (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA CDRs can be defined according to various numbering schemes known in the art, such as those defined in [End Page 110] (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one of skill in the art can readily identify the CDRs defined in each numbering scheme. Furthermore, the correspondence between different numbering schemes is well known to those of skill in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0144] In the present invention, the CDRs contained in an antibody or antigen-binding fragment thereof can be defined according to various numbering schemes known in the art, such as those defined by the Kabat, Chothia, IMGT, or AbM numbering schemes. In some embodiments, the CDRs contained in an antibody or antigen-binding fragment thereof are defined according to the Chothia numbering scheme.

[0145] The following general rules (published at www.bioinf.org.uk; by Professor Andrew CMartin's research team) can be used to define CDRs in antibody sequences, which contain amino acids that interact specifically with amino acids comprising the antigen epitope that the antibody binds to. In rare cases, these general constant features do not appear. However, Cys residues are the most conservative features.

[0146] [Table 1]

[0147] The amino acid sequence of the entire VH is generally numbered according to Kabat, and the three CDRs within the variable region can be defined according to any of the above numbering schemes. In a specific embodiment, the amino acid positions in the VH can be numbered sequentially from the first amino acid to the end of the sequence, and may be numbered according to Kabat. Unless otherwise specified, the amino acid positions in the VH and VL herein are defined according to SEQ ID NOs.

[0148] Amino acid positions in the heavy chain constant region can be numbered sequentially from the first amino acid to the end of the sequence, or may be numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region contains 330 amino acids, which are numbered sequentially from 1 to 330. The sequence according to Eu numbering starts at position 118 and ends at position 447. Unless otherwise specified, the amino acid positions in the heavy and light chains described herein are defined according to SEQ ID NOs.

[0149] The term "framework region" or "FR" residues refers to amino acid residues other than the above-defined CDR residues in antibody variable regions.

[0150] The term "antigen-binding fragment" of an antibody refers to a polypeptide that is a fragment of an antibody, e.g., a polypeptide that is a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen as the full-length antibody and / or specifically binds to the antigen in competition with the full-length antibody, also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), the entire contents of which are incorporated herein by reference for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)' fragments, F(ab)' fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAbs, nanobodies), and polypeptides comprising at least a portion of an antibody sufficient to confer polypeptide-specific antigen-binding ability. Genetically engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0151] The term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by disulfide bonds in the hinge region; and the term "Fab' fragment" refers to a fragment obtained after reducing the disulfide bond linking the two heavy chain fragments in the F(ab')2 fragment, and consists of one complete light chain and an Fd fragment of a heavy chain (consisting of the VH and CH1 domains).

[0152] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of one arm of an antibody. An Fv fragment is generally considered the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can identify and bind to an antigen, although possibly with lower affinity than the complete binding site.

[0153] The term "Fc" refers to an antibody fragment formed by disulfide bonding between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain of an antibody. The Fc fragment of an antibody has several different functions but is not involved in antigen binding.

[0154] The term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, although variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56; and Roovers et al. (2001), Cancer Immunol. In some circumstances, a disulfide bond may additionally be present between the VH and VL of the scFv. In some embodiments, the VH and VL domains may be positioned opposite each other in any suitable arrangement, e.g., NH2-VH-VH-COOH, NH 2- It is an scFv containing VL-VL-COOH.

[0155] The term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single domains are also called nanobodies.

[0156] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody and / or specifically binds to the antigen in competition with the full-length antibody.

[0157] In this specification, unless the context clearly indicates otherwise, reference to the term "antibody" includes not only complete antibodies but also antigen-binding fragments of antibodies.

[0158] Those skilled in the art can obtain antigen-binding fragments of antibodies (e.g., the antibody fragments described above) from a given antibody (e.g., an antibody provided by the present invention) using known conventional techniques (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and can screen for specific antigen-binding fragments of antibodies in the same manner as with intact antibodies.

[0159] The terms "monoclonal antibody" and "mAb" have the same meaning and can be used interchangeably to refer to a single antibody molecule derived from a population of highly homologous antibody molecules or antibody fragments (i.e., a population of antibodies). Except for natural mutations that may occur naturally, all antibody molecules are identical. mAbs have high specificity for a single epitope on an antigen. Compared to monoclonal antibodies, polyclonal antibodies typically contain at least two or more different antibodies, and these different antibodies typically identify different epitopes on the antigen. Additionally, the modifier "monoclonal" merely indicates that the characteristics of the antibody are obtained from a population of highly homologous antibodies and should not be understood as requiring that the antibody be prepared by any particular method.

[0160] The term "chimeric antibody" refers to an antibody in which a portion of its light and / or heavy chain is derived from an antibody (which may be from a particular species or belong to a particular antibody type or subtype) and another portion of the light and / or heavy chain is derived from another antibody (which may be from the same or a different species or belong to the same or a different antibody type or subtype), while still retaining binding activity for a target antigen. For example, the term "chimeric antibody" may include an antibody whose heavy and light chain variable regions are derived from a first antibody (e.g., human) and whose heavy and light chain constant regions are derived from a second antibody (e.g., mouse). For example, an antibody produced by immunizing a fully human transgenic mouse can be called a chimeric antibody and consists of a fully human variable region and a mouse constant region.

[0161] The term "mouse antibody" refers to an antibody obtained by fusing B cells and myeloma cells from an immunized mouse, screening for mouse hybridomas that can grow indefinitely and secrete antibodies, followed by screening, antibody preparation, and antibody purification; or an antibody secreted and produced by plasma cells that are formed by the differentiation and proliferation of B cells after an antigen has entered the mouse body.

[0162] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered to modify its amino acid sequence to increase its homology with that of a human antibody. Typically, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies typically retain the expected properties of the donor antibody, such as antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and the ability to enhance an immune response. The donor antibody may be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody that has the expected properties (e.g., antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and / or the ability to enhance an immune response).

[0163] The term "identity" refers to the sequence identity between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences, divided by the number of positions compared, and multiplied by 100. For example, if six of ten positions in two sequences are identical, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three of six total positions are identical). Generally, the comparison is performed when the two sequences are compared to produce the greatest identity. Such comparisons can be accomplished, for example, using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). Additionally, the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which is integrated into the ALIGN program (version 2.0), may be used to measure percent identity between two amino acid sequences using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The algorithm of Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) in the GAP program of the GCG software package (available from www.gcg.com) may also be used to measure percent identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0164] As used herein, the term "variant," in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide that contains an amino acid sequence that has been modified by introducing substitutions, deletions, or alterations into the amino acid sequence by introducing amino acid residues. In some cases, the term "variant" also refers to a polypeptide or peptide that has been further modified (i.e., by covalently attaching any type of molecule to the polypeptide or peptide). For example, but not limited to, a polypeptide may be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Derivatives of polypeptides or peptides may also be produced by chemical modification using techniques known to those of skill in the art, including specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Variants also have similar, identical, or improved functions as the polypeptide or peptide from which they are derived.

[0165] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (KD) or half maximal effective concentration (EC 50 ) can be expressed as

[0166] The specific binding properties between two molecules can be measured using methods known in the art. One method involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex. The "association rate constant" (k or k) and the "dissociation rate constant" (k or k) can both be calculated from the concentrations and the actual rates of binding and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of k / k is equal to the dissociation constant, K (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The values ​​of K, k, and k may be measured by any valid method. In some embodiments, dissociation constants may be measured using biolayer interferometry (e.g., ForteBio Octet). Alternatively, dissociation constants may be measured using surface plasmon resonance technology (e.g., Biacore) or Kinexa.

[0167] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide containing the amino acid sequence. Conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include replacing an amino acid residue with an amino acid residue having a similar side chain, such as substituting a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), polar uncharged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0168] The descriptions of the 20 conventional amino acids referred to herein follow conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0169] The terms "comprise," "have," "contain," or "such as," and other similar forms herein, are inclusive or open-ended and do not exclude unrecited elements or method steps.

[0170] The term "alkyl group" refers to groups such as "C 1~20 alkyl group," "C 1~10 alkyl group," "C 1~6 alkyl group," "C 1~4 alkyl group," "C 1~3 and the like. Specific examples include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, a 2-methylbutyl group, a neopentyl group, a 1-ethylpropyl group, an n-hexyl group, an isohexyl group, a 3-methylpentyl group, a 2-methylpentyl group, a 1-methylpentyl group, a 3,3-dimethylbutyl group, a 2,2-dimethylbutyl group, a 1,1-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1,3-dimethylbutyl group, a 2,3-dimethylbutyl group, a 2-ethylbutyl group, a 1,2-dimethylpropyl group, and the like.

[0171] The term "alkylene group" refers to, for example, "C 1~20 alkylene group," "C 1~10 alkylene group," "C 3~10 alkylene group," "C 5~8alkylene group," "C 1~6 alkylene group," "C 1~4 alkylene group," "C 1~3 It represents a group obtained by removing two hydrogen atoms from a straight-chain or branched-chain hydrocarbon group, such as an alkylene group, and specific examples include a methylene group, an ethylene group, a 1,3-propylene group, a 1,4-butylene group, a 1,5-pentylene group, and a 1,6-hexylene group.

[0172] The term "alkenylene group" refers to a divalent group obtained by the loss of two hydrogen atoms from a straight-chain or branched-chain hydrocarbon group containing at least one carbon-carbon double bond, and examples thereof include "C 2~20 alkenylene group," "C 3~10 alkenylene group," "C 5~8 Examples thereof include vinylene group, 1-propenylene group, 2-propenylene group, 1-butenylene group, 2-butenylene group, 1,3-butadienylene group, 1-pentenylene group, 2-pentenylene group, 3-pentenylene group, 1,3-pentadienylene group, 1,4-pentadienylene group, 1-hexenylene group, 2-hexenylene group, 3-hexenylene group, 1,4-hexadienylene group, and the like.

[0173] The term "alkynylene group" refers to a divalent group obtained by the loss of two hydrogen atoms from a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond. For example, "C 2~20 alkynylene group," "C 3~10 alkynylene group," "C 5~8 Examples thereof include ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 1,3-butadinylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 1,3-pentadinylene, 1,4-pentadinylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 1,4-hexadinylene, and the like.

[0174] The term "aliphatic heterocycle" refers to a saturated or partially saturated cyclic structure containing at least one (e.g., 1, 2, or 3) ring member selected from N, O, and S. Specific examples include 5- to 6-membered aliphatic heterocycles, 5- to 6-membered nitrogen-containing aliphatic heterocycles, and 5- to 6-membered oxygen-containing aliphatic heterocycles, such as tetrahydrofuran, pyrrolidine, piperidine, and tetrahydropyran.

[0175] The term "heteroaromatic ring" refers to an aromatic ring structure containing at least one ring member selected from N, O, and S. Specific examples include 5- to 6-membered aromatic heterocycles, 5- to 6-membered nitrogen-containing aromatic heterocycles, and 5- to 6-membered oxygen-containing aromatic heterocycles, such as furan, thiophene, pyrrole, thiazole, isothiazole, thiadiazole, oxazole, isoxazole, oxadiazole, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine, pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,3,5-triazine, and 1,2,4,5-tetrazine.

[0176] The term "aromatic ring system" refers to a monocyclic or polycyclic ring system containing at least one aromatic ring (e.g., a benzene ring, etc.) or heteroaromatic ring (e.g., a 5- to 6-membered aromatic heterocycle, such as a 5- to 6-membered nitrogen-containing aromatic heterocycle, such as a pyrimidine ring), where two or more aromatic and / or heteroaromatic rings form a fused ring or are connected by a single bond (e.g., a dipyrimidine phenyl group, etc.). The aromatic ring system may be divalent or higher (e.g., trivalent or tetravalent), for example, a 5- to 20-membered aromatic ring system.

[0177] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to carriers and / or excipients known in the art that are pharmacologically and / or physiologically compatible with the subject and the active ingredient (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parahydroxybenzoates, chlorobutanol, phenol, and sorbic acid. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents for delaying absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parahydroxybenzoates, chlorobutanol, phenol, and sorbic acid. Stabilizers have the meaning commonly understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug, including sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, glucan, or glucose), amino acids (such as glutamic acid and glycine), proteins (such as dried whey, albumin, or casein), or their degradation products (such as lactalbumin hydrolysate).

[0178] As used herein, the term "solvate" refers to a physical association of an ADC disclosed herein with one or more solvent molecules. Such physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, the solvate may be capable of isolation, for example, when one or more solvent molecules are incorporated into the lattice of a crystalline solid. "Solvate" includes solution-phase and isolable solvates. Non-limiting examples of solvates include ethanolates, methanolates, and the like. A "hydrate" is a solvate in which water is the solvent molecule.

[0179] One or more ADCs disclosed herein can optionally be converted to a solvate. The preparation of solvates is well known. Thus, for example, M. Caira et al., J. Pharmaceutical Sci., 93(3), 601-611 (2004) describe the preparation of antifungal fluconazole solvates in ethyl acetate and water. EC van Tonder et al., AAPS PharmSciTechours., 5(1), article 12 (2004) describe the similar preparation of solvates, hemisolvates, hydrates, etc., and A.L. Bingham et al., Chem. Commun., 603-604 (2001). A typical, non-limiting method involves dissolving a compound of the invention in the required amount of the required solvent (organic solvent or water, or a mixture thereof) at a temperature above room temperature, cooling the solution at a rate sufficient to form crystals, and then isolating the crystals by standard methods. Analytical techniques such as, for example infrared spectroscopy, show the presence of the solvent (or water) in the crystals in the form of a solvate (or hydrate).

[0180] As used herein, the terms "DAR" or "drug-antibody ratio" or "drug-antibody conjugation ratio" (used interchangeably herein) refer to the average number of linker / payload moieties linked to each antibody present in a composition. For a composition comprising an ADC of the invention, the DAR of the composition is the average number of linker-payload moieties of all ADC molecules present in the composition, and the DAR value can be expressed as a decimal or an integer. As used herein, the term "prevention" refers to a method performed to prevent or delay the occurrence of a disease, illness, or condition (e.g., tumor) in a subject's body. As used herein, the term "treatment" refers to a method performed to achieve a beneficial or desired clinical result. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or alleviation of disease progression, improvement or relief of the disease state, and remission of symptoms (whether partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0181] As used herein, the term "subject" refers to a mammal, e.g., a primate mammal, such as a human. In some embodiments, the subject (e.g., a human) has a tumor or is at risk of having such a disease.

[0182] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired effect, or at least partially achieve it. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, inhibit, or delay the onset of the disease (e.g., a tumor), and an effective amount for treating a disease refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient already suffering from the disease. Determining such effective amounts is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use depends on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the method of drug administration, and other concurrent treatments.

[0183] The terms "cancer" and "tumor" are used interchangeably and refer to a large category of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that invade neighboring tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant tumors as well as dormant tumors or micrometastases. Cancer also includes blood cancers.

[0184] The term "blood cancer" includes lymphoma, leukemia, myeloma or malignant lymphoma, as well as splenic cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphoma and T-cell lymphoma. B-cell lymphomas include, for example, Hodgkin's lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphoma. Blood cancers further include leukemias, such as, for example, secondary leukemia or acute lymphocytic leukemia. Blood cancers further include myeloma (e.g., multiple myeloma) and other cancers associated with the blood and / or B-cells or T-cells. [Brief explanation of the drawings]

[0185] [Figure 1A1] 1. Binding Detection of Anti-human B7-H3 Antibody-drug Conjugate and HT29 Cells [Figure 1A2] 2. Binding Detection of Anti-human B7-H3 Antibody-drug Conjugate and HT29 Cells [Figure 1B]Binding detection of anti-human B7-H3 antibody-drug conjugates to NCI-N87 cells. [Figure 1C] Binding detection of anti-human B7-H3 antibody-drug conjugates to HCC1806 cells. [Figure 1D1] 1. Binding Detection of Anti-human B7-H3 Antibody-drug Conjugate and HCC827 Cells [Figure 1D2] 2. Binding Detection of Anti-human B7-H3 Antibody-drug Conjugate and HCC827 Cells [Figure 1E] Binding detection of anti-human B7-H3 antibody-drug conjugates to CHOS-human B7-H3-4Ig cells. [Figure 1F] Binding detection of anti-human B7-H3 antibody-drug conjugates to CHOS-human B7-H3-2Ig cells. [Figure 2A] Binding detection of anti-human B7-H3 antibody-drug conjugates to CHOS-rat B7-H3 cells. [Figure 2B] Binding detection of anti-human B7-H3 antibody-drug conjugates to CHOS-monkey B7-H3 cells. [Figure 3A] Anti-human B7-H3 antibody-drug conjugate and endocytosis detection in NCI-N87 cells. [Figure 3B] Anti-human B7-H3 antibody-drug conjugate and endocytosis detection in HCC1806 cells. [Figure 3C] Anti-human B7-H3 antibody-drug conjugate and endocytosis detection in HCC827 cells. [Figure 4A] A375 cell killing detection of anti-human B7-H3 drug conjugates. [Figure 4B] Calu6-B7-H3 cell killing detection of anti-human B7-H3 drug conjugates. [Figure 4C] U87MG-B7-H3 cell killing detection of anti-human B7-H3 drug conjugates. [Figure 5A] Efficacy measurements of various antibody-drug conjugates in the HT29 model. [Figure 5B] Body weight measurements of the HT29 model for various antibody-drug conjugates. [Figure 6A] Efficacy measurements of various antibody-drug conjugates in the HCC1806 model. [Figure 6B]Body weight measurements of the HCC1806 model with various antibody-drug conjugates. [Figure 7A] Pharmacological efficacy of various drug conjugates of 2#8890 in the HCC1806 model. [Figure 7B] Body weight measurements of HCC1806 model with various drug conjugates of 2#8890. [Figure 8A] Efficacy measurements of various doses of antibody-drug conjugates in the HCC1806 model. [Figure 8B] Body weight measurements of HCC1806 models at various doses of antibody drug conjugates. [Figure 9A] Efficacy measurements of various antibody-drug conjugates in the NCI-N87 model. [Figure 9B] Body weight measurements of NCI-N87 models of various antibody-drug conjugates. [Figure 10] ADC, total antibody (Tab) blood drug concentration-time curve in cynomolgus monkeys. [Figure 11] Payload blood drug concentration-time curve in cynomolgus monkeys. DETAILED DESCRIPTION OF THE INVENTION

[0186] The present invention will be further explained below through the description of specific embodiments, which are not intended to limit the present invention. Those skilled in the art can make various modifications or variations in accordance with the teachings of the present invention without departing from the spirit and scope of the present invention.

[0187] The sequence information of the present invention is set forth in the following table.

[0188] [Table 2]

[0189] [Table 3]

[0190] [Table 4]

[0191] [Table 5]

[0192] [Table 6]

[0193] [Table 7]

[0194] The abbreviations used herein have the following meanings: Abbreviation Meaning CDR Complementarity-Determining Region in Immunoglobulin Variable Region FR Antibody framework region: Amino acid residues other than CDR residues in the antibody variable region VH antibody heavy chain variable region VL antibody light chain variable region IgG immunoglobulin G IMGT: A numbering scheme based on the international ImMunoGeneTics information system® (IMGT) initiated by Lefranc et al. See Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. The immunoglobulin comparison and numbering scheme proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The immunoglobulin numbering scheme proposed by Chothia et al. is a classical rule for identifying the boundaries of CDR regions based on the location of structural ring regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). A CDR definition method derived from related work by AbM Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). mAb monoclonal antibody EC 50 Concentration that produces 50% efficacy or binding I C 50 Concentration producing 50% inhibition ELISA Enzyme-linked immunosorbent assay PCR polymerase chain reaction HRP Horseradish Peroxidase K D equilibrium dissociation constant Ka binding rate constant Kd dissociation rate constant ADCC antibody-dependent cytotoxicity CDC Complement-dependent cytotoxicity FACS flow cytometry CDR-H1 Complementarity-determining region 1 in the immunoglobulin heavy chain variable region CDR-H2 Complementarity-determining region 2 in the immunoglobulin heavy chain variable region CDR-H3 Complementarity-determining region 3 in the immunoglobulin heavy chain variable region CDR-L1 Complementarity-determining region 1 in the immunoglobulin light chain variable region CDR-L2 Complementarity-determining region 2 in the immunoglobulin light chain variable region CDR-L3 Complementarity-determining region 3 in the immunoglobulin light chain variable region

[0195] [Table 8]

[0196] [Table 9]

[0197] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 1 H NMR) or mass spectrometry (MS). [Example]

[0198] Nuclear magnetic resonance ( 1 H NMR measurements were performed using a Bruker 400 MHz nuclear magnetic resonance spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6) as the deuterium reagent and tetramethylsilane (TMS) as the internal standard.

[0199] The abbreviations used in the examples for nuclear magnetic resonance (NMR) spectra are as follows: s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0200] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0201] Example 1: N-((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl-6-(2,5-dioxo-2,5-dihydro-1-H-pyrrol-1-yl)hexanamide (M-01) [ka] Compound IM-1 (0.40 g, 640.59 μmol; see Patent Document CN111936169A for its synthesis) and exatecan mesylate (0.37 g, 704.65 μmol) were dissolved in DMF (8 mL), and HATU (0.32 g, 832.77 μmol) and DIPEA (0.25 g, 1.92 mmol) were added. The mixture was allowed to react at 25°C for 4 hours. The DIPEA was removed under reduced pressure, and the mixture was lyophilized after adding water. The majority of the DMF was removed to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography (conditions are as follows) to obtain 273 mg of compound M-01. Column: Waters XBridge Prep C18 OBD 45mm x 450mm x 8.0μm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% trifluoroacetic acid)

[0202] [Table 10]

[0203] The structural property data of M-01 was as follows: ESI-MS(m / z):1034.4[M+H] + .

[0204] Example 2: N-((S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxy-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-05) [ka] Under nitrogen gas protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (IM-2, 0.66 g, 1.80 mmol) and (R)-16-amino-10-benzyl-6,9,12,15-tetraoxa-3-oxa-5,8,11,14-tetraazahexadecanoic acid (IM-3, 0.75 g, 1.77 mmol) were added to DMF (19 mL), and the temperature was raised to 35°C and the reaction was carried out for 16 hours. After that, (1S,9S )-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (1-4, 1.00 g, 1.77 mmol) was added, and the mixture was cooled to 5-15 °C with ice water. DMTMM (0.98 g, 3.53 mmol) was added, followed by dropwise addition of DIPEA (1.14 g, 8.84 mmol), and the mixture was allowed to react at 25 °C for 16 h. The reaction mixture was poured into a mixture of DCM (600 mL), IPA (60 mL), and water (100 mL). The mixture was stirred for 10 min. The DCM phase was separated, washed with brine (100 mL), and concentrated to give the crude product. After purification by preparative high performance liquid chromatography, the product was freeze-dried to obtain 0.98 g of compound A-05.

[0205] The separation and purification method for A-05 was as follows. Column: Waters SunFire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0206] [Table 11]

[0207] The structural property data of A-05 was as follows: MS m / z (ESI): 1107.3 [M+H]+ 1 H NMR (400 MHz, DMSO) δ 9.10 (s, 2H), 8.66 - 8.63 (m, 1H), 8.51 (d, J = 8.8 Hz, 1H), 8.34 - 8.31 (m, 1H), 8.21 - 8.19 (m, 1H), 8.17 - 8.09 (m, 2H), 8.08 - 8.04 (m, 1H), 7.30 (s, 1H), 7.26 - 7.15 (m, 5H), 6.55 (s, 1H), 5.56 - 5.55 (m, 1H), 5.48 - 5.35 (m, 2H), 5.25 - 5.10 (m, 2H), 4.64 (d, J = 6.4 Hz, 2H), 4.45 - 4.44 (m, 1H), 4.06 - 3.98 (m, 2H), 3.77 - 3.52 (m, 6H), 3.41 (s, 3H), 3.25 - 3.12 (m, 2H), 3.03 - 3.00 (m, 1H), 2.83 - 2.72 (m, 1H), 2.58 - 2.56 (m, 2H), 2.48 (s, 3H), 2.33 - 2.30 (m, 2H), 2.21 - 2.13 (m, 2H), 1.91 - 1.76 (m, 4H), 0.87 (t, J = 7.2 Hz, 3H).

[0208] Example 3: N-((S)-10-benzyl-1-(((1S,9S)-5-fluoro-9-ethyl-9-hydroxy-4-chloro-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxy-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-07) [ka] Under nitrogen gas protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (IM-2, 21.6 mg, 0.059 mmol) and (R)-16-amino-10-benzyl-6,9,12,15-tetraoxa-3-oxa-5,8,11,14-tetraazahexadecanoic acid (IM-3, 24.5 mg, 0.058 mmol) were added to DMF (1 mL), and the mixture was heated to 35°C and reacted for 16 hours. (1S,9S)-1-amino-5-fluoro-9-ethyl-9-hydroxy-4-chloro-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione trifluoroacetate (30.0 mg, 0.053 mmol), HATU (30 mg, 0.079 mmol), and DIPEA (27.2 mg, 0.21 mmol) were added to the reaction mixture, and the reaction mixture was allowed to react for 16 hours at 25°C. The reaction mixture was purified directly by preparative high-performance liquid chromatography and then lyophilized to obtain 26.4 mg of A-07 compound.

[0209] The separation and purification method for A-07 was as follows. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0210] [Table 12]

[0211] The structural property data of A-07 was as follows: ESI-MS (m / z): 1111.3 [M + H] + .

[0212] Example 4: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxa-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxa-3-oxa-5,8,11-triazatetradecan-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-14) [ka] Step 1: Compound IM-4 (657 mg, 1.22 mmol) and compound 1-4 (500 mg, 1.11 mmol) were dissolved in N,N-dimethylformamide (10 mL), followed by the addition of HATU (630.67 mg, 1.66 mmol) and N,N-diisopropylethylamine (428 mg, 3.32 mmol) and stirring at room temperature for 1 hour. After completion of the reaction, the reaction mixture was purified directly by preparative high-performance liquid chromatography and then lyophilized to obtain 700 mg of compound IM-5.

[0213] The separation method for high performance liquid chromatography was as follows. Column: Waters SunFire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0214] [Table 13]

[0215] Step 2: Compound IM-5 (500 mg, 0.513 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (75.05 mg, 1.03 mmol) was added. The mixture was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction mixture was purified by preparative high-performance liquid chromatography and then lyophilized to obtain 307 mg of compound IM-6.

[0216] The separation method for high performance liquid chromatography was as follows. Column: Waters SunFire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0217] [Table 14]

[0218] Step 3: IM-6 (170 mg, 0.226 mmol) and compound IM-2 (90.83 mg, 0.249 mmol) were dissolved in N,N-dimethylformamide (10 mL), and N,N-diisopropylethylamine (29.21 mg, 0.226 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was purified directly by preparative high-performance liquid chromatography and then lyophilized to obtain 50.56 mg of compound A-14.

[0219] The structural property data were as follows: MS m / z(ESI):1002.4[M+H]+

[0220] The separation method for high performance liquid chromatography was as follows. Column: Waters SunFire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0221] [Table 15]

[0222] 1 H NMR (400 MHz, DMSO) δ 9.11 (s, 2H), 8.68 (t, J = 6.4 Hz, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.16 (s, 1H), 8.10 (d, J = 7.2 Hz, 1H), 8.01 (d, J = 7.2 Hz, 1H), 7.91 (d, J = 6.8 Hz, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.65-5.55 (m, 1H), 5.43 (s, 2H), 5.21 (s, 2H), 4.67-4.55 (m, 2H), 4.29-4.15 (m, 3H), 3.98 (s, 2H), 3.41 (s, 3H), 3.25-3.15 (m, 2H), 2.57-2.56 (m, 2H), 2.35-2.27 (m, 2H), 2.22-2.12 (m, 2H), 1.91-1.75 (m, 4H), 1.23-1.09 (m, 9H), 0.87 (t, J = 7.2 Hz, 3H).

[0223] Example 5 Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (B-01) [ka] Step 1: At room temperature, compound B-01-1 (413.40 mg, 0.251 mmol, its synthesis see Patent No. CN111295389B specification) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), copper bromide (I) (72.95 mg, 0.503 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (95.10 mg, 0.302 mmol) were added, and the mixture was stirred for 1 hour, then filtered. The filtrate was purified by preparative high performance liquid chromatography (conditions are as follows), obtaining 30.00 mg of compound B-01-2. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water

[0224] [Table 16]

[0225] Step 2: Compound B-01-2 (30.00 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL) and trifluoroacetic acid (0.2 mL) was added. The mixture was allowed to react at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure and then purified by preparative high-performance liquid chromatography (conditions are as follows) to obtain 20.00 mg of the trifluoroacetate salt of compound B-01. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile, Mobile phase B: water (0.05% trifluoroacetic acid)

[0226] [Table 17]

[0227] The structural characterization data was as follows: ESI-MS (m / z): 1631.7 [M+H] + , 816.0[M / 2+H] + .

[0228] Example 6: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxy-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((1S,9R)-9-ethyl-5-fluoro-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-02) [ka] Step 1: At 25 ° C., the mesylate salt of 1-1 (30.00 mg, 56.44 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (58.74 mg, 112.88 μmol), N,N-diisopropylethylamine (43.76 mg, 338.63 μmol), and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (26.62 mg, 84.66 μmol) were added in this order. The temperature was maintained at 25 ° C. for 1 hour. The reaction was monitored by liquid chromatography-mass spectrometry. After completion of the reaction, water was added to the reaction solution, extracted with ethyl acetate, and the organic phases were combined, dried over sodium sulfate, and then concentrated under reduced pressure. The crude product and crude raw materials were separated by thin layer chromatography (dichloromethane:methanol = 15:1) to obtain 27.00 mg of B-02-1 compound.

[0229] Step 2: At 0°C, B-02-1 (20 mg, 27.33 μmol) was dissolved in dichloromethane (2 mL), and the solution was added in this order to a dichloromethane solution (0.5 mL) of 4-dimethylaminopyridine (26.71 mg, 218.61 μmol) and triphosgene (8.11 mg, 27.33 μmol). The mixture was allowed to react for 0.5 hours while maintaining the temperature at 0°C. After purging the residual triphosgene with nitrogen gas, a dichloromethane solution (1 mL) of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-3,9-diazapentatriacontanamido)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (43.46 mg, 40.99 μmol) was added dropwise and the reaction was continued for 0.5 hours at 0 °C. The reaction was monitored by liquid chromatography-mass spectrometry. After completion of the reaction, the reaction solution was concentrated, and the crude product and raw materials were separated by thin-layer chromatography (dichloromethane:methanol = 15:1) and purified to obtain 30.00 mg of B-02-2 compound.

[0230] Step 3: At 25 ° C, B-02-2 (250.00 mg, 137.51 μmol) was dissolved in a mixed solvent of DMSO (2 mL) and water (0.4 mL), and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (62.98 mg, 206.26 μmol) and copper(I) bromide (39.45 mg, 275.01 μmol) were added. The temperature was maintained at 25 ° C and the reaction was continued for 1 hour. The reaction was monitored by liquid chromatography-mass spectrometry, and after completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography (conditions are as follows), and the aliquot was lyophilized to obtain 150.00 mg of B-02-3 compound. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0231] [Table 18]

[0232] Step 4: At 25°C, B-02-3 (150 mg, 49.45 μmol) was dissolved in tetrahydrofuran (1 mL), and a mixture of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) and glacial acetic acid (v / v = 13 / 1) (50 μL) was added dropwise. The reaction was carried out for 0.5 hours while maintaining the temperature at 25°C. The reaction was monitored by liquid chromatography-mass spectrometry, and after completion of the reaction, the reaction solution was purified by preparative high-performance liquid chromatography (conditions are as follows), and the collected solution was lyophilized to obtain 50.00 mg of B-02-4 compound. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0233] [Table 19]

[0234] Step 5: B-02-4 (50 mg, 26.52 μmol) was dissolved in dichloromethane (1 mL) at 25°C, trifluoroacetic acid (60.49 mg, 530.49 μmol) was added, and the reaction was carried out for 0.5 hours while maintaining the temperature at 25°C. The reaction was monitored by liquid chromatography-mass spectrometry, and after completion of the reaction, the reaction solution was concentrated, and the crude product was purified by preparative high-performance liquid chromatography (conditions are as follows), and the collected solution was freeze-dried to obtain 23.69 mg of B-02 compound. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0235] [Table 20]

[0236] The structural property data of B-02 was as follows: ESI-MS(m / z):1613.6[M+H] + .

[0237] Example 7: N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxa-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxa-3,17,20,23-tetraoxa-5,8,11,14-tetraazapentan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzeneamide (C-07) [ka] Step 1: The raw material C-07-1 (4.80 g, 16.33 mmol), tributyl(2-methylsulfanylpyrimidin-4-yl)tin (16.27 g, 39.19 mmol), and dichlorobis(triphenylphosphine)palladium(II) (2.29 g, 3.27 mmol) were dissolved in 1,4-dioxane (100 mL), and the reaction mixture was stirred at 110 °C for 5 hours under a nitrogen gas atmosphere. The reaction was monitored by LC-MS, and the reaction mixture was concentrated and purified by column chromatography (EA / PE = 0 to 50%) to obtain 1.36 g of C-07-2 compound.

[0238] Step 2: Compound C-07-2 (510 mg, 1.33 mol), NaOH (212.24 mg, 5.31 mmol) were dissolved in THF (12.5 mL), MeOH (12.5 mL), and HO (2.5 mL). The mixture was stirred at 25 °C for 2 hours and monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 3N HCl to precipitate a large amount of solid, which was then filtered. The filter cake was collected and dried to obtain 380 mg of compound C-07-3.

[0239] Step 3: Compound C-07-3 (315 mg, 850.32 μmol), tert-butyl 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxyacetate (246.31 mg, 935.35 μmol), HATU (484.99 mg, 1.28 mmol), and DIPEA (329.69 mg, 2.55 mmol) were added to DMF (3 mL) and reacted at 25 °C for 2 h. The reaction was monitored by LC-MS. The reaction mixture was purified by preparative high-performance liquid chromatography and then lyophilized to obtain 40 mg of compound C-07-3.

[0240] The separation method for high performance liquid chromatography was as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0241] [Table 21]

[0242] Step 4: Compound C-07-4 (40 mg, 64.96 μmol) was dissolved in DCM (3 mL) and TFA (1.5 mL) and reacted at 25° C. for 1.5 hours. The reaction was monitored by LC-MS, and the reaction mixture was concentrated to dryness to give 36 mg of compound C-07-5.

[0243] Step 5: Compound C-07-5 (26 mg, 46.46 μmol), sodium periodate (99.37 mg, 464.57 μmol), and RuCl3·H2O (9.64 mg, 46.46 μmol) were dissolved in ACN (15 mL) and water (7.5 mL) and reacted at 25 °C for 40 minutes. The reaction was monitored by LC-MS, and the mixture was extracted with water and ethyl acetate. The ethyl acetate layer was concentrated to give 28 mg of compound C-07-6.

[0244] Step 6: Compound exatecan mesylate (600 mg, 1.13 mmol), (5S,8S,11S)-1-(9H-fluoren-9-yl)-5,8,11-trimethyl-3,6,9,12-tetraoxy-2,15-dioxy-4,7,10,13-tetraazaheptan-17-oic acid (IM-4, 610.17 mg, 1.13 mmol), HATU (643.81 mg, 1.69 mmol), and DIPEA (437.65 mg, 3.39 mmol) were added to DMF (6 mL) and reacted at 25 °C for 16 h. The reaction was monitored by LC-MS. Water was added to the reaction mixture to precipitate a large amount of solid. The solid was collected by filtration, dissolved in DCM, and concentrated to obtain the crude product. The residue was purified by column chromatography (DCM / MeOH=0 to 10%) to obtain 660 mg of C-07-7 compound.

[0245] Step 7: Compound C-07-7 (660 mg, 688.94 μmmol) was dissolved in N,N-dimethylformamide (6 mL), and diethylamine (251.94 mg, 3.44 mmol) was added and reacted at room temperature for 1 hour. After the reaction was completed, the reaction solution was purified directly by preparative high-performance liquid chromatography and then lyophilized to obtain 325 mg of compound C-07-8.

[0246] The separation method for high performance liquid chromatography was as follows. Column: Waters SunFire Prep C18 OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0247] [Table 22]

[0248] Step 8: Compounds C-07-6 (15.95 mg, 25.58 μmol), C-07-8 (20 mg, 25.58 μmol), HATU (14.59 mg, 38.37 μmol), and DIPEA (9.92 mg, 76.75 μmol) were added to DMF (3 mL) and reacted at 25 °C for 2 h. The reaction was monitored by LC-MS. The reaction mixture was purified by preparative high-performance liquid chromatography and then lyophilized to obtain 7 mg of compound C-07.

[0249] The structural property data were as follows: ESI-MS(m / z):1342.4[M+H] + .

[0250] The separation method for high performance liquid chromatography was as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0251] [Table 23]

[0252] Example 8: N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxa-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxa-3,17,20,23-tetraoxa-5,8,11,14-tetraazapentan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzeneamide (C-10) [ka] Step 1: The starting material C-10-1 (720 mg, 2.45 mmol), 2-methylthiopyrimidine-5-boronic acid (874 mg, 5.14 mmol), XPhosPd G3 (207 mg, 245 μmol), and K3PO4 (1.56 g, 7.35 mmol) were added to dioxane (12 mL) and HO (4 mL). The reaction mixture was stirred at 90 °C under a nitrogen atmosphere for 3 h. The reaction was monitored by LC-MS, filtered through diatomaceous earth, and the filtrate was extracted with water and ethyl acetate. The crude product was concentrated to give 710 mg of C-10-1. The crude product was purified by column chromatography (EA / PE = 0-25%).

[0253] Step 2: Compound C-10-1 (650 mg, 1.69 mol) and lithium hydroxide (121 mg, 5.07 mmol) were dissolved in THF (2 mL), MeOH (2 mL), and HO (2 mL). The mixture was stirred at 25 °C for 2 hours and monitored by LC-MS. The pH of the system was adjusted to about 2 with 1N HCl, and a large amount of solid precipitated. The solid was filtered, and the filter cake was collected and dried to obtain 560 mg of compound C-10-2.

[0254] Step 3: Compound C-10-2 (450.80 mg, 1.22 mmol) was dissolved in DCM (10 mL), and m-CPBA (2.46 g, 12.1 mmol, 85% purity) was added to the reaction mixture. The mixture was reacted at 25 °C for 12 hours and monitored by LC-MS. The solvent was dried under a stream of nitrogen gas to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography and then lyophilized to obtain 153 mg of compound C-10-3.

[0255] The separation method for high performance liquid chromatography was as follows. Column: Phenomenex Luna C18 200 x 40 mm x 10 um. Mobile phase A: acetonitrile; Mobile phase B: water (0.05% hydrochloric acid) Mobile phase: [water(HCl)-ACN]; B%: 13%~43%, 10min).

[0256] [Table 24]

[0257] Step 4: Compound C-10-3 (140 mg, 322.25 μmol), tert-butyl 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxyacetate (84.86 mg, 322.25 μmol), HATU (183.80 mg, 483.37 μmol), and DIPEA (124.94 mg, 966.75 μmol) were added to DMF (4 mL) and reacted at 25 °C for 2 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative high-performance liquid chromatography and then lyophilized to obtain 51 mg of compound C-10-4.

[0258] The separation method for high performance liquid chromatography was as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0259] [Table 25]

[0260] Step 5: Compound C-10-4 (50 mg, 73.56 μmol) was added to DCM (2 mL) and TFA (1 mL) and reacted at 25 °C for 1 h. The reaction was monitored by LC-MS, and the reaction mixture was concentrated to dryness to give 45 mg of compound C-10-5.

[0261] Step 6: Compound C-10-5 (31.91 mg, 51.17 μmol), C-07-8 (40 mg, 51.17 μmol), HATU (29.18 mg, 76.75 μmol), and DIPEA (19.84 mg, 153.50 μmol) were added to DMF (3 mL) and reacted at 25 °C for 2 h. The reaction was monitored by LC-MS. The reaction mixture was purified by preparative high-performance liquid chromatography and then lyophilized to obtain 13 mg of compound C-10.

[0262] The structural property data were as follows: ESI-MS(m / z): 1342.5 [M+H]+.

[0263] The separation method for high performance liquid chromatography was as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0264] [Table 26]

[0265] Example 9: N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxa-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxa-3,17,20,23-tetraoxa-5,8,11,14-tetraazapentan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzeneamide (C-17) [ka] Step 1: C-10-2 (3.00 g, 8.10 mmol) and tert-butyl 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]-propionate (2.25 g, 8.10 mmol) were added to DMF (3 mL), and HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol), and DIPEA (4.19 g, 32.4 mmol, 5.64 mL) were added in this order. The mixture was heated to 60 °C and reacted for 2 hours. Water (50 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give crude C-17-1 (3.8 g, 4.75 mmol). This was used directly in the next step without further purification.

[0266] Step 2: C-17-1 (3.40 g, 5.40 mmol) was dissolved in dichloromethane (30 mL) and trifluoroacetic acid (10.8 g, 94.2 mmol, 7 mL) was added. The reaction mixture was stirred at 25 °C for 2 hours, concentrated, purified by preparative high-performance liquid chromatography, and lyophilized to obtain C-17-2 (2.09 g, 3.64 mmol).

[0267] The separation method for high performance liquid chromatography was as follows. Column: Phenomenex luna C18 (250 mm x 70 mm x 10 μm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0268] [Table 27]

[0269] Step 3: C-17-2 (56 mg, 97.61 μmol) was added to acetonitrile (6 mL) and water (3 mL), and then sodium periodate (208.79 mg, 976.15 μmol) and ruthenium(III) chloride hydrate (8.10 mg, 39.05 μmol) were added to the reaction system, and the mixture was stirred at 25°C for 30 minutes. The reaction was monitored by LC-MS, and the mixture was extracted with water and ethyl acetate and concentrated to give C-17-3 (60 mg).

[0270] Step 4: IM-6 (20 mg, 25.06 μmol), C-17-3 (16 mg, 25.06 μmol), HATU (19.05 mg, 50.11 μmol), and DIPEA (16.19 mg, 125.28 μmol) were added to DMF (3 mL) in this order, and the reaction system was reacted at 25°C for 1 hour. The reaction solution was purified directly by preparative high-performance liquid chromatography and then lyophilized to obtain C-17 (16 mg).

[0271] The structural property data were as follows: ESI-MS(m / z):1371.4[M+H] + .

[0272] The separation method for high performance liquid chromatography was as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0273] [Table 28]

[0274] Example 10: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24-tetraoxo-5,8,11,14-tetraazahexan-26-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-19) [ka] Step 1: C-19-1 (5.00 g, 16.9 mmol), (2-(methylthio)pyrimidin-5-yl)boronic acid (6.34 g, 37.3 mmol), XPhos Pd G3 (1.44 g, 1.70 mmol), and potassium phosphate (10.80 g, 50.9 mmol) were added to 1,4-dioxane (51.0 mL) and water (17.0 mL). The reaction mixture was purged with nitrogen gas three times and then reacted at 100 °C for 5 hours. After cooling to room temperature, water (50.0 mL) was added to the reaction mixture, which was then filtered. The filtrate was concentrated to obtain the crude product. The mixture was triturated with petroleum ether, filtered again, and the filter cake was dried under vacuum to obtain C-19-2 (5.65 g).

[0275] Step 2: C-19-2 (5.26 g, 13.7 mmol) was dissolved in THF (30 mL), MeOH (30 mL), and water (30 mL), and LiOH·HO (1.72 g, 40.9 mmol) was added and stirred at 25 °C for 2 hours. The pH of the reaction mixture was adjusted to 3 with 1N aqueous hydrochloric acid, and the precipitated solid was filtered. The filter cake was dried under vacuum to obtain C-19-3 (4.20 g).

[0276] Step 3: C-19-3 (1.50 g, 4.04 mmol) and tert-butyl 3-(2-(2-aminoethoxy)ethoxyethoxyethyl)propionate (1.12 g, 4.04 mmol) were dissolved in DMF (20 mL). HOBt (1.64 g, 12.1 mmol), EDCI (2.32 g, 12.1 mmol), and DIPEA (2.09 g, 16.2 mmol) were added in this order, and the mixture was heated to 60 °C and stirred for 2 h. The reaction mixture was cooled to room temperature, and water (10 mL) and ethyl acetate (20 mL) were added. The aqueous phase was extracted twice with ethyl acetate (25 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude C-19-4 (2.50 g). This was used directly in the next step without further purification.

[0277] Step 4: C-19-4 (2.50 g, 3.96 mmol) was dissolved in dichloromethane (3.00 mL), and TFA (4.61 g, 40.4 mmol) was added. The reaction mixture was stirred at 25° C. for 12 hours. The reaction mixture was concentrated, purified by preparative high-performance liquid chromatography, and lyophilized to obtain C-19-5 (1.20 g).

[0278] The separation method for high performance liquid chromatography was as follows. Column: Phenomenex luna C18 (150 mm x 25 mm x 10 μm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0279] [Table 29]

[0280] Step 5: C-19-5 (1.10 g, 1.91 mmol) was dissolved in a mixed solvent of acetonitrile (30 mL) and water (15 mL), and ruthenium(III) chloride hydrate (39.70 mg, 0.19 mmol) and sodium periodate (4.09 g, 19.14 mmol) were added. The reaction mixture was incubated at 25 °C for 1 h, then extracted with water (50 mL) and ethyl acetate (80 mL). The organic phase was concentrated to give the crude product. The crude product was purified by column chromatography (MeOH / DCM = 10-20%) and concentrated to give C-19-6 (130 mg).

[0281] Step 6: IM-6 (20.0 mg, 0.025 mmol) and C-19-6 (16.0 mg, 0.025 mmol) were added to DMF (1 mL) and dissolved by stirring. HATU (19.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were added and the mixture was allowed to react at room temperature for 2 hours. The reaction mixture was purified directly by preparative high-performance liquid chromatography and then lyophilized to obtain C-19 (20.4 mg).

[0282] The structural property data were as follows: ESI-MS(m / z):1372.4[M+H] + .

[0283] The separation method for high performance liquid chromatography was as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0284] [Table 30]

[0285] Example 11: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,1,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24,27,30,33,36,39-nonyloxy-5,8,11,14-tetraazabutan-41-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-21) [ka] Step 1: C-10-2 (3.00 g, 8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptan-27-oate (4.03 g, 8.10 mmol) were added to DMF (40 mL), and HOBt (3.28 g, 24.3 mmol), EDCI (4.66 g, 24.3 mmol), and DIPEA (4.19 g, 32.4 mmol, 5.64 mL) were added in that order. The reaction mixture was stirred at 60 °C for 2 h. The reaction mixture was extracted with water (100 mL) and ethyl acetate (60 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give C-21-1 (4.20 g, 4.14 mmol). This was used directly in the next step without further purification.

[0286] Step 2: C-21-1 (3.60 g, 4.24 mmol) was dissolved in dichloromethane (30 mL), TFA (15.3 g, 134 mmol, 10 mL) was added, and the reaction mixture was stirred at 25 °C for 6 hours. The reaction mixture was extracted with water (60 mL) and ethyl acetate (40 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The crude product was purified by preparative high-performance liquid chromatography and then lyophilized to give C-21-2 (2.93 g, 3.63 mmol).

[0287] The separation method for high performance liquid chromatography was as follows. Column: Phenomenex luna C18 (250 mm x 70 mm x 10 μm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0288] [Table 31]

[0289] Step 3: C-21-2 (148 mg, 0.186 mmol) was added to acetonitrile (15 mL) and water (7.5 mL), and then sodium periodate (398.71 mg, 1.86 mmol) and ruthenium(III) chloride hydrate (15.47 mg, 74.56 μmol) were added to the reaction mixture, which was stirred for 30 minutes at 25° C. The reaction mixture was extracted with water and ethyl acetate and concentrated to give C-21-3 (155 mg).

[0290] Step 4: IM-6 (27.91 mg, 34.97 μmol), C-21-3 (30 mg, 34.97 μmol), HATU (26.59 mg, 69.93 μmol), and DIPEA (22.60 mg, 174.84 μmol) were added to DMF (3 mL), and the reaction mixture was incubated at 25°C for 1 hour. The reaction mixture was purified by high-performance liquid chromatography and then lyophilized to obtain C-21 (15 mg).

[0291] The structural property data were as follows: ESI-MS(m / z):1591.7[M+H] + .

[0292] The separation method for high performance liquid chromatography was as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0293] [Table 32]

[0294] Example 12: N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-7,1,13-trimethyl-1,6,9,12,15-pentaoxo-3,18,21,24,27,30,33,36,39-nonyloxy-5,8,11,14-tetraazabutan-4-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-23) [ka] Step 1: C-19-3 (1.50 g, 4.04 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptan-27-oate (2.01 g, 4.04 mmol) were added to DMF (20 mL), and HOBt (1.64 g, 12.1 mmol), EDCI (2.32 g, 12.1 mmol), and DIEA (2.09 g, 16.2 mmol) were added in that order. The mixture was heated to 60 °C and stirred for 2 h. After cooling to room temperature, the reaction mixture was separated with water (10 mL) and ethyl acetate (20 mL). The aqueous phase was extracted twice with ethyl acetate (25 mL × 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give crude product C-23-1 (3.00 g), which was used directly in the next step.

[0295] Step 2: C-23-1 (3.00 g, 3.53 mmol) was added to dichloromethane (10.0 mL), and TFA (15.4 g, 134 mmol) was added, followed by stirring at 25 °C for 12 hours. The reaction mixture was concentrated directly to obtain a crude product. The crude product was purified by preparative high-performance liquid chromatography and then lyophilized to obtain C-23-2 (1.20 g).

[0296] The separation method for high performance liquid chromatography was as follows. Column: Welch Ultimate C18 (150 mm x 25 mm x 5 μm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0297] [Table 33]

[0298] Step 3: C-23-2 (500 mg, 0.63 mmol) was added to acetonitrile (10 mL) and water (5 mL), followed by the addition of ruthenium(III) chloride hydrate (13.0 mg, 0.063 mmol) and sodium periodate (1.35 g, 6.29 mmol). The mixture was reacted at 25 °C for 1 hour, then extracted with water (10 mL) and ethyl acetate (40 mL). The organic phase was concentrated to give the crude product. The crude product was purified by column chromatography (MeOH / DCM = 10-20%) and concentrated to give C-23-3 (350 mg).

[0299] Step 4: IM-6 (20.0 mg, 0.025 mmol) and C-23-3 (21.5 mg, 0.025 mmol) were dissolved in DMF (1 mL), and HATU (19.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were added. The mixture was then reacted at room temperature for 2 hours. The reaction mixture was purified by preparative high-performance liquid chromatography and lyophilized to give C-23 (17.0 mg).

[0300] The structural property data were as follows: ESI-MS(m / z):1592.6[M+H] + .

[0301] The separation method for high performance liquid chromatography was as follows. Column: Waters XBridge Prep C18OBD (5 μm × 19 mm × 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0302] [Table 34]

[0303] Example 13: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl ((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03) [ka] Step 1: Preparation of 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl acetate (B-03-1) (1S,9S)-1-Amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2 g, 3.65 mmol) was dissolved in DMF (50 mL), DIPEA (1.18 g, 9.12 mmol, 1.59 mL) was added dropwise, and acetoxyacetyl chloride (548.12 mg, 4.01 mmol, 431.59 μL) was added dropwise under stirring in an ice bath. The reaction mixture was stirred for 1 hour. The reaction solution was added to 0.1 M dilute aqueous hydrochloric acid, and the precipitated solid was filtered. The filter cake was dissolved in dichloromethane and methanol, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The residue was purified on a silica gel column (methanol / dichloromethane = 0% to 5%) and concentrated again to give the title compound (1.7 g, 3.077 mmol).

[0304] Its structural property data were as follows: ESI-MS(m / z): 552.2 [M+1] + .

[0305] Step 2: Preparation of 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidine[1,2-b]quinolin-1-yl)amino)-2-oxoethyl acetate (B-03-2) Ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetate (500 mg, 0.905 mmol) and DMAP (885.33 mg, 7.25 mmol) were dissolved in dry dichloromethane (5 mL). The solution was cooled to 0°C under nitrogen gas protection, and a dichloromethane solution (5 mL) of triphosgene (268.81 mg, 0.905 mmol) was added dropwise. The mixture was then stirred for 0.5 hours while maintaining the temperature. A solution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azotrinitroamino)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (1.44 g, 1.36 mmol) in dichloromethane was slowly added dropwise, allowed to warm to room temperature, and reacted for 4 hours. The reaction was quenched by adding water, extracted three times with dichloromethane (100 ml × 3), and the combined organic phases were washed with saturated brine, dried, and concentrated. Purification using a silica gel column (MeOH / DCM = 0% to 5%) afforded the title compound (498 mg, 0.304 mmol).

[0306] Its structural property data were as follows: ESI-MS(m / z): 1352.8[M+1] + .

[0307] Step 3: Preparation of 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl ((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03-3) 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamide)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,10,1 Ethyl 3,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidine[1,2-b]quinolin-1-yl)amino-2-oxoacetate (200 mg, 0.122 mmol) was dissolved in THF (3 mL) and MeOH (3 mL), and aqueous sodium carbonate (25.88 mg, 0.224 mmol) (1 mL) was added dropwise with stirring. After the addition was complete, stirring was continued for 1 hour. The reaction mixture was neutralized by adding dilute hydrochloric acid dropwise, concentrated under reduced pressure, and then directly carried on to the next step.

[0308] Its structural property data were as follows: ESI-MS(m / z):1596.7[M+1] + .

[0309] Step 4: Preparation of 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl ((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03-4) 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamide)benzyl ((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl) carbonate (190 mg, 119.04 μmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (0.5 mL) was added, followed by continued reaction for 1 hour. The reaction mixture was neutralized by adding saturated aqueous sodium bicarbonate solution, and the organic phase was concentrated to give the crude product, which was purified by reverse-phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0% to 50%) and then lyophilized to give the title compound (95 mg, 69.35 μmol).

[0310] Its structural property data were as follows: ESI-MS(m / z): 1323.6[M+1] + .

[0311] Step 5: Preparation of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl ((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03) 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamide)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4' [6,7] Indolizino[1,2-b]quinolin-9-yl)carbonate (90 mg, 0.066 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (24.07 mg, 0.079 mmol) were dissolved in DMSO (2 mL) and water (0.2 mL), and copper(I) bromide (9.42 mg, 0.066 mmol) was added and stirred for 2 hours. The reaction mixture was filtered, and the crude product was concentrated and purified by preparative high-performance liquid chromatography. The product was then lyophilized to give the title compound (42.2 mg, 24.69 μmol).

[0312] The structural property data were as follows: ESI-MS(m / z):1628.7[M+1] + .

[0313] The separation method for high performance liquid chromatography was as follows. Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0314] [Table 35]

[0315] II. Preparation of antibodies We immunized fully humanized mice with human B7-H3-4Ig-His protein and measured serum titers by ELISA and flow cytometry. Based on the titer results, we selected optimal mice. Spleen cells were then fused, screened, and subcloned to measure the activity of various monoclonal human / monkey proteins and cells. The optimal clone, 20G11G6 / 2#, was obtained. After modifying the antibody sequence to eliminate PTM sites, reduce PI, and eliminate ADCC activity in the heavy chain constant region, we finally obtained fully human antibody 2#8890 (heavy chain variable region, SEQ ID NO: 3; light chain variable region, SEQ ID NO: 4), which contains a sequence (SEQ ID NO: 31) for modifying the human IgG1 heavy chain constant region to enhance ADCC activity, and a human κ light chain constant region (SEQ ID NO: 32). This fully humanized antibody was constructed (see Table 1). The antibody was codon-optimized and gene-synthesized by Nanjing GenScript Biotechnology Co., Ltd., and constructed into the pTT5 plasmid. The heavy and light chain plasmids were co-transfected into CHO-S cells, and the expressed antibody in the supernatant was purified using Protein A to obtain the corresponding antibody protein 2#8890. The heavy and light chain amino acid sequences of 2#8890 are set forth in SEQ ID NO:42 and SEQ ID NO:43, respectively.

[0316] hIgG1 is an anti-chicken lysozyme antibody, whose heavy chain variable region was fused to a mutated human IgG1 heavy chain constant region (SEQ ID NO: 31) and whose light chain variable region was fused to a wild-type human κ light chain constant region (SEQ ID NO: 32). The antibody hIgG1 was obtained by expression and purification using the method described above.

[0317] The B7-H3 control antibody DS7300 was derived from Patent No. CN103687945A. After codon optimization, the antibody heavy chain variable region nucleotide sequence was synthesized and cloned into the human IgG1 heavy chain constant region (sequence number 31) containing mutations. The light chain variable region nucleotide sequence was synthesized into a pTT5 vector containing the wild-type kappa light chain constant region (sequence number 32). The antibody DS7300 was obtained by expressing and purifying it using the above method.

[0318] [Table 36]

[0319] 3. Conjugation of antibodies with compounds containing cellular biologically active molecules and linkers The antibodies 2#8890, DS7300, and hIgG1 for which antibody-drug conjugates are prepared in the following examples are the corresponding antibodies described in Part 2 above.

[0320] 1. Preparation of ADC 1 (DS7300-M-01, DAR 4) 38.041 ml of homemade DS7300 antibody (26.287 mg / mL) was taken, the pH adjusted to 7.4 with 1 M NaHPO, and the antibody was diluted to 3 mg / mL with 20 mM PB. 4 mM ZnCl (3.413 mL) and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 4.096 mL, pH 7.4) solutions were added in that order in an ice bath, mixed evenly, and left to stand at 4 °C overnight. A 6-fold volume of M-01 (4.18 mL, 10 mM) solution containing dimethyl sulfoxide was added and mixed evenly. The mixture was incubated at 4 °C for 4 hours, after which 6.826 mL of 10 mM cysteine ​​solution was added. After 1.5 hours of incubation, the reaction mixture was transferred to room temperature and 6.826 mL of 10 mM EDTA solution was added. After 30 minutes, additional DHAA solution (10 mM, 6.826 mL) was added and the reaction continued for 30 minutes. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 1 (DS7300-M-01). The DAR value measured by mass spectrometry was 3.8.

[0321] 2. Preparation of ADC 2 (hIgG1-M-01, DAR 4) 1.2245 ml of hIgG1 antibody (24.5 mg / mL) was taken, the pH adjusted to 7.3 with 1 M NaHPO, and the antibody was diluted to 3 mg / mL with 20 mM PB. 4 mM ZnCl (52.04 μL) and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 124.89 μL, pH 7.3) solutions were added in this order in an ice bath, mixed evenly, and left to stand overnight at 4°C. A 6-fold volume of 171 (127.44 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added and mixed evenly. The mixture was incubated at 4°C for 4 hours, after which cysteine ​​solution (10 mM, 208.15 μL) was added. After incubation for 1.5 hours, the reaction mixture was transferred to room temperature and EDTA solution (10 mM, 208.15 μL) was added. After 30 minutes, additional DHAA solution (10 mM, 208.15 μL) was added and the reaction continued for 30 minutes. Upon completion, the buffer was replaced with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain ADC 2 (i.e., hIgG1-M-01). The DAR value measured by mass spectrometry was 4.46.

[0322] 3. Preparation of ADC 3 (2#8890-M-01, DAR 4) 9.36 ml of antibody 2#8890 (3.205 mg / mL) was taken, the pH adjusted to 7.4 with 1 M NaHPO, and the antibody was diluted to 3 mg / mL with 20 mM PB. 4 mM ZnCl (57.31 μL) and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 124.9 μL, pH 7.4) solutions were added in that order in an ice bath, mixed evenly, and left to stand at 4°C overnight. A 6-fold volume of M-01 (124.9 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added and mixed evenly. The mixture was incubated at 4°C for 4 hours, after which 208.2 μL of 10 mM cysteine ​​solution was added. After 1.5 hours of incubation, the reaction mixture was transferred to room temperature and 208.2 μL of 10 mM EDTA solution was added. After 30 minutes, 208.2 μL of 10 mM DHAA solution was added and the reaction continued for another 30 minutes. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 3 (2#8890-M-01). The DAR value measured by mass spectrometry was 3.89.

[0323] 4. Preparation of ADC 4 (hIgG1-A-05, DAR 8) 0.943 ml of hIgG1 antibody (11 mg / mL) was diluted with 47 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 57 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of A-05 (103 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, i.e., ADC 4 (hIgG1-A-05). The DAR measured by mass spectrometry was 8.03.

[0324] 5. Preparation of ADC 5 (2#8890-A-05, DAR 8) 3.052 ml of 2#8890 antibody (9.83 mg / mL) was diluted with 153 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 114.5 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of A-05 (219.1 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 5 (2#8890-A-05). The DAR measured by mass spectrometry was 7.90.

[0325] 6. Preparation of ADC 6 (hIgG1-A-07, DAR 8) 0.518 ml of hIgG1 antibody (19.3 mg / mL) was diluted with 25.9 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 38.1 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of A-07 (69.2 μL, 10 mM) dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 6 (hIgG1-A-07). The DAR measured by mass spectrometry was 8.04.

[0326] 7. Preparation of ADC 7 (2#8890-A-07, DAR 8) 1.017 ml of 2#8890 antibody (9.83 mg / mL) was diluted with 50.85 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 38.2 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 12-fold volume of A-07 (87.6 μL, 10 mM) dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 7 (2#8890-A-07). The DAR measured by mass spectrometry was 7.76.

[0327] 8. Preparation of ADC 8 (hIgG1-A-14, DAR 8) 1.9126 mL of hIgG1 antibody (18.3 mg / mL) was diluted with 95.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 66.86 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of A-14 (260.53 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, i.e., ADC 8 (hIgG1-A-14). The DAR measured by mass spectrometry was 8.0.

[0328] 9. Preparation of ADC 9 (2#8890-A-14, DAR 8) 3.6788 ml of 2#8890 antibody (10.873 mg / mL) was diluted with 183.94 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 152 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of A-14 (292.26 μL, 10 mM) dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 9 (2#8890-A-14). The DAR value determined by mass spectrometry was 7.22.

[0329] 10. Preparation of ADC 10 (hIgG1-B-01, DAR 8) 1.533 mL of hIgG1 antibody (19.57 mg / mL) was diluted with 76.65 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 114.5 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of B-01 (212.4 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer solution, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, i.e., ADC 10 (hIgG1-B-01). The DAR measured by mass spectrometry was 8.03.

[0330] 11. Preparation of ADC 11 (2#8890-B-01, DAR 8) 3.052 ml of 2#8890 antibody (9.83 mg / mL) was diluted with 152.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 114.5 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of B-01 (212.4 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 11 (2#8890-B-01). The DAR value determined by mass spectrometry was 7.75.

[0331] The conjugated ADC samples were subjected to LC-MS molecular weight analysis.

[0332] Chromatographic measurement conditions: Liquid phase column: Thermo MAbPac RP 3.0 × 100 mm; Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN; Flow rate: 0.25 ml / min; chamber temperature: 8°C; column temperature: 60°C; sample injection volume: 2 μl;

[0333] [Table 37]

[0334] Mass spectrometry measurement conditions: Mass spectrometry model: AB Sciex Triple TOF 5600+; GS1 35;GS2 35;CUR 30;TEM 350;ISVF 5500;DP 200;CE 10;Cumulative time 0.5s; m / z 600–4000; total time bin 40.

[0335] 12. Preparation of ADC 12 (2#8890-C-07, DAR 4) 0.2274 ml of 2#8890 antibody (10.994 mg / mL) was diluted with 11.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 9.5 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. An 8-fold volume of C-07 (14.6 μL, 10 mM) solution dissolved in dimethyl sulfoxide was slowly added, mixed evenly, and allowed to stand at room temperature overnight. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 12 (2#8890-C-07). The DAR value determined by mass spectrometry was 4.12.

[0336] 13. Preparation of ADC 13 (2#8890-C-10, DAR 4) 0.2274 mL of 2#8890 antibody (10.994 mg / mL) was diluted with 11.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO solution. 9.5 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. An 8-fold volume of C-10 (14 μL, 10 mM) dissolved in dimethyl sulfoxide was slowly added, mixed evenly, and allowed to stand at room temperature overnight. After completion, the buffer was replaced with 20 mM histidine buffer solution, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 13 (2#8890-C-10). The DAR measured by mass spectrometry was 4.17.

[0337] The conjugated ADC samples were subjected to LC-MS molecular weight analysis.

[0338] Chromatographic measurement conditions: Liquid phase column: ACQUITY UPLC MAbPac BEH SEC; Mobile phase A: 20mM NH4Ac; Flow rate: 0.1 ml / min; chamber temperature: 8°C; column temperature: 60°C; sample injection volume: 2 μl;

[0339] [Table 38]

[0340] Mass spectrometry measurement conditions: Mass spectrometry model: AB Sciex Triple TOF 5600+; GS1 55;GS2 55;CUR 30;TEM 450;ISVF 5500;DP 75;CE 5;Cumulative time 0.5s; m / z 900–7000; total time bin 40.

[0341] 14. Preparation of ADC 14 (2#8890-C-17, DAR 4) 0.292 ml of 2#8890 antibody (8.565 mg / mL) was diluted with 14.6 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 4.77 μL of 20 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, the mixture was mixed evenly, and the mixture was left to stand at room temperature for 1.5 h. A 5-fold volume of C-17 (8.76 μL, 10 mM) dissolved in dimethyl sulfoxide was slowly added, the mixture was mixed evenly, and the mixture was left to stand at room temperature overnight. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 14 (2#8890-C-17). The DAR value measured by mass spectrometry was 3.86.

[0342] 15. Preparation of ADC 15 (2#8890-C-19, DAR 4) 0.584 ml of 2#8890 antibody (8.565 mg / mL) was diluted with 29.2 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 9.54 μL of 20 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 5.5-fold volume of C-19 (17.9 μL, 10 mM) dissolved in dimethyl sulfoxide was slowly added, mixed evenly, and allowed to stand at room temperature overnight. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 15 (2#8890-C-19). The DAR value measured by mass spectrometry was 4.05.

[0343] 16. Preparation of ADC 16 (2#8890-C-21, DAR 4) 0.584 ml of 2#8890 antibody (8.565 mg / mL) was diluted with 29.2 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 9.54 μL of 20 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 5.5-fold volume of C-21 (17.9 μL, 10 mM) dissolved in dimethyl sulfoxide was slowly added, mixed evenly, and allowed to stand at room temperature overnight. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 16 (2#8890-C-21). The DAR value measured by mass spectrometry was 3.92.

[0344] 17. Preparation of ADC 17 (2#8890-B-03, DAR 8) 0.867 ml of 2#8890 antibody (34.6 mg / mL) was diluted with 93.4 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 112.65 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. An 11-fold volume of B-03 (227.58 μL, 10 mM) solution dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 17 (2#8890-B-03, DAR 8). The DAR value determined by mass spectrometry was 7.82.

[0345] 18. Preparation of ADC 18 (2#8890-B-03, DAR 8) 0.867 ml of 2#8890 antibody (34.6 mg / mL) was diluted with 58.35 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was adjusted to 7.60 with 1 M NaHPO. 112.65 μL of 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added, mixed evenly, and allowed to stand at room temperature for 1.5 h. A 10-fold volume of B-03 (211.1 μL, 10 mM) dissolved in dimethyl sulfoxide was added, mixed evenly, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with 20 mM histidine buffer, pH 6.0, using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 18 (2#8890-B-03, DAR 8). The DAR value determined by mass spectrometry was 7.23.

[0346] 4. Antibody-drug conjugate activity detection 1. Dynamic affinity detection of anti-human B7-H3 antibodies and their complexes The dynamic affinity of anti-human B7-H3 antibodies and their complexes with human B7-H3-4Ig-his, human B7-H3-2Ig-his, rat B7-H3-his, and monkey B7-H3-his proteins was determined using ForteBio (Pall Life Sciences). The antibody and its complexes were diluted to 5 μg / ml in PBST (0.02% Tween-20). Human B7-H3-4Ig-his, human B7-H3-2Ig-his, rat B7-H3-his, and monkey B7-H3-his proteins were serially diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, and 0 nM. The antibody and its complexes were then captured in PBST (0.02% Tween-20) using a Protein A sensor (Pall Life Sciences). The four proteins were then allowed to bind for 60 s and then dissociated for 180 s. The results were analyzed using Data Analysis 11.0 software, 1:1 mode, and global fitting to obtain affinity constants. The results are shown in Tables 2-1 and 2-2, and show that antibody 2#8890 and its complex bind to monkey B7-H3 but do not bind to rat B7-H3.

[0347] [Table 39]

[0348] [Table 40]

[0349] 2. Cell affinity detection of anti-human B7-H3 antibodies and their complexes A flow cytometer (Beckman, model Cytoflex) was used to detect the affinity of the anti-human B7-H3 fully human antibody and its conjugates with human colon cancer cells HT29 (Cell Bank, Chinese Academy of Sciences), human gastric cancer cells NCI-N87 (ATCC), human breast squamous cell carcinoma cells HCC1806 (ATCC), and human non-small cell lung cancer cells HCC827 (ATCC). The affinity of the anti-human B7-H3 fully human antibody and its conjugates with CHOS-human B7-H3-4Ig and CHOS-human B7-H3-2Ig was also detected. The species cross-reactivity of the anti-human B7-H3 fully human antibody and its conjugates with CHOS-rat B7-H3 and CHOS-monkey B7-H3 was also detected. Adherent cells were digested with trypsin-EDTA (0.25%) (Thermo) solution and counted to a cell density of 4.0 × 10 cells. 6 The cells were resuspended in 1% BSA solution, and 50 μl of the cell suspension was added to each well of a 96-well V-bottom plate (2 × 10 cells). 5 (cells / well). Antibodies and their conjugates were diluted in 1% BSA (starting at a final concentration of 10 μg / ml and diluted in 11 3-fold increments), with hIgG1 antibody and its conjugates as controls (final concentration 10 μg / ml). 50 μl of the diluted antibody was added to the V-bottom plate containing the cells and incubated for 60 min at 4°C. The cells were washed twice with 1% BSA, and 50 μl of diluted secondary antibody was added to each well, mixed evenly, and incubated for 30 min at 4°C. The cells were washed twice with 1% BSA, resuspended in 200 μl of 1% BSA, and detected by flow cytometer. Data processing: After the median PE value was calculated, the data was entered into GraphPad Prism 6 software, and EC 50 was calculated.

[0350] The affinity of anti-human B7-H3 antibodies and their conjugates to HT29, NCI-N87, HCC1806, and HCC827 tumor cells is shown in Figures 1A1, 1A2, 1B, 1C, 1D1, 1D2, and Tables 3-1 and 3-2, respectively. 2#8890 exhibited stronger tumor cell affinity than DS7300, demonstrating a match between the ADC and the corresponding antibody. The affinity of anti-human B7-H3 fully human antibodies and their conjugates to CHOS-human B7-H3-4Ig and CHOS-human B7-H3-2IG cells is shown in Figures 1E and 1F and Table 4. 2#8890 and DS7300 exhibited comparable affinity to the two overexpressing cells, demonstrating a match between the ADC and the corresponding antibody. The affinity of the anti-human B7-H3 antibody and its conjugates to CHOS-rat B7-H3 and CHOS-monkey B7-H3 cells is shown in Figure 2A, Figure 2B, and Table 5. The results indicate that 2#8890 and its conjugates bind to monkey B7-H3-overexpressing cells but not to rat B7-H3-overexpressing cells.

[0351] [Table 41]

[0352] [Table 42]

[0353] [Table 43]

[0354] [Table 44]

[0355] 3. Detection of endocytosis of anti-human B7-H3 antibody and its complex Endocytosis of anti-human B7-H3 antibody and its conjugates in human gastric cancer cells NCI-N87 (ATCC), human breast squamous cell carcinoma cells HCC1806 (ATCC), and human non-small cell lung cancer cells HCC827 (ATCC) was detected using a flow cytometer (Thermo, model Attune NxT). Adherent cells were digested with trypsin-EDTA (0.25%) (Thermo) solution, counted, and adjusted to a cell density of 1 × 10 using complete medium. 5 The cell suspension was adjusted to 1 × 10 cells / ml, and 100 μl of the cell suspension was added to each well of a 96-well plate (cell count: 1 × 10 4 (cells / well). The 96-well plate was incubated at 37°C in a CO2 incubator and cultured for 24 hours. The 96-well plate was removed, the medium was aspirated and discarded, and 50 μl of fresh complete medium was added to each well. The antibody and its conjugate were serially diluted in six steps using complete medium. hIgG1 antibody and its conjugate were used at a single concentration. pHrodo reagent (Thermo, Cat# Z25612) at 300 μg / ml was diluted to 12 μg / ml in complete medium (final pHrodo concentration: 3 μg / ml). The serially diluted antibody and diluted pHrodo reagent were mixed equally (30 μl:30 μl) at room temperature, avoiding light, for 30 minutes. 50 μl of the mixture of antibody and pHrodo reagent was added to the 96-well plate and cultured at 37°C in 5% CO2 for 24 hours. The 96-well plate was removed, the medium was aspirated and discarded, and washed once with sterile PBS. 100 μl of trypsin-EDTA (0.25%) was added to each well to digest the cells, and 100 μl of complete medium was added to neutralize them. The cells in the wells were dispersed by spraying and then detected by FACS. Data processing: The median YL-1H values ​​were calculated and entered into GraphPad Prism 6 software, and the EC 50 The results are shown in Figures 3A, 3B, and 3C and Table 6. The endocytosis of 2#8890 was stronger than that of DS7300, and the cellular endocytosis of the ADC and the corresponding antibody was consistent.

[0356] [Table 45]

[0357] 4. In vitro cell killing detection of anti-human B7-H3 antibody-drug conjugates Adherent cells of A375, Calu6-B7-H3, and U87MG-B7-H3 were digested using trypsin-EDTA (0.25%) (Thermo) solution, counted, and cultured at a cell density of 1 × 10 using complete medium. 4 , 5×10 4 , 1×10 4 The cell suspension was adjusted to 1000, 5000, or 1000 cells / ml, and 100 μl of the cell suspension was added to each well of a 96-well plate (cell counts of 1000, 5000, and 1000 cells / well, respectively). The 96-well plate was incubated at 37°C in a CO2 incubator and cultured for 24 hours. The ADC to be measured was diluted in complete medium, starting at a final concentration of 3333.3 nM, with 12 four-fold dilutions. 100 μl of the diluted ADC was added to the 96-well plate and cultured at 37°C and 5% CO2 for 4 to 7 days. The 96-well plate was removed, and 20 μl of CCK8 reagent was added to each well. The plate was then incubated at 37°C for 2 to 3 hours. The OD was measured using a microplate reader. 450nm After detecting the signal values, they were input into GraphPad Prism 6 software and calculated as IC 50 was calculated. The results are shown in Table 7 and Figures 4A to 4C. The killing activity of the conjugate was significantly stronger than that of the negative antibody conjugate, indicating that the killing effect was target-mediated.

[0358] [Table 46]

[0359] 5. In vivo efficacy measurement of various antibody-drug conjugates in the HT29 model Human colon cancer cells HT29 (Cell Bank of the Chinese Academy of Sciences) were cultured in vitro as a monolayer. The culture conditions were as follows: 10% fetal bovine serum was added to McCoy's 5a medium, and the cells were cultured in an incubator at 37°C with 5% CO2. Subculture was performed every 2 to 3 weeks using pancreatin-EDTA for digestion. When the cells showed an exponential growth phase, the culture medium was taken for mycoplasma detection, and then the cells were harvested and counted. 5×10 6 HT29 cells were subcutaneously inoculated into the right scapula of each mouse and suspended in 0.05 ml of PBS + 0.05 ml of Matrigel. When the average volume of the tumor grew to 100 - 200 mm 3 , mice with irregular or too small or too large tumor volumes were excluded, and the remaining mice were randomly assigned into 4 groups according to tumor volume and animal body weight, with 6 mice in each group. Intravenous injection (i.v.) was administered once a week (QW) for 2 doses (10 mg / kg). After administration, the tumor was measured twice a week using calipers, and the tumor volume was calculated using the formula V = 0.5a×b 2 . In the formula, a and b represent the major and minor diameters of the tumor, respectively. The antitumor effect was evaluated by the tumor growth inhibition rate TGI (%). Calculation formula: TGI (%) (tumor volume) = [1 - (T<​​​​​​​​​​​​​​​​​​​​​​​​​​ [Table 47]

[0361] As a result, all treatment groups showed significant efficacy and TGI (%), except for the negative control ADC group 2. The efficacy of ADC group 3 was superior to that of the positive control ADC group 1, and each group was well tolerated by animals, demonstrating the good efficacy and safety of antibody 2#8890.

[0362] 6. In vivo efficacy measurement of various antibody-drug conjugates in the HCC1806 model Human breast squamous cell carcinoma cells HCC1806 (ATCC) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in an incubator containing 5% CO2. When the cells reached the exponential growth phase, the culture medium was sampled for mycoplasma detection, and the cells were then harvested and counted. 2 × 10 cells were placed on the right scapula of each mouse. 6 HCC1806 cells were subcutaneously inoculated and suspended in 0.1 ml of PBS. The average tumor volume was 100–200 mm. 3 When tumors grew to 1000 mcg / kg, mice with tumors that were too small or too large were removed. The remaining mice were randomly assigned to four groups (6 mice per group) based on tumor volume and body weight. A single dose of 10 mg / kg was administered via tail vein injection. Tumor volume and body weight were measured twice weekly after administration. See Table 9 and Figures 6A-B for specific results.

[0363] [Table 48]

[0364] As a result, the efficacy of the ADC 3 group in terms of TGI (%) was superior to that of the positive control ADC 1 group, and the animals in each group were well tolerated, demonstrating the good efficacy and safety of the 2#8890 antibody.

[0365] 7.2 In vivo efficacy of various conjugates of #8890 in HCC1806 model Human breast squamous cell carcinoma cells HCC1806 (ATCC) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in an incubator containing 5% CO2. When the cells reached the exponential growth phase, the culture medium was sampled for mycoplasma detection, and the cells were then harvested and counted. 2 × 10 cells were placed on the right scapula of each mouse. 6 HCC1806 cells were subcutaneously inoculated and suspended in 0.1 ml of PBS. The average tumor volume was 100–200 mm. 3 When tumors grew to 1000 mg / kg, mice with tumors that were too small or too large were removed. The remaining mice were randomly assigned to six groups, each consisting of six mice, based on tumor volume and body weight. A single dose of the drug was administered via tail vein injection (DAR4 group: 10 mg / kg, DAR8 group: 5 mg / kg). Tumor volume and body weight were measured twice weekly after administration. See Table 10 and Figures 7A-B for specific results.

[0366] [Table 49]

[0367] The mice in the treatment groups showed stable weight gain and good tolerability. At comparable toxin doses, the efficacy of ADC 3 and ADC 11 was similar and weaker than that of ADC 5. At 95 days after a single dose, the mice in the ADC 5 group showed complete tumor response.

[0368] 8. Efficacy Measurement of Various Doses of Antibody-Drug Conjugates in the HCC1806 Model Human breast squamous cell carcinoma cells HCC1806 (ATCC) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37°C in an incubator containing 5% CO2. When the cells reached the exponential growth phase, the culture medium was sampled for mycoplasma detection, and the cells were then harvested and counted. 2 × 10 cells were placed on the right scapula of each mouse. 6 HCC1806 cells were subcutaneously inoculated and suspended in 0.1 ml of PBS. The average tumor volume was 100–200 mm. 3When tumors grew to 1000 mcg, mice with tumors that were too small or too large were removed. The remaining mice were randomly assigned to 12 groups, each consisting of 6 mice, based on tumor volume and body weight. A single dose was administered via tail vein injection. Tumor volume and body weight were measured twice weekly after administration. See Table 11 and Figures 8A-B for specific results.

[0369] [Table 50]

[0370] The results showed that the ADC 5 and ADC 9 dose groups had similar efficacy, achieving partial remission (PR) at a dose of 5 mg / kg. The efficacy of the ADC 1 3 mg / kg group was weaker than that of the ADC 5 and ADC 9 1.5 mg / kg groups with equivalent toxin doses (P<0.001). In the ADC 1 1.5 mg / kg group, two mice experienced hypothermia, decreased activity, and subsequently died. One mouse each died in the ADC 4 and ADC 8 groups. The mice in the other groups maintained stable weights and were well tolerated.

[0371] 9. Drug efficacy measurement of various antibody-drug conjugates in the NCI-N87 model Human gastric cancer cells NCI-N87 (ATCC) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in an incubator containing 5% CO2. When the cells reached the exponential growth phase, the culture medium was sampled for mycoplasma detection, and the cells were then harvested and counted. Each mouse was injected with 5 × 10 cells into the right scapula. 6 NCI-N87 cells were subcutaneously inoculated and suspended in 0.1 ml of PBS-based Matrigel. 3 When tumors grew to 100% tumor volume, mice with tumors that were too small or too large were removed. The remaining mice were randomly assigned to eight groups, each consisting of six mice, based on tumor volume and body weight. Administration was via tail vein injection twice daily. Tumor volume and body weight were measured twice weekly after administration. See Table 12 and Figures 9A-B for specific results.

[0372] [Table 51]

[0373] As a result, mice in each treatment group had stable body weight and were well tolerated. ADC 3 and ADC 1 had similar efficacy. ADC 11, ADC 5, and ADC 9 had similar efficacy but were significantly more effective than ADC 1.

[0374] 10. Hydrophilicity Detection of Anti-Human B7-H3 Antibodies and Complexes The hydrophilicity of antibodies and conjugates was determined using an Agilent 1260 with an analytical column, TSKgel Butyl-NPR. An appropriate amount of sample was diluted with diluent (0.75 mol / L (NH4)2SO4) to prepare a 1.0 mg / ml solution, which served as the sample solution. Control antibodies (hydrophilic control, tagitanlimab; hydrophobic control, sacituzumab, both manufactured by Sichuan Kernelbotai Biopharmaceutical Co., Ltd.) were prepared as 1 mg / ml solutions with diluent, which served as system suitability solutions. Approximately 40 μg of sample was added, and the gradient elution was analyzed. After detection, the sample hydrophobicity value was calculated using the control sample. The calculation formula was: (sample retention time - hydrophilic control retention time) / (hydrophobic control retention time - hydrophilic control retention time). The smaller the retention time and hydrophobicity value, the better the antibody's hydrophilicity. See Tables 13 to 15 for the results. Antibody 2#8890 had good hydrophilicity, and was more hydrophilic than the control antibody DS7300. The hydrophilicity of the 2#8890-antibody conjugates was ADC 9 > ADC 11 > ADC 5, with ADC 9 being particularly good.

[0375] [Table 52]

[0376] [Table 53]

[0377] [Table 54]

[0378] 11. Detection of binding activity of anti-human B7-H3 antibody-drug conjugate to Fc receptors The dynamic affinity of antibodies DS7300, 2#8890, and DS7300 and 2#8890 antibody-drug conjugates to the human Fc receptor proteins CD16a, CD32a, CD32b, C1q, and FcRn was measured using ForteBio (Pall Life Sciences). The specific method was as follows: Biotinylated proteins were captured in PBST solution using an SA Sensor (Pall Life Sciences). The antibodies and conjugates were diluted to an initial concentration of 5000 nM in PBST, and then diluted two-fold in seven steps to allow binding and dissociation. The measurement results were analyzed in Data Analysis 11.0 software using 1:1 mode and global fitting to obtain binding rates, dissociation rates, and affinity constants. The results are shown in Table 16.

[0379] [Table 55]

[0380] As a result, the mutated 2#8890 and its complexes did not bind to the Fc receptors CD16a, CD32a, CD32b, and C1q proteins, thereby reducing Fc receptor-mediated nonspecific killing and improving drug safety. At the same time, 2#8890 and its complexes retained their FcRn protein binding activity and did not affect the half-life of the drug.

[0381] 12. Pharmacokinetic study of total antibody (Tab), ADC, and payload in serum after multiple intravenous administration of anti-human B7-H3 antibody-drug conjugates to cynomolgus monkeys ELISA and LC-MS / MS were used to quantitatively detect ADCs (ADC5, ADC9), total antibodies (TAb), and payloads in cynomolgus monkey serum. The standard curve quantitative ranges for ADCs (ADC5, ADC9) and total antibodies (TAb) were 11.72–3000.00 ng / mL, and the linear range for payloads was 0.1–40 ng / mL. Both ADCs (ADC5, ADC9) and total antibodies (TAb) used B7-H3 protein as the capture protein. After incorporation into a 96-well microplate, total antibodies (TAb) were detected using goat anti-human IgG-HRP; ADCs (ADC5, ADC9) used anti-toxin mouse antibody and goat anti-mouse IgG as the secondary and detection antibodies, respectively. Color development was achieved by the action of the enzyme and substrate, and the readings were performed using a SpectraMax i3x (Molecular Devices) microplate reader. The concentration of each sample was calculated by fitting a standard curve using the 4-P parameter method. The color depth and the concentration of ADC (ADC 5, ADC 9) and total antibody (TAb) showed a positive correlation. LC-MS / MS analysis was performed using a Shimadzu LC 30-AD flow unit coupled to a SCIEX QTRAP 5500+ (SCIEX) mass spectrometer, using (+)ESI ionization with multiple reaction monitoring (MRM) selected. The column was an Xbridge C18 50 x 4.6 mm, 5 μm column. The analyte compound 1-10 ion pair was 510.2 / 435.2. Sample pretreatment involved protein precipitation using acetonitrile. Results: After multiple intravenous administration of ADC5 and ADC9 to cynomolgus monkeys, measurements showed that the ADC molecules (e.g., ADC5 and ADC9) exhibited favorable pharmacokinetic properties, were relatively stable in the systemic circulation, and released little free toxin. Among them, ADC9 had a longer half-life in cynomolgus monkeys and faster clearance of free toxin.

[0382] [Table 56]

[0383] [Table 57]

[0384] [Table 58]

[0385] 13. Repeated dose toxicity test The repeat-dose toxicity study included a four-dose repeat toxicity study in which ADC5 and ADC9 were administered intravenously to cynomolgus monkeys.

[0386] This study consisted of three groups, one animal per group per sex. Each group received 30 mg / kg of ADC 5, ADC 9, and saline (volume: 10 mL / kg) intravenously, once weekly for a total of two doses. Subsequently, ADC 5, ADC 9, and saline at doses increased to 50 mg / kg were administered once weekly for a total of two doses. No drug-related deaths or moribundity were observed during the study. Animals in the ADC 5 group experienced decreased appetite, alopecia, skin pigmentation, and weight loss during the administration period. Female monkeys experienced decreases in WBC, NEUT, LYM, and MONO. Male monkeys experienced decreases in RBC, HGB, and HCT, and increases in FGB, which tended to recover during the recovery period. Animals in the ADC 9 group experienced decreased appetite during the administration period. Female monkeys experienced decreases in WBC, NEUT, LYM, and MONO, and increases in FGB. In male monkeys, elevated FBG and decreased RBC, HGB, and HCT were observed. During the recovery period, pigmentation and slight hair loss were additionally observed at the injection site, but all other changes showed a tendency to recover. Under these experimental conditions, cynomolgus monkeys were intravenously injected with 30 mg / kg of ADC 5 and ADC 9 once a week for two consecutive weeks. The dose was then increased to 50 mg / kg of ADC 5 and ADC 9 once a week for two consecutive weeks. All animals tolerated the treatment. The maximum non-severely toxic dose (HNSTD) was 50 mg / kg.

[0387] 14. Tumor growth inhibition of antibody-drug conjugates in subcutaneously transplanted tumor models in mice Formulations containing the ADC of the present invention were administered via tail vein injection to a mouse CDX model in which human breast squamous cell carcinoma cells HCC1806 had been subcutaneously transplanted. Changes in tumor volume and animal body weight were measured twice a week, and the tumor-suppressing effect of the ADC of the present invention on cancer-bearing mice was calculated.

[0388] Experimental animal: Balb / c Nude mouse (Chengdu Pharmacological Biotechnology Co., Ltd.)

[0389] Cell line: Human breast squamous cell carcinoma cell line HCC1806 (ATCC)

[0390] Experimental Method: HCC1806 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in 5% CO2. HCC1806 cells in the exponential growth phase were harvested, resuspended in PBS to an appropriate concentration, and subcutaneously inoculated into female Balb / c nude mice to establish a breast squamous cell carcinoma model. The average tumor volume was approximately 200 mm3. 3 At approximately 10 days, mice were randomly assigned according to tumor size and administered the respective drugs. The groups and their doses were as follows: Solvent control group (i.e., negative control, vehicle group): 0.9% NaCl injection was administered. ADC 5: dose 3 mg / kg. ADC 9: dose 3 mg / kg. ADC 11: dose 3.16 mg / kg. ADC 17: dose 3.32 mg / kg. Each group was administered via tail vein injection (iv) on Day 0, for a total of one dose. After administration, the mice were weighed twice a week, and the long and short diameters of the tumor were measured with calipers. The tumor volume was calculated using the following formula: V=0.5a×b 2 In the formula, a and b represent the long and short diameters of the tumor, respectively. The mortality of the animals was observed daily and recorded.

[0391] The tumor growth inhibition rate (TGI) (%) was calculated using the following formula: V T末 >V T0 , TGI(%)=[1-(V T末 -V T0 ) / (V C末 -V C0)] × 100% or V T末 ≦V T0 , TGI(%)=[1-(V T末 -V T0 ) / V T0 ]×100%. In the formula, V T末 : Mean tumor volume at the end of the experiment for each treatment group V T0 : Mean tumor volume at the start of treatment V C末 : Mean tumor volume at the end of the experiment in the negative control group V C0 : Mean tumor volume at the start of administration in the negative control group

[0392] The relative tumor growth rate T / C (%) was calculated using the following formula: T / C=(V T末 / V T0 ) / (V C末 / V C0 )

[0393] The ADCs of the present invention exhibited significant tumor growth inhibition in an HCC1806 breast squamous cell carcinoma xenograft tumor model. On day 14, the tumor growth inhibition rates (TGI) of ADC 5, ADC 9, ADC 11, and ADC 17 of the present invention were 96.68%, 98.50%, 82.61%, and 86.69%, respectively, compared with the vehicle group, demonstrating significant differences compared with the control group. During the treatment period, no deaths or significant weight loss were observed in animals in any treatment group, and no significant drug toxicity was observed. The ADCs of the present invention were well tolerated by mice. See Table 20 for specific results.

[0394] [Table 59]

[0395] Although the specific embodiments of the present invention have been described in detail above, it is understood that those skilled in the art can make various modifications and substitutions to the details according to all the teachings already disclosed, and all of these modifications fall within the protection scope of the present invention. The full scope of the present invention is provided by the appended claims and any equivalents thereof.

Claims

1. Formula Ab-[MMME� ｘ (In the formula, Ab is an antibody or antigen-binding fragment thereof that specifically binds to the B7-H3 antigen; M is a connecting site that links the antibody or antigen-binding fragment thereof; L is a linker connecting the connecting moieties M and E, E is a structural fragment connecting L and D, D is a payload fragment, x is selected from 1 to 10 An antibody-drug conjugate having the structure shown below.

2. The antibody or antigen-binding fragment thereof has the following complementarity determining regions (CDRs): (a) CDR-H1, CDR-H2, CDR-H3 contained in the heavy chain variable region (VH) represented by SEQ ID NO: 1, and / or CDR-L1, CDR-L2, CDR-L3 contained in the light chain variable region (VL) represented by SEQ ID NO: 2; (b) CDR-H1, CDR-H2, CDR-H3 contained in the heavy chain variable region (VH) represented by SEQ ID NO: 3, and / or CDR-L1, CDR-L2, CDR-L3 contained in the light chain variable region (VL) represented by SEQ ID NO: 4; or (c) CDR-H1, CDR-H2, CDR-H3 contained in the heavy chain variable region (VH), and / or CDR-L1, CDR-L2, CDR-L3 contained in the light chain variable region (VL), which contain mutations in at least one CDR that are substitutions, deletions, or additions of one or several amino acids (e.g., substitutions, deletions, or additions of one, two, or three amino acids) compared to the heavy chain variable region and / or light chain variable region described in either (a) or (b), and preferably the substitutions are conservative substitutions. Including; Preferably, the CDRs are defined according to the IMGT, Kabat, Chothia, or AbM numbering scheme.

3. The antibody or antigen-binding fragment thereof is (1) A heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the IMGT numbering scheme as follows: (1a) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 5 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 6 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 8 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 9 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; (1b) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 18 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 19 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 20 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 8 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 9 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; Or, (2) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the Chothia numbering scheme as follows: (2a) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 11 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 12 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 15 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; (2b) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 21 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 22 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 15 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; Or, (3) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the Kabat numbering scheme as follows: (3a) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 16 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 17 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 15 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; (3b) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 24 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 25 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 15 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; Or, (4) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the AbM numbering scheme as follows: (4a) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 26 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 27 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 15 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof; (4b) A heavy chain variable region (VH) comprising three CDRs: CDR-H1 whose sequence is SEQ ID NO: 28 or a variant thereof, CDR-H2 whose sequence is SEQ ID NO: 29 or a variant thereof, and CDR-H3 whose sequence is SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 whose sequence is SEQ ID NO: 14 or a variant thereof, CDR-L2 whose sequence is SEQ ID NO: 15 or a variant thereof, and CDR-L3 whose sequence is SEQ ID NO: 10 or a variant thereof. Including; The antibody-drug conjugate of claim 1 or 2, wherein the variant described in any of (1a), (1b), (2a), (2b), (3a), (3b), (4a), and (4b) has one or several amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, and preferably the substitutions are conservative substitutions.

4. The antibody or antigen-binding fragment thereof is (a) a VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; or (b) a VH comprising the sequence shown in SEQ ID NO: 3 or a variant thereof and / or a VL comprising the sequence shown in SEQ ID NO: 4 or a variant thereof Including; The antibody-drug conjugate of any one of claims 1 to 3, wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) with respect to the sequence from which it is derived, and preferably the substitutions are conservative substitutions.

5. The antibody-drug conjugate of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

6. the antibody or antigen-binding fragment thereof further comprises a constant region originating from or derived from a human immunoglobulin; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region originating from or derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4), and preferably the antibody or antigen-binding fragment thereof comprises a wild-type Fc region or a mutated or chemically modified Fc region with altered effector function compared to the wild-type Fc region; Preferably, the antibody or antigen-binding fragment thereof comprises a variant of a human IgG1 heavy chain constant region, the variant having substitutions of Leu234Ala, Leu235Ala and Gly237Ala (positions according to the EU numbering scheme) compared to the wild-type sequence from which it is derived; Preferably, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region originating from or derived from a human immunoglobulin (e.g., kappa or lambda), Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 30 or a variant thereof, wherein the variant has 20 or fewer conservative amino acid substitutions (e.g., 15 or fewer, 10 or fewer, or 5 or fewer conservative amino acid substitutions, e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared to SEQ ID NO: 30; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 31 or a variant thereof, wherein the variant has 20 or fewer conservative amino acid substitutions (e.g., 15 or fewer, 10 or fewer, or 5 or fewer conservative amino acid substitutions, e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared to SEQ ID NO: 31; Preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) set forth in SEQ ID NO: 32 or a variant thereof, wherein the variant has 20 or fewer conservative amino acid substitutions (e.g., 15 or fewer, 10 or fewer, or 5 or fewer conservative amino acid substitutions, e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared to SEQ ID NO: 32; More preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) represented by SEQ ID NO: 30 and a light chain constant region (CL) represented by SEQ ID NO:

32. Alternatively, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) represented by SEQ ID NO: 31 and a light chain constant region (CL) represented by SEQ ID NO:

32. The antibody-drug conjugate of any one of claims 1 to 5.

7. The antibody or antigen-binding fragment thereof is (1) A heavy chain comprising a VH represented by SEQ ID NO: 1 and a heavy chain constant region (CH) represented by SEQ ID NO: 30, and a light chain comprising a VL represented by SEQ ID NO: 2 and a light chain constant region (CL) represented by SEQ ID NO: 32; (2) A heavy chain comprising a VH represented by SEQ ID NO: 3 and a heavy chain constant region (CH) represented by SEQ ID NO: 30, and a light chain comprising a VL represented by SEQ ID NO: 4 and a light chain constant region (CL) represented by SEQ ID NO: 32; (3) A heavy chain comprising a VH represented by SEQ ID NO: 1 and a heavy chain constant region (CH) represented by SEQ ID NO: 31, and a light chain comprising a VL represented by SEQ ID NO: 2 and a light chain constant region (CL) represented by SEQ ID NO: 32; Or, (4) A heavy chain comprising a VH represented by SEQ ID NO: 3 and a heavy chain constant region (CH) represented by SEQ ID NO: 31, and a light chain comprising a VL represented by SEQ ID NO: 4 and a light chain constant region (CL) represented by SEQ ID NO:

32. Including; Preferably, the antibody or antigen-binding fragment thereof is: (1) a heavy chain having the sequence set forth in SEQ ID NO: 40 and a light chain having the sequence set forth in SEQ ID NO: 41; or (2) The antibody-drug conjugate according to any one of claims 1 to 6, comprising a heavy chain having the sequence shown in SEQ ID NO: 42 and a light chain having the sequence shown in SEQ ID NO:

43.

8. The antibody-drug conjugate of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof is selected from ScFv, Fab, Fab', Fab'-SH, (Fab')2, Fv fragment, disulfide-linked Fv (dsFv), a diabody, a bispecific antibody, and a multispecific antibody.

9. The antibody or antigen-binding fragment thereof is (a) (i) an antibody heavy chain variable region encoded by a nucleic acid molecule having a nucleotide sequence set forth in SEQ ID NO: 33, (ii) a sequence essentially the same as SEQ ID NO: 33 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 33, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above, and / or (iv) a nucleotide sequence set forth in SEQ ID NO: 34, (v) a sequence essentially the same as SEQ ID NO: 34 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 34, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v); or (b) (i) an antibody heavy chain variable region encoded by a nucleic acid molecule having a nucleotide sequence set forth in SEQ ID NO: 35, (ii) essentially the same sequence as SEQ ID NO: 35 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 35, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above; and / or (iv) a nucleotide sequence set forth in SEQ ID NO: 36, (v) essentially the same sequence as SEQ ID NO: 36 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 36, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v) above. The antibody-drug conjugate of any one of claims 1 to 8, comprising:

10. M is, 【Chemistry 1】 wherein ring A is a 5- to 6-membered aliphatic heterocycle or a 5- to 20-membered aromatic ring system, and the aliphatic heterocycle and aromatic ring system optionally contain oxygen groups (═O), halogens, cyano groups, amino groups, carboxyl groups, thiol groups, and C １～６ substituted with one or more groups selected from alkyl groups, M １ is a single bond and C １～２０ Alkylene group, C ２～２０ Alkenylene group, C ２～２０ an alkynylene group or an amine group; Preferably, M is 【Chemistry 2】 In the formula, ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycycle formed by connecting one or more (for example, two) 6-membered aromatic heterocycles with a benzene ring or a 6-membered heteroaromatic ring via a single bond, and the aliphatic heterocycle may optionally contain an oxygen group (═O), a halogen atom, and C １～４ substituted with one or more groups selected from alkyl groups, M １ is a single bond, C １～２０ Alkylene group, C ２～２０ Alkenylene group, C ２～２０ selected from an alkynylene group or an amine group; Preferably, M is 【Transformation 3】 (Wherein, ring A is 【Chemistry 4】 is selected from M １ is a single bond and C １～６ Alkylene group, C ２～６ Alkenylene group, C ２～６ an alkynylene group or an amine group; Preferably, M is 【Transformation 5】 is selected from Preferably, M is 【Transformation 6】 is selected from Preferably, M is 【Transformation 7】 is selected from Preferably, M is 【Transformation 8】 The antibody-drug conjugate according to any one of claims 1 to 9, wherein the antibody-drug conjugate is selected from the group consisting of:

11. L is C １～６ Alkylene groups, -N(R')-, carbonyl groups, -O-, natural amino acids or unnatural amino acids and analogs thereof (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH ２ CH ２ (OCH ２ CH ２ ) ｒ OCH ３ )), and short peptides consisting of amino acids (e.g., Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Gly u-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val -Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), 【Chemistry 9】 In the formula, R′ is hydrogen, C １～６ represents an alkyl group or a polyethylene glycol fragment having 1 to 10 EO units, and s is selected from an integer of 1 to 20; Preferably, L is C １～６ an alkylene group, a carbonyl group, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly, 【Chemistry 10】 wherein s is selected from an integer from 1 to 20; Preferably, L is 【Chemistry 11】 【Chemistry 12】 and selected from the structure consisting of one or more of: Preferably, L is 【Chemistry 13】 is selected from the structure Preferably, L is 【Chemistry 14】 is selected from the structure Preferably, L is 【Chemistry 15】 is selected from the structure Preferably, L is 【Chemistry 16】 The antibody-drug conjugate according to any one of claims 1 to 10, wherein the structure is selected from the following:

12. E is a single bond, -NHCH ２ - or 【Chemistry 17】 is selected from the structure Preferably, E is a single bond, —NHCH ２ -, [Chemistry 18] and Preferably, E is —NHCH ２ -or 【Chemistry 19】 and Preferably, E is —NHCH ２ - and Preferably, E is a single bond, Preferably, E is 【Chemistry 20】 The antibody-drug conjugate according to any one of claims 1 to 11, wherein 【Request Item 13】 【Chemistry 21】 teeth, 【Chemistry 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemistry 27】 【Chemistry 28】 is selected from the structure Preferably, 【Chemistry 29】 teeth, 【Transformation 30】 【Chemistry 31】 【Chemistry 32】 The antibody-drug conjugate according to any one of claims 1 to 12, wherein the structure is selected from the following:

14. the payload is selected from a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor; preferably, the tubulin inhibitor is an auristatin compound or a maytansine compound; preferably, the DNA intercalator is a pyrrolobenzodiazepine (PBD); preferably, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, nogitecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., adriamycin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); preferably, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analog thereof; Preferably, the payload is of Formula I, Formula II 【Transformation 33】 (In the formula, R １ , R ２ are each independently C １～６ selected from alkyl groups and halogens; R ３ is H and -CO-CH ２ OH, R ４ and R ５ are each independently selected from H, halogen and hydroxyl groups, or R ４ and R ５ forms a 5- to 6-membered oxygen-containing heterocycle together with the carbon atom to which it is attached, R ６ is hydrogen or -C １～４ Alkylene group -NR ａ R ｂ is selected from R ７ is C １～６ Alkyl group and -C １～４ Alkylene group -NR ａ R ｂ is selected from In the formula, R ａ , R ｂ are independently H, C for each occurrence. １～６ Alkyl group, —SO ２ -C １～６ Alkyl group and —CO—C １～６ alkyl group), or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer or prodrug of a compound of Formula I or Formula II, Preferably, the payload is one of the following compounds: 【Transformation 34】 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer or prodrug of said compound; The fragment corresponding to the payload obtained after the payload is linked to the linker is D in the general formula, and preferably D is -OH, -NH on the payload. ２ or a monovalent structure obtained by losing one H from the secondary amine group, Preferably, the payload is one of the following compounds: 【Chemistry 35】 or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer or prodrug of said compound.

15. ADC A-01 to ADC A-34, ADC B-01 to ADC B-07 shown below, or 【Transformation 36】 【Chemistry 37】 【Transformation 38】 【Chemistry 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 【Chemistry 43】 【Chemistry 44】 【Chemistry 45】 【Chemistry 46】 (wherein a thiol group on the antibody forms a sulfide bond with a linker compound of a drug through an addition reaction or a substitution reaction to obtain a complete antibody-drug conjugate; x represents the drug-antibody ratio; Preferably, the Ab in each antibody-drug conjugate represents an antibody or antigen-binding fragment thereof comprising a VH set forth in SEQ ID NO: 3 and a VL set forth in SEQ ID NO: 4, such as an antibody or antigen-binding fragment thereof comprising a VH set forth in SEQ ID NO: 3 and a CH set forth in SEQ ID NO: 31, and a VL set forth in SEQ ID NO: 4 and a VL set forth in SEQ ID NO: 51; During the ceremony, 【Chemistry 47】 The antibody-drug conjugate according to any one of claims 1 to 14, wherein:

16. The DAR value (drug-antibody binding ratio) is 1 to 10, for example, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 up to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10, preferably 3 to 9, for example, 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.0 to 5.5, 3.0 to 6.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 3.5 to 5.5, 3.5 to 6.0, 3.5 to 6.5, 3.5 to 7.0, 3.5 to 7.5, 3.5-8.0, 4.0-4.5, 4.0-5.0, 4.0-5.5, 4.0-6.0, 4.0-6.5, 4.0-7.0, 4.0-7.5, 4.0-8.0, 4.5-5.0, 4.5-5.5, 4.5-6.0, 4.5-6.5, 4.5-7.0, 4.5-7.5, 4.5-8.0, 5.0-5.5, 5.0-6.0, 5.0-6.5, 5.0-7.0, 5.0-7.5, 5 0 to 8.0, 5.5 to 6.0, 5.5 to 6.5, 5.5 to 7.0, 5.5 to 7.5, 5.5 to 8.0, 6.0 to 6.5, 6.0 to 7.0, 6.0 to 7.5, 6.0 to 8.5, 6.5 to 7.0, 6.5 to 7.5, 6.5 to 8.5, 7.0 to 7.5, 7.0 to 9.0 or 7.5 to 9.0, more preferably 4 to 8.

17. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 16 and one or more pharmaceutical additives.

18. Use of the antibody-drug conjugate of any one of claims 1 to 16 or the pharmaceutical composition of claim 19 in a medicament for treating B7-H3-positive tumors, comprising: Preferably, the B7-H3-positive tumor includes a solid tumor or a blood cancer, such as colon cancer, gastric cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, melanoma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, etc.), kidney cancer, bladder cancer, thyroid cancer, mesothelioma, pancreatic cancer, ovarian cancer, endometrial cancer, esophageal cancer, liver cancer, salivary gland cancer, bile duct cancer, meningioma, etc.

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