Antibody drug conjugate as well as preparation method and application thereof

Abstract

The invention relates to an antibody drug conjugate as well as a preparation method and application thereof. In particular to an antibody drug conjugate for treating B7-H3 positive tumors. The antibody drug conjugate provided by the invention comprises a B7-H3 antibody, and the antibody has excellent binding activity to B7-H3 positive cells and can efficiently deliver a drug to the B7-H3 positive cells. The antibody drug conjugate has an excellent drug-antibody coupling ratio, and has a good targeted killing effect on tumors such as lung cancer, breast cancer, gastric cancer and colon cancer. Therefore, the invention further provides a preparation method of the antibody drug conjugate and application of the antibody drug conjugate in treatment of B7-H3 positive tumors.

Description

Antibody drug conjugates and preparation methods and uses thereof Technical Field

[0001] This application relates to the field of targeted therapy, and more specifically, to an antibody-drug conjugate for treating B7-H3-positive tumors. Specifically, this application provides an antibody-drug conjugate comprising a B7-H3 antibody, which has excellent binding activity against B7-H3-positive cells and can efficiently deliver the drug to B7-H3-positive cells. The resulting antibody-drug conjugate has an excellent drug-antibody coupling ratio and exhibits excellent targeted killing effects against tumors such as breast cancer, colon cancer, and gastric cancer. Therefore, this application further provides a method for preparing the antibody-drug conjugate and its use in treating B7-H3-positive tumors. Background Art

[0002] Targeting the major disease area of ​​oncology, anti-tumor drug development has become a global focus and challenge. B7-H3's potential indications include 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, meningioma, and other high-incidence solid tumors. These diseases present significant clinical needs and are a hot area for major pharmaceutical companies to develop.

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

[0004] Research has shown that B7-H3 mRNA is overexpressed in a variety of tumor types, including breast cancer, colorectal cancer, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, and thyroid cancer. Multiple studies have demonstrated that B7-H3 plays a crucial role in tumor progression, including promoting tumor proliferation and migration, mediating EMT (epitope-mediated metastasis) in tumor cells, and influencing tumor cell metabolism. B7-H3 expression is regulated by oncogenic genes, and upregulation of B7-H3 promotes tumor growth and metastasis through multiple signaling pathways. Currently, there is a relatively concentrated pipeline of companies targeting this target, including Macrogenics, which has monoclonal antibodies, bispecific antibodies, and ADCs; Daiichi Sankyo, which has monoclonal antibodies and ADCs; Y-mAbs, which has monoclonal antibodies, bispecific antibodies, and ADCs; and AbbVie and GT Biopharma, which have ADCs and factor-carrying bispecific antibodies, respectively. Preclinical studies have shown that B7-H3 expression is upregulated in tumors, macrophages, and DC cells of cancer patients compared with normal tissues. In mice with B7-H3 knockout, tumor growth was significantly inhibited (~50%) in a tumor cell challenge. DS7300, an ADC drug targeting B7-H3 developed by Daiichi Sankyo, has demonstrated good safety and preliminary efficacy in Phase I clinical trials.

[0005] Biomacromolecule drugs have become a crucial component of anti-tumor drugs and are experiencing rapid growth. ADCs, consisting of monoclonal antibodies coupled to highly toxic drugs via a bioactive linker, are potent anti-cancer drugs that specifically target cancer cells. Their precise recognition of targets significantly enhances efficacy and minimizes toxic side effects, making them a key research area for future anti-tumor drugs.

[0006] Summary of the Invention

[0007] The present application relates to an antibody-drug conjugate for treating B7-H3 positive tumors, and exemplarily discloses an antibody-drug conjugate with the general formula Ab-[MLED] using the fully humanized antibody 2#8890 as the targeting moiety. x The results showed that the conjugate had a relatively good drug-antibody coupling ratio and excellent binding activity to B7-H3-positive cells, and had a good targeted killing effect on B7-H3-positive tumors, such as non-small cell lung cancer, brain glioma, breast cancer, gastric cancer, colon cancer, etc. Therefore, the present application provides an antibody-drug conjugate for treating B7-H3-positive tumors, a pharmaceutical composition containing the antibody-drug conjugate, and their use in treating B7-H3-positive tumors.

[0008] Antibody Drug Conjugates

[0009] In one aspect, the present application provides an antibody drug conjugate having the formula Ab-[MLED] x The structure shown, wherein:

[0010] Ab is an antibody or antigen-binding fragment thereof that specifically binds to B7-H3;

[0011] M is a linker site with an antibody or antigen-binding fragment thereof;

[0012] L is a linker between the linkers M and E;

[0013] E is a structural fragment connecting L and D;

[0014] D is the cytotoxic drug fragment;

[0015] x is selected from 1 to 10.

[0016] In the present application, the inventors have developed a high-affinity fully human antibody with excellent properties, which can specifically recognize / bind to B7-H3, does not bind or essentially does not bind to B7-1, B7-2, B7-H1, B7-H2 and / or B7-H4, and has no ADCC activity, effectively avoiding side effects caused by ADCC function.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):

[0018] (a) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 1; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 2;

[0019] (b) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 3; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 4; or

[0020] (c) CDR-H1, CDR-H2 and CDR-H3 contained in the following heavy chain variable region (VH), and / or CDR-L1, CDR-L2 and CDR-L3 contained in the following light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation compared to the heavy chain variable region and / or light chain variable region described in any one of (a) or (b), and the mutation is a substitution, deletion or addition of one or several amino acids (for example, a substitution, deletion or addition of 1, 2 or 3 amino acids).

[0021] In certain embodiments, the substitutions are conservative substitutions.

[0022] In certain embodiments, the CDRs are defined according to the IMGT, Kabat, Chothia, or AbM numbering systems.

[0023] In certain embodiments, the B7-H3 comprises human B7-H3 and / or monkey B7-H3. In certain embodiments, the monkey is a rhesus monkey (Macaca mulatta).

[0024] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0025] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 5 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 6 or a variant thereof; CDR-H3 having a sequence of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 8 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 9 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 10 or a variant thereof; or

[0026] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof; CDR-H2 of SEQ ID NO: 19 or a variant thereof; CDR-H3 of SEQ ID NO: 20 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof; CDR-L2 of SEQ ID NO: 9 or a variant thereof; CDR-L3 of SEQ ID NO: 10 or a variant thereof;

[0027] Wherein, the variant described in any one of (1a) and (1b) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0028] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Chothia numbering system:

[0029] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof; CDR-H2 of SEQ ID NO: 12 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0030] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 21 or a variant thereof; CDR-H2 of SEQ ID NO: 22 or a variant thereof; CDR-H3 of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof;

[0031] Wherein, the variant described in any one of (2a) and (2b) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0032] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:

[0033] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 16 or a variant thereof; CDR-H2 of SEQ ID NO: 17 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0034] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 24 or a variant thereof; CDR-H2 of SEQ ID NO: 25 or a variant thereof; CDR-H3 of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof;

[0035] Wherein, the variant described in any one of (3a) and (3b) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0036] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0037] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof; CDR-H2 of SEQ ID NO: 27 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0038] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof; CDR-H2 of SEQ ID NO: 29 or a variant thereof; CDR-H3 of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof;

[0039] Wherein, the variant described in any one of (4a) and (4b) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0040] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL):

[0041] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence of SEQ ID NO: 5 or a variant thereof; CDR-H2 having a sequence of SEQ ID NO: 6 or a variant thereof; CDR-H3 having a sequence of SEQ ID NO: 7 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence of SEQ ID NO: 8 or a variant thereof; CDR-L2 having a sequence of SEQ ID NO: 9 or a variant thereof; CDR-L3 having a sequence of SEQ ID NO: 10 or a variant thereof; or

[0042] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18 or a variant thereof; CDR-H2 of SEQ ID NO: 19 or a variant thereof; CDR-H3 of SEQ ID NO: 20 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8 or a variant thereof; CDR-L2 of SEQ ID NO: 9 or a variant thereof; CDR-L3 of SEQ ID NO: 10 or a variant thereof;

[0043] Wherein, the variant described in any one of (1a) and (1b) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0044] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL):

[0045] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11 or a variant thereof; CDR-H2 of SEQ ID NO: 12 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0046] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 21 or a variant thereof; CDR-H2 of SEQ ID NO: 22 or a variant thereof; CDR-H3 of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof;

[0047] Wherein, the variant described in any one of (2a) and (2b) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0048] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL):

[0049] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 16 or a variant thereof; CDR-H2 of SEQ ID NO: 17 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0050] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 24 or a variant thereof; CDR-H2 of SEQ ID NO: 25 or a variant thereof; CDR-H3 of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof;

[0051] Wherein, the variant described in any one of (3a) and (3b) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0052] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL):

[0053] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26 or a variant thereof; CDR-H2 of SEQ ID NO: 27 or a variant thereof; CDR-H3 of SEQ ID NO: 13 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; CDR-L3 of SEQ ID NO: 10 or a variant thereof; or

[0054] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 28 or a variant thereof; CDR-H2 of SEQ ID NO: 29 or a variant thereof; CDR-H3 of SEQ ID NO: 23 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14 or a variant thereof; CDR-L2 of SEQ ID NO: 15 or a variant thereof; and CDR-L3 of SEQ ID NO: 10 or a variant thereof;

[0055] Wherein, the variant described in any one of (4a) and (4b) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0056] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL):

[0057] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 5; CDR-H2 of SEQ ID NO: 6; and CDR-H3 of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8; CDR-L2 of SEQ ID NO: 9; and CDR-L3 of SEQ ID NO: 10; or

[0058] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18; CDR-H2 of SEQ ID NO: 19; and CDR-H3 of SEQ ID NO: 20; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8; CDR-L2 of SEQ ID NO: 9; and CDR-L3 of SEQ ID NO: 10;

[0059] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL):

[0060] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11; CDR-H2 of SEQ ID NO: 12; and CDR-H3 of SEQ ID NO: 13; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14; CDR-L2 of SEQ ID NO: 15; and CDR-L3 of SEQ ID NO: 10; or

[0061] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 21; CDR-H2 of SEQ ID NO: 22; and CDR-H3 of SEQ ID NO: 23; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14; CDR-L2 of SEQ ID NO: 15; and CDR-L3 of SEQ ID NO: 10;

[0062] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL):

[0063] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 16; CDR-H2 of SEQ ID NO: 17; and CDR-H3 of SEQ ID NO: 13; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14; CDR-L2 of SEQ ID NO: 15; and CDR-L3 of SEQ ID NO: 10; or

[0064] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 24; CDR-H2 of SEQ ID NO: 25; and CDR-H3 of SEQ ID NO: 23; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14; CDR-L2 of SEQ ID NO: 15; and CDR-L3 of SEQ ID NO: 10;

[0065] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL):

[0066] (4a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26; CDR-H2 of SEQ ID NO: 27; and CDR-H3 of SEQ ID NO: 13; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14; CDR-L2 of SEQ ID NO: 15; and CDR-L3 of SEQ ID NO: 10; or

[0067] (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 28; CDR-H2 with a sequence of SEQ ID NO: 29; CDR-H3 with a sequence of SEQ ID NO: 23; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 14; CDR-L2 with a sequence of SEQ ID NO: 15; and CDR-L3 with a sequence of SEQ ID NO: 10.

[0068] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 5; CDR-H2 with a sequence of SEQ ID NO: 6; CDR-H3 with a sequence of SEQ ID NO: 7; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 8; CDR-L2 with a sequence of SEQ ID NO: 9; and CDR-L3 with a sequence of SEQ ID NO: 10.

[0069] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 18; CDR-H2 of SEQ ID NO: 19; and CDR-H3 of SEQ ID NO: 20; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 8; CDR-L2 of SEQ ID NO: 9; and CDR-L3 of SEQ ID NO: 10.

[0070] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 11; CDR-H2 of SEQ ID NO: 12; and CDR-H3 of SEQ ID NO: 13; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14; CDR-L2 of SEQ ID NO: 15; and CDR-L3 of SEQ ID NO: 10; or

[0071] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 21; CDR-H2 with a sequence of SEQ ID NO: 22; and CDR-H3 with a sequence of SEQ ID NO: 23; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 14; CDR-L2 with a sequence of SEQ ID NO: 15; and CDR-L3 with a sequence of SEQ ID NO: 10.

[0072] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 16; CDR-H2 of SEQ ID NO: 17; CDR-H3 of SEQ ID NO: 13; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14; CDR-L2 of SEQ ID NO: 15; and CDR-L3 of SEQ ID NO: 10.

[0073] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 24; CDR-H2 with a sequence of SEQ ID NO: 25; CDR-H3 with a sequence of SEQ ID NO: 23; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 14; CDR-L2 with a sequence of SEQ ID NO: 15; and CDR-L3 with a sequence of SEQ ID NO: 10.

[0074] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 26; CDR-H2 of SEQ ID NO: 27; CDR-H3 of SEQ ID NO: 13; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 14; CDR-L2 of SEQ ID NO: 15; and CDR-L3 of SEQ ID NO: 10.

[0075] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 28; CDR-H2 with a sequence of SEQ ID NO: 29; CDR-H3 with a sequence of SEQ ID NO: 23; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 14; CDR-L2 with a sequence of SEQ ID NO: 15; and CDR-L3 with a sequence of SEQ ID NO: 10.

[0076] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0077] (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

[0078] (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;

[0079] wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0080] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments may comprise a constant region from or derived from a human immunoglobulin.

[0081] In certain embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises from or is derived from the heavy chain constant region of human immunoglobulin (such as IgG1, IgG2, IgG3 or IgG4).In certain embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises wild-type Fc district, or comprises mutated or chemically modified Fc district, which has the effector function (such as reduced ADCC activity) of change compared with wild-type Fc district.In certain exemplary embodiments, the antibody or its antigen-binding fragment of the present invention comprises the variant of human IgG1 heavy chain constant region, and the variant has the following displacement compared with the wild-type sequence from which it is derived: Leu234Ala, Leu235Ala and Gly237Ala (according to the position of EU numbering system).In such embodiments, the antibody or its antigen-binding fragment of the present invention has reduced ADCC activity. In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a sequence as set forth in SEQ ID NO: 30 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared thereto (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids). In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a sequence as set forth in SEQ ID NO: 31 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared thereto (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids).

[0082] In certain embodiments, the light chain of the antibody or its antigen-binding fragment comprises a light chain constant region from or derived from a human immunoglobulin (e.g., κ or λ). In certain embodiments, the light chain of the antibody or its antigen-binding fragment comprises a sequence as shown in SEQ ID NO: 32 or a variant thereof, the variant having a conservative substitution of up to 20 amino acids compared thereto (e.g., a conservative substitution of up to 15, up to 10, or up to 5 amino acids; e.g., a conservative substitution of 1, 2, 3, 4, or 5 amino acids).

[0083] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0084] (1) a heavy chain comprising the VH of SEQ ID NO: 1 and the heavy chain constant region (CH) of SEQ ID NO: 30, and a light chain comprising the VL of SEQ ID NO: 2 and the light chain constant region (CL) of SEQ ID NO: 32;

[0085] (2) a heavy chain comprising the VH of SEQ ID NO: 3 and the heavy chain constant region (CH) of SEQ ID NO: 30, and a light chain comprising the VL of SEQ ID NO: 4 and the light chain constant region (CL) of SEQ ID NO: 32;

[0086] (3) a heavy chain comprising the VH of SEQ ID NO: 1 and the heavy chain constant region (CH) of SEQ ID NO: 31, and a light chain comprising the VL of SEQ ID NO: 2 and the light chain constant region (CL) of SEQ ID NO: 32;

[0087] or,

[0088] (4) A heavy chain comprising the VH represented by SEQ ID NO: 3 and the heavy chain constant region (CH) represented by SEQ ID NO: 31, and a light chain comprising the VL represented by the sequence represented by SEQ ID NO: 4 and the light chain constant region (CL) represented by SEQ ID NO: 32.

[0089] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0090] (1) a heavy chain having the sequence shown in SEQ ID NO: 40 and a light chain having the sequence shown in SEQ ID NO: 41; or,

[0091] (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.

[0092] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0093] In certain embodiments, the variable region of the antibody or antigen-binding fragment thereof described in any of the above embodiments is of human origin.

[0094] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments is selected from ScFv, Fab, Fab', (Fab')2, Fab'-SH, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody and multispecific antibody.

[0095] In specific embodiments of the antibodies or antibody drug conjugates disclosed herein, the heavy chain constant region may comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

[0096] In some embodiments, the N-terminal amino acid of the variable region of the antibody or antigen-binding fragment thereof can be cyclized to pyroglutamic acid.

[0097] Thus, the composition can comprise a population of antibody drug conjugates, wherein the antibody or antigen-binding fragment thereof of each antibody drug conjugate can independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid or the N-terminal amino acid is cyclized to pyroglutamic acid.

[0098] Thus, in specific embodiments, the present invention further provides a composition comprising an antibody drug conjugate disclosed herein, wherein the primary antibody drug conjugate in the composition comprises (i) an antibody wherein the heavy chain C-terminus lacks a lysine residue; (ii) an antibody wherein the heavy chain N-terminus is glutamine, glutamic acid, or pyroglutamic acid; (iii) an antibody wherein the heavy chain C-terminus lacks a lysine residue and the heavy chain N-terminus is glutamine, glutamic acid, or pyroglutamic acid; (iv) an antibody wherein the heavy chain C-terminus lacks a lysine residue and the heavy chain N-terminus is a pyroglutamic acid residue; or, (v) an antibody wherein the heavy chain C-terminus lacks a lysine residue and the heavy chain N-terminus is a glutamine or glutamic acid residue.

[0099] 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 also provides a composition comprising one or more antibody drug conjugates as described in any one of the above.

[0100] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region encoded by the following nucleic acid molecule: (i) the nucleotide sequence set forth in SEQ ID NO:33, (ii) a sequence substantially identical to SEQ ID NO:33 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:33), 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 substantially identical to SEQ ID NO:34 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:34), or (vi) a degenerate sequence of (iv) or (v) above.

[0101] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the present invention, or a heavy chain variable region and / or a light chain variable region thereof. According to codon degeneracy in the art, in certain embodiments, the nucleotide sequence can be replaced based on codon degeneracy. In certain embodiments, the nucleotide sequence is codon-optimized.

[0102] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence set forth in SEQ ID NO:33, (ii) a sequence substantially identical to SEQ ID NO:33 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:33), 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 substantially identical to SEQ ID NO:34 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:34), or (vi) a degenerate sequence of (iv) or (v) above.

[0103] In certain embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence set forth in SEQ ID NO:35, (ii) a sequence substantially identical to SEQ ID NO:35 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:35), 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:36, (v) a sequence substantially identical to SEQ ID NO:36 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to SEQ ID NO:36), or (vi) a degenerate sequence of (iv) or (v) above. In certain embodiments, the antibody or antigen-binding fragment thereof having the features of any one of (1a), (2a), (3a), (4a) or (a) above further has a feature selected from the following:

[0104] (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, the EC50 is measured by ELISA;

[0105] (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, the KD is determined by biofilm interferometry (BLI) (e.g., ForteBio ) measured;

[0106] (3) does not bind or does not substantially bind to B7-1, B7-2, B7-H1, B7-H2, and / or B7-H4; for example, as determined by ELISA;

[0107] (4) having CDC activity, such as inducing killing of cells expressing B7-H3 (e.g., tumor cells) through CDC;

[0108] (5) No ADCC activity;

[0109] (6) inducing B7-H3 internalization, e.g., as measured by flow cytometry;

[0110] (7) inhibiting cell (such as tumor cell) proliferation; and / or

[0111] (8) Inhibit tumor growth.

[0112] In certain embodiments, the antibody or antigen-binding fragment thereof having the features of any one of (1b), (2b), (3b), (4b) or (b) above further has a feature selected from the following:

[0113] (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, the EC50 is measured by ELISA;

[0114] (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, the KD is determined by biofilm interferometry (BLI) (e.g., ForteBio ) measured;

[0115] (3) does not bind or does not substantially bind to B7-1, B7-2, B7-H1, B7-H2, and / or B7-H4; for example, as determined by ELISA;

[0116] (4) having CDC activity, such as inducing killing of cells expressing B7-H3 (e.g., tumor cells) through CDC;

[0117] (5) No ADCC activity;

[0118] (6) inducing B7-H3 internalization, e.g., as measured by flow cytometry;

[0119] (7) inhibiting cell (such as tumor cell) proliferation; and / or

[0120] (8) Inhibit tumor growth.

[0121] In certain embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments is labeled. In certain embodiments, the antibody or antigen-binding fragment thereof is labeled with a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

[0122] Derivatized antibodies

[0123] The antibodies or antigen-binding fragments thereof of the present invention may be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof will not adversely affect its binding to B7-H3 (particularly human B7-H3). Therefore, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms. For example, an antibody or antigen-binding fragment thereof of the present invention may be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical agent, and / or a protein or polypeptide capable of mediating binding of the antibody or antigen-binding fragment to another molecule (e.g., an avidin or polyhistidine tag).

[0124] As one of the derivatives of the antibody, the present invention provides a conjugate comprising the antibody or antigen-binding fragment thereof of the present invention and a conjugated moiety.

[0125] In certain embodiments, the coupling moiety is selected from a detectable label. The detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In certain embodiments, the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme. In certain embodiments, the detectable label as described above can be linked to the antibody or antigen-binding fragment thereof of the present invention by linkers of varying lengths to reduce potential steric hindrance.

[0126] In certain embodiments, the conjugated moiety is selected from a therapeutic agent. In certain embodiments, the therapeutic agent is preferably an anti-tumor agent, such as a cytotoxic agent, a cytokine, a toxin or a radionuclide.

[0127] In certain embodiments, the conjugated moiety is selected from substances that can improve the biological properties of the antibody (eg, increase serum half-life), for example, a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a sugar group.

[0128] As one of the antibody derivatives, the present invention provides a multispecific antibody comprising the antibody of the present invention or an antigen-binding fragment thereof.

[0129] In certain embodiments, the multispecific antibody comprises an antibody of the present invention or an antigen-binding fragment thereof as a first antigen-binding domain and further comprises at least one second antigen-binding domain directed against another target.

[0130] In certain embodiments, each antigen-binding domain of the multispecific antibody retains its respective original binding specificity.

[0131] In certain embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0132] As one of the derivatives of the antibody, the present invention provides a chimeric antigen receptor, which includes an antibody or antigen-binding fragment thereof of the present invention. In certain embodiments, the chimeric antigen receptor includes an antibody or antigen-binding fragment thereof (e.g., ScFv) of the present invention as an extracellular antigen-binding domain that specifically binds to B7-H3, as well as a transmembrane domain and one or more intracellular T cell signaling domains. The present invention also provides a host cell (e.g., an immune cell, such as a T lymphocyte, NK cell, DC cell, macrophage) containing or expressing the chimeric antigen receptor.

[0133] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0134] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture fluid. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0135] In the antibody-drug conjugate, the cytotoxic drug can be linked to the antibody or antigen-binding fragment thereof via a linker (such as the "MLE" fragment shown in this application).

[0136] In some embodiments, M is Wherein ring A is a 5-6 membered alicyclic heterocyclic ring, or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl group substituted; M1 is selected from single bond and C 1-20 Alkylene, C 2-20 Alkenylene, C 2- 20 Alkynylene or amino.

[0137] In some embodiments, M is wherein Ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more (e.g., 2) 6-membered heteroaromatic rings to a benzene ring or a 6-membered heteroaromatic ring via a single bond, wherein the alicyclic heterocycle is optionally substituted by one or more selected from oxy (=O), halogen and C 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkynylene or amino.

[0138] In some embodiments, M is wherein ring A is selected from M1 is selected from a single bond and C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 an alkynylene or amine group;

[0139] In some embodiments, M is selected from

[0140] In some embodiments, M is selected from

[0141] In some embodiments, M is selected from

[0142] In some embodiments, M is selected from

[0143] In some embodiments, L is selected from the structure consisting of one or more of the following: 1-6 Alkylene, -N(R')-, carbonyl, -O-, natural or unnatural amino acids and their analogs (such as 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)), and short peptides composed of amino acids (such as 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, Val- Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly),

[0144] Where R' represents hydrogen, C 1-6 Alkyl or polyethylene glycol fragment containing 1-10 EO units (i.e., -(CH2CH2O) r -alkyl, wherein r is selected from an integer of 1-10, and alkyl is methyl or ethyl); s is selected from an integer of 1-20;

[0145] In some embodiments, L is selected from the structure consisting of one or more of the following: 1-6 Alkylene, carbonyl, -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-L ys, 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, wherein s is an integer selected from 1 to 20;

[0146] In some embodiments, L is selected from a structure consisting of one or more of the following:

[0147] In some embodiments, L is selected from the following structures:

[0148] In some embodiments, L is selected from the following structures:

[0149] In some embodiments, L is selected from the following structures:

[0150] In some embodiments, L is selected from the following structures:

[0151] In some embodiments, E is a single bond, -NHCH2-, or is selected from the following structures:

[0152] In some embodiments, E is a single bond, -NHCH2-, In some embodiments, E is -NHCH2- or

[0153] In some embodiments, E is -NHCH2-.

[0154] In some embodiments, E is a single bond. In some embodiments, E is In some embodiments, Selected from the following structures:

[0155] In certain embodiments, Selected from the following structures:

[0156] In some embodiments, the cytotoxic drug is selected from In some embodiments, the cytotoxic drug is selected from microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors and RNA polymerase inhibitors. In some embodiments, the microtubule 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, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, multicarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester or analog thereof.

[0157] The cytotoxic drugs disclosed in this application generally contain a variety of functional groups, such as hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH), primary amino (-NH2), secondary amine (-NR A H) or tertiary amine (-NR B R C ), where R A 、R B 、R C These represent only non-hydrogen substituents on N, through which the cytotoxic drug can be attached to the linker in the conjugate.

[0158] In some embodiments, the cytotoxic drug is linked to E in the antibody-drug conjugate through a -OH, -SH, primary amino group, secondary amine group, or tertiary amine group on the cytotoxic drug.

[0159] In some embodiments, the cytotoxic drug is selected from a compound of Formula I, Formula II, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, or prodrug of a compound of Formula I or Formula II:

[0160] Wherein, R1, R2 are each independently selected from C 1-6 Alkyl and halogen;

[0161] R3 is selected from H and -CO-CH2OH;

[0162] R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring;

[0163] R6 is selected from hydrogen or -C 1-4 Alkylene-NRa R b ;

[0164] R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ;

[0165] where R a 、R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6 alkyl;

[0166] In some embodiments, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:

[0167] The corresponding fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is D in the general formula; preferably, D is a monovalent structure obtained by losing one H from the -OH, -NH2 or secondary amine group on the cytotoxic drug.

[0168] In some embodiments, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:

[0169] In some embodiments, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:

[0170] In some embodiments, D is selected from the following structures:

[0171] 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 definition of Ab in the following diagram is as described above, wherein the thiol group on the antibody and the drug linker compound form a thioether bond through an addition reaction or a substitution reaction to obtain a complete antibody-drug conjugate, and x represents the amount of drug loading:

[0172] Wherein, Ab in the antibody-drug conjugate represents any one of the antibodies or antigen-binding fragments described above; wherein, Indicates the specific connection method between the thiol group in the antibody or its antigen-binding fragment and the linker.

[0173] In certain embodiments, Ab in each antibody-drug conjugate represents an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO: 3 and VL as shown in SEQ ID NO: 4, for example, an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO: 3 and CH as shown in SEQ ID NO: 31, and VL as shown in SEQ ID NO: 4 and CL as shown in SEQ ID NO: 32;

[0174] In certain embodiments, each antibody drug conjugate comprises a heavy chain as set forth in SEQ ID NO:42 and a light chain as set forth in SEQ ID NO:43.

[0175] In certain embodiments, the antibody drug conjugate is:

[0176] Wherein, Ab in the antibody-drug conjugate represents an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO: 3 and VL as shown in SEQ ID NO: 4; x is an integer from 1 to 10;

[0177] in, Indicates the specific connection method between the thiol group in the antibody or antigen-binding fragment thereof and the linker. In certain embodiments, the antibody drug conjugate is:

[0178] Wherein, Ab in the antibody-drug conjugate represents an antibody, and the antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0179] (i) the heavy chain variable region comprises the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 18; CDR-H2 with a sequence of SEQ ID NO: 19; and CDR-H3 with a sequence of SEQ ID NO: 20; and / or, wherein the light chain variable region comprises the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 8; CDR-L2 with a sequence of SEQ ID NO: 9; and CDR-L3 with a sequence of SEQ ID NO: 10; or

[0180] (ii) the heavy chain variable region comprises the following three CDRs: CDR-H1 of SEQ ID NO: 21; CDR-H2 of SEQ ID NO: 22; and CDR-H3 of SEQ ID NO: 23; and / or the light chain variable region comprises the following three CDRs: CDR-L1 of SEQ ID NO: 14; CDR-L2 of SEQ ID NO: 15; and CDR-L3 of SEQ ID NO: 10; or

[0181] (iii) the heavy chain variable region comprises the following three CDRs: CDR-H1 of SEQ ID NO: 24; CDR-H2 of SEQ ID NO: 25; and CDR-H3 of SEQ ID NO: 23; and / or the light chain variable region comprises the following three CDRs: CDR-L1 of SEQ ID NO: 14; CDR-L2 of SEQ ID NO: 15; and CDR-L3 of SEQ ID NO: 10;

[0182] (iv) the heavy chain variable region comprises the following three CDRs: CDR-H1 of SEQ ID NO: 28; CDR-H2 of SEQ ID NO: 29; and CDR-H3 of SEQ ID NO: 23; and / or the light chain variable region comprises the following three CDRs: CDR-L1 of SEQ ID NO: 14; CDR-L2 of SEQ ID NO: 15; and CDR-L3 of SEQ ID NO: 10;

[0183] in, Indicates the specific connection method between the thiol group in the antibody or its antigen-binding fragment and the linker.

[0184] In certain embodiments, the antibody drug conjugate is:

[0185] Wherein, Ab in the antibody-drug conjugate represents an antibody or an antigen-binding fragment thereof, wherein: the antibody has 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 from 1 to 10; and Ab is conjugated through one or more thiol groups to form a conjugate.

[0186] 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.

[0187] In some embodiments, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0188] In some embodiments, antibody disclosed herein is genetically engineered to include one or more cysteines or amino acid whose non-conventional amino acid replacements at the restricted position in the antibody.Then, these cysteine ​​residues or non-conventional amino acid residues can be coupled to a drug linker by the sulfhydryl group of cysteine ​​residues or the reactive group of non-conventional amino acid.Therefore, the antibody drug conjugate of the present invention can include one or more replacements of the amino acid in the heavy chain or light chain of the antibody with cysteine ​​residues or non-conventional amino acid residues, which are then coupled to drug-connector disclosed herein.In a specific embodiment, the amino acid position that can be substituted is selected from position 152,153,171,172,173 and 375 (numbering according to Eu numbering scheme) of heavy chain constant domain and position 165 and 168 (numbering starting from the amino acid 1 of N-terminal) of light chain constant domain. In a specific embodiment, cysteine ​​can be substituted for one or more of the amino acids at positions 152, 153, 171, 172, 173, and 375 of the heavy chain constant domain (numbered according to the Eu numbering scheme) and positions 165 and 168 of the light chain constant domain (numbered starting from the N-terminal amino acid 1). In a specific embodiment, the antibody drug conjugate comprises an S375C amino acid substitution coupled 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 coupled 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 coupled to a drug linker disclosed herein.

[0189] In some embodiments, the DAR value (drug-antibody conjugate ratio) of the antibody drug conjugate is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4- 10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-9, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3.5 ~6.0,3.5~6.5,3.5~7.0,3.5~7.5,3.5~8.0,4.0~4.5,4.0~5.0,4.0~5.5,4.0~6.0,4.0~6.5,4.0~7.0,4.0~7.5,4.0~8.0,4.5~5.0,4.5~5.5,4.5~6.0,4.5~6.5,4.5~7.0,4.5~7.5,4.5~8.0,5.0~5 .5, 5.0-6.0, 5.0-6.5, 5.0-7.0, 5.0-7.5, 5.0-8.0, 5.5-6.0, 5.5-6.5, 5.5-7.0, 5.5-7.5, 5.5-8.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.5, 6.5-7.0, 6.5-7.5, 6.5-8.5, 7.0-7.5, 7.0-9.0 or 7.5-9.0. In some embodiments, the DAR value of the antibody drug conjugate is 4-8.

[0190] It will be understood by those skilled in the art that the antibody-drug conjugates described herein can be prepared by modularizing the drug-linker. For example, first obtain the free form of the "drug-linker" (which can be understood as GM-[LED] x , wherein GM is the structural form before covalently linking to the antibody or antigen-binding fragment thereof), which is then covalently linked to the antibody or antigen-binding fragment thereof to obtain the antibody-drug conjugate described herein. Accordingly, the GM in the free form of the "drug-linker" is linked to one or more sulfhydryl (-SH), amino (-NH2), or carboxyl (-COOH) groups on the antibody or antigen-binding fragment thereof via a substitution reaction (e.g., removal of structures such as -SO2Me or -Br) or an addition reaction.

[0191] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the present invention. In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a lentivirus, or the like. In certain embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof of the present invention in a subject (e.g., a mammal, such as a human).

[0192] In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding the light chain or light chain variable region thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0193] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention can be used to construct a chimeric antigen receptor (CAR), which comprises an extracellular antigen binding domain (e.g., ScFv) specifically binding to 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 include a nucleotide sequence encoding a chimeric antigen receptor, which further includes a nucleotide sequence encoding an antibody of the present invention or its antigen-binding fragment (e.g., ScFv). In certain embodiments, the isolated nucleic acid 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.

[0194] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention can be used to construct chimeric antigen receptor-modified immune cells, wherein the chimeric antigen receptor-modified immune cells comprise a chimeric antigen receptor (CAR) and an immune cell (e.g., T lymphocytes, NK cells, DC cells, macrophages).

[0195] The present invention further provides a host cell comprising an isolated nucleic acid molecule of the present invention or a vector of the present invention. The host cell can be a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44, CHO-EBNA).

[0196] In certain embodiments, the host cell of the present invention may be a chimeric antigen receptor T cell (CAR-T). In such embodiments, the isolated nucleic acid molecule contained in the host cell may include a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further includes a nucleotide sequence encoding an antibody of the present invention or its antigen-binding fragment (e.g., ScFv). In certain embodiments, the isolated nucleic acid molecule contained in the host cell encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention.

[0197] In another aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell of the present invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0198] Composition

[0199] In another aspect, the present application provides a composition of an antibody drug conjugate (ADC) as described herein. Such a composition may comprise a plurality of ADCs as described herein, wherein each ADC comprises a drug-linker as described herein, wherein 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 in that the "drug-antibody" ratio (DAR) is in the range of about 1 to about 10. Methods for determining DAR are well known to those skilled in the art, including methods using reverse phase chromatography or HPLC-MS.

[0200] For example, in any embodiment, the ADC compositions described herein have a DAR of about 1 to about 10, or any subrange therebetween, e.g., 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 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.

[0201] In certain 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 8.5, about 7.0 to 7.5.

[0202] Pharmaceutical composition

[0203] 1. Pharmaceutical compositions of antibody-drug conjugates

[0204] In another aspect, the present application provides a pharmaceutical composition comprising the antibody-drug conjugate described in any one of the foregoing items, and one or more pharmaceutical excipients.

[0205] The antibody drug conjugates described herein are generally formulated in a unit injectable form together with a pharmaceutically acceptable parenteral vehicle for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc. Optionally, the antibody drug conjugate having the desired purity is mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer in the form of a lyophilized agent or solution (Remington's Pharmaceutical Sciences (1980) 16 th The antibody drug conjugates described herein or pharmaceutical compositions containing the same can be administered by any route appropriate to the subject to be treated.

[0206] 2. Pharmaceutical compositions of antibodies or antigen-binding fragments thereof

[0207] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or chimeric antigen receptor or host cell expressing the chimeric antigen receptor of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0208] In certain embodiments, the pharmaceutical compositions of the present invention comprise the antibodies or antigen-binding fragments thereof of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0209] In certain embodiments, the pharmaceutical composition of the present invention comprises a vector or host cell of the present invention, and a pharmaceutically acceptable carrier and / or excipient. In such embodiments, the isolated nucleic acid molecule contained in the vector comprises a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further comprises a nucleotide sequence encoding an antibody of the present invention or its antigen-binding fragment (e.g., ScFv); the host cell comprises an isolated nucleic acid molecule or vector as described above. In certain embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention. In certain embodiments, the host cell is an immune cell, such as a T cell. In certain embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0210] In certain embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent. In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity. In certain embodiments, the additional pharmaceutically active agent is selected from B7-H3 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutics or any combination thereof. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention and the additional pharmaceutically active agent are provided as independent components or as mixed components. Therefore, the antibody or antigen-binding fragment thereof of the present invention and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

[0211] In certain embodiments, the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or chimeric antigen receptor or host cell expressing the chimeric antigen receptor in the pharmaceutical composition of the present invention is sufficient (e.g., in a subject):

[0212] (a) Inhibit cell (such as tumor cell) proliferation;

[0213] (b) inhibiting tumor growth;

[0214] (c) inducing and / or increasing antibody-dependent cellular cytotoxicity;

[0215] (d) inhibiting B7-H3-mediated signaling;

[0216] (e) preventing and / or treating B7-H3 mediated diseases / disorders; or

[0217] (f) Any combination of (a)-(e).

[0218] In certain embodiments, the B7-H3 mediated disease / disorder is a tumor, e.g., a tumor expressing B7-H3. In certain embodiments, the tumor is selected from breast cancer, colorectal cancer, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, thyroid cancer, or any combination thereof.

[0219] The antibodies or antigen-binding fragments thereof and the pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc.

[0220] application

[0221] 1. Application of Antibody Drug Conjugates

[0222] The antibody drug conjugates or pharmaceutical compositions described herein can be used to treat a variety of diseases or conditions, such as B7-H3 positive tumors.

[0223] Therefore, the present application provides use of any of the above-mentioned antibody-drug conjugates or pharmaceutical compositions containing the same in the preparation of drugs for preventing and / or treating and / or adjuvant treating B7-H3-positive tumors.

[0224] At the same time, the present application also provides a method for preventing and / or treating and / or adjuvant treating B7-H3 positive tumors, which comprises the step of administering an effective amount of any of the above-mentioned antibody-drug conjugates or a pharmaceutical composition containing the same to a subject in need thereof.

[0225] The present application also provides the use of any of the above-described antibody drug conjugates or pharmaceutical compositions in inhibiting the proliferation of B7-H3-positive tumor cells. In certain embodiments, the antibody drug conjugate or pharmaceutical composition is administered to cells in vitro or in vivo; for example, applied to a subject to inhibit the proliferation of tumor cells in the subject; or, applied to in vitro tumor cells (e.g., cell lines or cells from a subject) to inhibit the proliferation of tumor cells in vitro.

[0226] In the present application, B7-H3-positive tumors include solid tumors or hematological malignancies, 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.), renal cancer, bladder cancer, thyroid cancer, mesothelioma, pancreatic cancer, ovarian cancer, endometrial cancer, esophageal cancer, liver cancer, salivary gland cancer, bile duct cancer, and meningioma.

[0227] In the present application, the subject is preferably a mammal, such as bovine, equine, porcine, canine, feline, rodent, or primate; for example, a human.

[0228] 2. Application of antibodies or their antigen-binding fragments

[0229] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention in the preparation of a medicament, wherein the medicament is used to inhibit cell proliferation, or prevent and / or treat and / or assist in the treatment of tumors.

[0230] In certain embodiments, the medicament is used to inhibit the proliferation of cells expressing B7-H3 (eg, tumor cells).

[0231] In another aspect, the present invention provides a method for inhibiting cell proliferation, comprising contacting the cell with an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor, or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention. In certain embodiments, the cell is a cell expressing B7-H3, such as a tumor cell.

[0232] In another aspect, the present invention provides a method for preventing and / or treating and / or adjuvant treating a tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention.

[0233] In certain embodiments, the method further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof. In certain embodiments, the second therapy can be applied simultaneously, separately, or sequentially with the above method.

[0234] In any of the above embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention can be any tumor type. In certain embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention is a B7-H3 positive tumor. In certain embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention is selected from breast cancer, colorectal cancer, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, thyroid cancer or any combination thereof.

[0235] Detection applications of antibodies or their antigen-binding fragments

[0236] The antibodies or antigen-binding fragments thereof of the present invention can specifically bind to B7-H3 and thus can be used to detect the presence or level of B7-H3 in a sample.

[0237] Therefore, in another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof of the present invention. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention carries a detectable label. In a preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the present invention. Preferably, the second antibody further comprises a detectable label.

[0238] In the present invention, the detectable label can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Particularly preferably, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. In certain embodiments, the detectable labels described above can be attached to the antibodies of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0239] In another aspect, the present invention provides a method for detecting the presence or level of B7-H3 in a sample, comprising the step of using an antibody or antigen-binding fragment thereof of the present invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention also carries 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 with a detectable label. The method can be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample rather than a sample from a patient).

[0240] In certain embodiments, the method comprises contacting the sample with the antibody or antigen-binding fragment thereof of the present invention under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and B7-H3, and detecting formation of the complex.

[0241] Given that B7-H3 is lowly expressed or absent in normal tissues, and is expressed or highly expressed in some cancers, tumors can be diagnosed by detecting the presence or level of B7-H3 in a sample. Therefore, in certain embodiments, the method is used to diagnose tumors, such as B7-H3-positive tumors, such as breast cancer, gastric cancer, lung cancer (such as 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.

[0242] In certain embodiments, the method comprises detecting the expression level of B7-H3 in a test sample from a subject, and comparing the expression level with a reference value (e.g., a healthy control), wherein an increase in the expression level compared to the reference value is indicative of a tumor.

[0243] In another aspect, provided is a use of the antibody or antigen-binding fragment thereof of the present invention in preparing a kit for detecting the presence or level of B7-H3 in a sample and / or diagnosing a tumor.

[0244] In another aspect, the present invention provides a diagnostic or therapeutic kit comprising an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, or multispecific antibody as described herein, and instructions for use. The kit may also include a drug delivery device for topical administration. The drug delivery device may include a prefilled syringe or a needle-free device.

[0245] definition

[0246] Unless otherwise defined below, the meanings of all technical and scientific terms used herein are intended to be the same as those generally understood by those skilled in the art. Reference to the techniques used herein is intended to refer to techniques generally understood in the art, including variations of those techniques that are obvious to those skilled in the art or replacements with equivalent techniques. Furthermore, laboratory procedures such as genomics, nucleic acid chemistry, and molecular biology used herein are conventional procedures widely used in the corresponding fields. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0247] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing a "D" region of approximately 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 is composed 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 is composed of one domain, CL. The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen binding site, respectively. The allocation of amino acids in each region or domain can follow various numbering systems known in the art.

[0248] The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, as defined in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0249] In the present invention, the CDRs contained in an antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art, such as those defined by the Kabat, Chothia, IMGT, or AbM numbering systems. In certain embodiments, the CDRs contained in an antibody or antigen-binding fragment thereof are defined by the Chothia numbering system.

[0250] The following general rules (published at www.bioinf.org.uk: Professor Andrew CR Martin's research group) can be used to define CDRs within an antibody sequence, which include amino acids that specifically interact with amino acids that make up the epitope to which the antibody binds. In rare cases, these normally constant features do not appear. However, Cys residues are the most conserved features.

[0251] The entire amino acid sequence of a VH is generally numbered according to Kabat, while the three CDRs within the variable region can be defined according to any of the aforementioned numbering systems. In specific embodiments, amino acid positions in a VH can be numbered sequentially starting from amino acid position 1 and continuing to the end of the sequence, or numbered according to Kabat. Unless otherwise indicated, amino acid positions in VH and VL herein are defined according to sequential numbering.

[0252] Amino acid positions in the heavy chain constant region can be numbered sequentially starting from amino acid position 1 and continuing to the end of the sequence, or numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence numbered according to Eu begins at position 118 and ends at position 447. Unless otherwise indicated, amino acid positions in the heavy and light chains described herein are defined according to sequential numbering.

[0253] The term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0254] The term "antigen-binding fragment" of an antibody refers to polypeptides that are fragments of an antibody, such as polypeptides that are fragments of a full-length antibody, which retain the ability to specifically bind to the same antigen bound by the full-length antibody and / or compete with the full-length antibody for specific binding to the antigen, and are also referred to as "antigen-binding portions." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAbs, nanobodies), and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0255] The term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0256] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0257] The term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but is not involved in antigen binding.

[0258] The term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, containing NH2-VH-VH-COOH, NH 2- VL-VL-COOH scFv.

[0259] The term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed 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 the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also called nanobodies.

[0260] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen.

[0261] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0262] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0263] The terms "monoclonal antibody," "single antibody," and "mAb" have the same meaning and are used interchangeably to refer to an antibody molecule from a group of highly homologous antibody molecules or antibody fragments (i.e., a population). Antibody molecules are identical except for natural mutations that may arise spontaneously. mAbs are highly specific for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically contain at least two or more different antibodies that typically recognize different epitopes on the antigen. Furthermore, the modifier "monoclonal" only indicates the characteristic of the antibody as being obtained from a highly homogeneous population of antibodies and should not be construed as requiring that the antibody be prepared by any particular method.

[0264] The term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from an antibody (which may be derived from a specific species or belong to a specific antibody class or subclass), while the other portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen. For example, the term "chimeric antibody" can include antibodies whose heavy chain and light chain variable regions are derived from a first antibody (e.g., human) and whose heavy chain and light chain constant regions are derived from a second antibody (e.g., mouse). For example, antibodies produced by immunizing fully human transgenic mice can be called chimeric antibodies, which consist of fully human variable regions and mouse constant regions.

[0265] The term "murine antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for murine hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, followed by screening, antibody preparation, and antibody purification; or refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells in mice after antigen invasion.

[0266] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0267] The term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 total positions match). Typically, two sequences are compared when aligned for maximum identity. Such alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0268] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived.

[0269] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured in terms of the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC). 50 )express.

[0270] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rates of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant, KD (see Davies et al., Annual Rev Biochem, 1990;59:439-473). KD, kon, and kdis values ​​can be measured using any valid method. In certain embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., the ForteBio Octet method). Surface plasmon resonance techniques (e.g., Biacore) or Kinexa can also be used to measure the dissociation constant.

[0271] The term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0272] The twenty conventional amino acids referred to herein are denoted according to 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 single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0273] The terms "comprises," "comprising," "having," "containing," or "involving," and other similar forms thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0274] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, for example, "C 1-20Alkyl", "C 1- 10 Alkyl", "C 1-6 Alkyl", "C 1-4 Alkyl", "C 1-3 alkyl”, etc., specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0275] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight or branched hydrocarbon group, for example, "C 1-20 Alkylene", "C 1-10 Alkylene", "C 3-10 Alkylene", "C 5-8 Alkylene", "C 1-6 Alkylene", "C 1-4 Alkylene", "C 1-3 Specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene.

[0276] The term "alkenylene" refers to a divalent group derived from a straight or branched hydrocarbon group containing at least one carbon-carbon double bond losing two hydrogen atoms, including, for example, "C 2-20 Alkenylene", "C 3-10 Alkenylene", "C 5-8 Examples include, but are not limited to, vinylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 1,3-butadienylene, 1-pentenylene, 2-pentenylene, 3-pentenylene, 1,3-pentadienylene, 1,4-pentadienylene, 1-hexenylene, 2-hexenylene, 3-hexenylene, 1,4-hexadienylene, and the like.

[0277] The term "alkynylene" refers to a divalent group derived from a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond that loses two hydrogen atoms. 2-20 Alkynylidene", "C 3-10 Alkynylidene", "C 5-8Examples include, but are not limited to, ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 1,3-butadiynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 1,3-pentadiynylene, 1,4-pentadiynylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 1,4-hexadiynylene, and the like.

[0278] 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, but are not limited to, 5-6 membered aliphatic heterocycles, 5-6 membered nitrogen-containing aliphatic heterocycles, 5-6 membered oxygen-containing aliphatic heterocycles, and the like, such as tetrahydrofuran, pyrrolidine, piperidine, tetrahydropyran, and the like.

[0279] 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, but are not limited to, 5-6 membered aromatic heterocycles, 5-6 membered nitrogen-containing aromatic heterocycles, 5-6 membered oxygen-containing aromatic heterocycles, and the like, 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, 1,2,4,5-tetrazine, and the like.

[0280] The term "aromatic ring system" refers to a monocyclic or polycyclic ring system comprising at least one aromatic ring (e.g., a benzene ring, etc.) or heteroaromatic ring (e.g., a 5-6 membered aromatic heterocycle, e.g., a 5-6 membered nitrogen-containing aromatic heterocycle, e.g., a pyrimidine ring, etc.); two or more aromatic rings and / or heteroaromatic rings may form a fused ring or be connected by a single bond (e.g., dipyrimidinylphenyl, etc.); the aromatic ring system may be divalent or higher valent (e.g., trivalent or tetravalent), e.g., a 5-20 membered aromatic ring system.

[0281] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), etc.

[0282] As used herein, the term "solvate" refers to a physical association of an ADC disclosed herein with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, the solvate will be capable of separation, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses both solution-phase and separable solvates. Non-limiting examples of solvates include ethanolates, methanolates, and the like. "Hydrate" is a solvate in which the solvent molecule is water.

[0283] One or more ADCs disclosed herein may optionally be converted into solvates. 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 solvates of the antifungal fluconazole in ethyl acetate and water. EC van Tonder et al., AAPS Pharm Sci Techours., 5(1), article 12 (2004) describe similar preparations of solvates, hemisolvates, hydrates, etc.; and ALBingham et al., Chemistry. Communications, 603-604 (2001). A typical non-limiting method comprises dissolving the compound of the invention in the desired amount of the desired 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 infrared spectroscopy show that the solvent (or water) in the crystals is present in the form of a solvate (or hydrate).

[0284] As used herein, the term "DAR" or "drug-antibody ratio" or "drug-antibody conjugation ratio" (as used interchangeably herein) refers to the average number of linker / payload moieties attached to each antibody present in a composition. For a composition comprising an ADC of the present 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, condition, or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization (i.e., no longer worsening) of the disease state, delaying or slowing the progression of the disease, improvement or alleviation of the disease state, and alleviation of symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0285] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) suffers from a tumor, or is at risk of suffering from the above-mentioned disease.

[0286] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0287] The terms "cancer" and "tumor" are used interchangeably to refer to a broad category of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors, or cells that invade neighboring tissues and may spread to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0288] The term "hematological malignancy" includes lymphoma, leukemia, myeloma or lymphoid malignancies, as well as spleen cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphomas and T-cell lymphomas. B-cell lymphomas include, for example, Hodgkin's lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphoma. Hematological malignancies also include leukemias, such as secondary leukemia or acute lymphoblastic leukemia. Hematological malignancies also include myeloma (e.g., multiple myeloma) and other hematological and / or B-cell or T-cell related cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0289] Figure 1A1: Binding assay of anti-human B7-H3 antibody and conjugate drug to HT29 cells

[0290] Figure 1A2: Binding assay of anti-human B7-H3 antibody and conjugate drug to HT29 cells II

[0291] Figure 1B: Binding assay of anti-human B7-H3 antibody and conjugate drug to NCI-N87 cells

[0292] Figure 1C: Binding assay of anti-human B7-H3 antibody and conjugate drug to HCC1806 cells

[0293] Figure 1D1: Binding assay of anti-human B7-H3 antibody and conjugate drug to HCC827 cells

[0294] Figure 1D2: Binding assay of anti-human B7-H3 antibody and conjugate drug to HCC827 cells II

[0295] Figure 1E: Binding assay of anti-human B7-H3 antibodies and conjugated drugs to CHOS-human B7-H3-4Ig cells

[0296] Figure 1F: Binding assay of anti-human B7-H3 antibody and conjugate drug to CHOS-human B7-H3-2Ig cells

[0297] Figure 2A: Binding assay of anti-human B7-H3 antibody and conjugated drug to CHOS-rat B7-H3 cells

[0298] Figure 2B: Binding assay of anti-human B7-H3 antibody and conjugated drug to CHOS-monkey B7-H3 cells

[0299] Figure 3A: Endocytosis activity assay of anti-human B7-H3 antibody and conjugated drug in NCI-N87 cells

[0300] Figure 3B: Endocytosis activity assay of anti-human B7-H3 antibody and conjugated drug in HCC1806 cells

[0301] Figure 3C: Endocytosis activity assay of anti-human B7-H3 antibody and conjugated drug in HCC827 cells

[0302] Figure 4A: Detection of anti-human B7-H3 conjugated drugs killing A375 cells

[0303] Figure 4B: Detection of anti-human B7-H3 conjugate drug killing of Calu6-B7-H3 cells

[0304] Figure 4C: Detection of U87MG-B7-H3 cell killing by anti-human B7-H3 conjugate drugs

[0305] Figure 5A: Efficacy testing of different antibody-drug conjugates in the HT29 model

[0306] Figure 5B: Body weight detection of HT29 model with different antibody-drug conjugates

[0307] Figure 6A: Efficacy testing of different antibody-drug conjugates in the HCC1806 model

[0308] Figure 6B: Body weight measurement of HCC1806 models with different antibody-drug conjugates

[0309] Figure 7A: Pharmacological efficacy testing of 2#8890 with different conjugated drugs in HCC1806 model

[0310] Figure 7B: Body weight test of HCC1806 model with different conjugated drugs of 2#8890

[0311] Figure 8A: Efficacy testing of different doses of antibody-drug conjugates in the HCC1806 model

[0312] Figure 8B: Body weight measurement of HCC1806 model with different doses of antibody-drug conjugates

[0313] Figure 9A: Efficacy testing of different antibody-drug conjugates in the NCI-N87 model

[0314] Figure 9B: Body weight test of NCI-N87 model with different antibody-drug conjugates

[0315] Figure 10: Drug concentration-time curves of ADC and total antibody (Tab) in cynomolgus monkey serum

[0316] Figure 11: Drug concentration-time curve of Payload in cynomolgus monkey serum DETAILED DESCRIPTION

[0317] The present invention will be further described below by describing specific embodiments, but this is not intended to limit the present invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic idea and scope of the present invention.

[0318] The information of the sequences involved in the present invention is described in the following table:

[0319] The abbreviations used in this document have the following meanings:

[0320] Abbreviation meaning

[0321] CDR Complementarity determining region in immunoglobulin variable region

[0322] FR Antibody framework region: amino acid residues in the variable region of an antibody other than CDR residues

[0323] VH antibody heavy chain variable region

[0324] VL Antibody light chain variable region

[0325] IgG immunoglobulin G

[0326] IMGT is based on the International Immunogenetics Information System (The

[0327] international ImMunoGeneTics information system (IMGT)) number

[0328] system, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003.

[0329] Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g.,

[0330] Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.

[0331] Public Health Service,National Institutes of Health,Bethesda,Md.,

[0332] 1991).

[0333] Chothia Immunoglobulin numbering system proposed by Chothia et al., which is based on the position of the structural loop regions.

[0334] Classical rules for identifying CDR region boundaries are set (see, e.g., Chothia & Lesk (1987) J.

[0335] Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0336] The definition of AbM CDR is derived from the relevant research of Martin (Martin ACR, Cheetham

[0337] JC,Rees AR(1989)Modelling antibody hypervariable loops:A

[0338] combined algorithm. Proc Natl Acad Sci USA 86:9268-9272).

[0339] mAb monoclonal antibody

[0340] EC 50Concentration that produces 50% efficacy or binding

[0341] IC 50 Concentration that produces 50% inhibition

[0342] ELISA enzyme-linked immunosorbent assay

[0343] PCR polymerase chain reaction

[0344] HRP horseradish peroxidase

[0345] K D Equilibrium dissociation constant

[0346] Ka binding rate constant

[0347] Kd dissociation rate constant

[0348] ADCC Antibody-dependent cell-mediated cytotoxicity

[0349] CDC Complement-dependent cytotoxicity

[0350] FACS flow cytometry technology

[0351] CDR-H1 Complementarity-determining region 1 in the immunoglobulin heavy chain variable region

[0352] CDR-H2 Complementarity-determining region 2 in the immunoglobulin heavy chain variable region

[0353] CDR-H3 Complementarity determining region 3 in the immunoglobulin heavy chain variable region

[0354] CDR-L1 Complementarity-determining region 1 in the immunoglobulin light chain variable region

[0355] CDR-L2 Complementarity determining region 2 in the immunoglobulin light chain variable region

[0356] CDR-L3 Complementarity determining region 3 in the immunoglobulin light chain variable region

[0357] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0358] Nuclear magnetic resonance (NMR) 1H NMR measurements were performed using a Bruker 400 MHz nuclear magnetic resonance instrument; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0359] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0360] 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.

[0361] Mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0362] 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-tetraazahexadec-16-yl-6-(2,5-dioxo-2,5-dihydro-1-H-pyrrol-1-yl)hexanamide (M-01)

[0363] Compound IM-1 (0.40 g, 640.59 μmol, its synthesis is described in patent CN 111936169A) and isotecan mesylate (0.37 g, 704.65 μmol) were dissolved in DMF (8 mL). HATU (0.32 g, 832.77 μmol) and DIPEA (0.25 g, 1.92 mmol) were added and reacted at 25°C for 4 hours. DIPEA was removed under reduced pressure, and the mixture was freeze-dried with water to remove most of the DMF to obtain a crude product. The crude product was purified by preparative HPLC (under the following conditions) to obtain 273 mg of compound M-01.

[0364] Column: Waters XBridge Prep C18OBD 45mm×450mm×8.0μm

[0365] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0366] The structural characterization data of M-01 are as follows:

[0367] ESI-MS (m / z): 1034.4 [M+H] + .

[0368] 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]indolizin[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxyl-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-05)

[0369] Under nitrogen protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (IM-2, 0.66 g, 1.80 mmol) and (R)-16-amino-10-benzyl-6,9,12,15-tetrahydro-3-oxo-5,8,11,14-tetraazahexadecanoic acid (IM-3, 0.75 g, 1.77 mmol) were added to DMF (19 mL), the temperature was raised to 35 ° C for 16 hours, and the reaction was stirred for 2 hours. (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (1-4, 1.00 g, 1.77 mmol) was added to the system, the mixture was cooled to 5-15°C with ice water, DMTMM (0.98 g, 3.53 mmol) was added, and DIPEA (1.14 g, 8.84 mmol) was added dropwise, and the mixture was reacted at 25°C for 16 hours. The reaction solution was poured into a mixture of DCM (600 mL), IPA (60 mL), and water (100 mL) and stirred for 10 minutes. The DCM phase was separated, washed with brine (100 ml), and concentrated to obtain a crude product, which was purified by preparative HPLC and freeze-dried to obtain 0.98 g of compound A-05.

[0370] A-05 separation and purification method is as follows:

[0371] Chromatographic column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0372] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0373] A-05 structural characterization data are as follows:

[0374] MS m / z(ESI):1107.3[M+H]+

[0375] 1 H NMR (400MHz, DMSO) δ9.10 (s, 2H), 8.66-8.63 (m, 1H), 8.51 (d, J = 8.8Hz, 1H), 8.34-8.3 1(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=6.4Hz,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=7.2Hz,3H).

[0376] 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]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxyl-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-07)

[0377] Under nitrogen protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (IM-2, 21.6 mg, 0.059 mmol) and (R)-16-amino-10-benzyl-6,9,12,15-tetrahydro-3-oxo-5,8,11,14-tetraazahexadecanoic acid (IM-3, 24.5 mg, 0.058 mmol) were added to DMF (1 mL) and the temperature was raised to 35 °C. After 16 hours of reaction, (1S,9S)-1-amino-5-fluoro-9-ethyl-9-hydroxy-4-chloro-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[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 system. The reaction system was reacted at 25°C for 16 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 26.4 mg of compound A-07.

[0378] The A-07 separation and purification method is as follows:

[0379] Chromatographic column: SunFire Prep C18OBD 19mm×150mm×5.0μm

[0380] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0381] A-07 structural characterization data are as follows:

[0382] ESI-MS (m / z): 1111.3 [M+H] + .

[0383] Example 4 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]indolizin[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetrahydro-3-oxo-5,8,11-triazatetradec-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-14)

[0384] Step 1:

[0385] 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). HATU (630.67 mg, 1.66 mmol) and N,N-diisopropylethylamine (428 mg, 3.32 mmol) were then added and stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 700 mg of compound IM-5.

[0386] The HPLC preparation method is as follows:

[0387] Chromatographic column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0388] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0389] Step 2:

[0390] 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 completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 307 mg of compound IM-6.

[0391] The HPLC preparation method is as follows:

[0392] Chromatographic column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0393] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0394] Step 3:

[0395] 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 directly purified by preparative HPLC and freeze-dried to obtain 50.56 mg of compound A-14.

[0396] The structural characterization data are as follows:

[0397] MS m / z(ESI):1002.4[M+H]+

[0398] The HPLC preparation method is as follows:

[0399] Chromatographic column: Waters SunFire Prep C18OBD (5μm*19mm*150mm)

[0400] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0401] 1 H NMR (400MHz, DMSO) δ9.11(s,2H),8.68(t,J=6.4Hz,1H),8.49(d,J=8.8Hz,1H),8.16(s,1H),8.10(d,J=7.2Hz,1 H),8.01(d,J=7.2Hz,1H),7.91(d,J=6.8Hz,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=7.2Hz,3H).

[0402] 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-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)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)

[0403] Step 1:

[0404] At room temperature, compound B-01-1 (413.40 mg, 0.251 mmol, its synthesis reference patent CN111295389B) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), and cuprous bromide (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. The reaction was stirred for 1 hour and then filtered. The filtrate was purified by preparative high performance liquid chromatography (conditions as follows) to obtain 30.00 mg of compound B-01-2.

[0405] Chromatographic column: SunFire Prep C18OBD 19mm×150mm×5.0μm

[0406] Mobile phase A: acetonitrile; mobile phase B: water

[0407] Step 2:

[0408] Compound B-01-2 (30.00 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL). Trifluoroacetic acid (0.2 mL) was added to the reaction mixture and allowed to react at room temperature for 30 min. The reaction mixture was concentrated under reduced pressure and purified by preparative HPLC (conditions as follows) to obtain 20.00 mg of the trifluoroacetic acid salt of compound B-01.

[0409] Chromatographic column: SunFire Prep C18OBD 19mm×150mm×5.0μm

[0410] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0411] The structural characterization data are as follows: ESI-MS (m / z): 1631.7 [M+H] + ,816.0[M / 2+H] + .

[0412] 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-diazapentatriacontamido)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)

[0413] Step 1:

[0414] At 25 ° C, the methanesulfonate of 1-1 (30.00 mg, 56.44 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 1H-benzotriazol-1-yloxytripyrrolidino 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 sequentially. The reaction was maintained at 25 ° C for 1 hour and the reaction was monitored by liquid chromatography-mass spectrometry. After completion of the reaction, water was added to the reaction solution and extracted with ethyl acetate. The organic phases were combined, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was separated by thin layer chromatography (dichloromethane: methanol = 15:1) to obtain 27.00 mg of compound B-02-1.

[0415] Step 2:

[0416] At 0°C, B-02-1 (20 mg, 27.33 μmol) was dissolved in dichloromethane (2 mL), and 4-dimethylaminopyridine (26.71 mg, 218.61 μmol) and triphosgene (8.11 mg, 27.33 μmol) in dichloromethane (0.5 mL) were added in sequence. The mixture was kept at 0°C for 0.5 h. After the residual triphosgene was replaced with nitrogen, (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,2 A solution of 7,30-nonaoxa-3,9-diazapentatriacontamide)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (43.46 mg, 40.99 μmol) in dichloromethane (1 mL) was maintained at 0°C for 0.5 hours; the reaction was monitored by liquid chromatography-mass spectrometry. After completion of the reaction, the reaction solution was concentrated and the crude product was purified by thin layer chromatography (dichloromethane:methanol = 15:1) to obtain 30.00 mg of compound B-02-2.

[0417] Step 3:

[0418] 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 cuprous bromide (39.45 mg, 275.01 μmol) were added. The reaction was maintained at 25°C for 1 hour; the reaction was monitored by liquid chromatography-mass spectrometry; after the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography (conditions as follows), and the prepared solution was lyophilized to obtain 150.00 mg of B-02-3 compound.

[0419] Chromatographic column: SunFire Prep C18OBD 19mm×150mm×5.0μm

[0420] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0421] Step 4:

[0422] 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) / glacial acetic acid (v / v = 13 / 1) (50 uL) was added dropwise. The reaction was maintained at 25 ° C for 0.5 hours and the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography (conditions as follows), and the prepared solution was lyophilized to obtain 50.00 mg of B-02-4 compound.

[0423] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0424] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0425] Step 5:

[0426] At 25°C, B-02-4 (50 mg, 26.52 μmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (60.49 mg, 530.49 μmol) was added, and the reaction was maintained at 25°C for 0.5 hours; the reaction was monitored by liquid chromatography-mass spectrometry; after the reaction was completed, the reaction solution was concentrated, and the crude product was purified by preparative high-performance liquid chromatography (conditions as follows), and the preparative solution was lyophilized to obtain 23.69 mg of B-02 compound.

[0427] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0428] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0429] The structural characterization data of B-02 are as follows:

[0430] ESI-MS (m / z): 1613.6 [M+H] + .

[0431] Example 7 N-((7S,10S,13S)-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]indolizin[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxo-5,8,11,14-tetraazapentacarbonyl-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzamide (C-07)

[0432] Step 1:

[0433] The raw material C-07-1 (4.80 g, 16.33 mmol), tributyl (2-methylsulfonylpyrimidin-4-yl) tin (16.27 g, 39.19 mmol) and bistriphenylphosphine palladium dichloride (2.29 g, 3.27 mmol) were dissolved in 1,4-dioxane (100 mL). The reaction system was stirred at 110 ° C. under a nitrogen atmosphere for 5 hours. The reaction was monitored by LC-MS. The reaction system was concentrated and purified by column chromatography (EA / PE = 0-50%) to obtain 1.36 g of C-07-2 compound.

[0434] Step 2:

[0435] Compound C-07-2 (510 mg, 1.33 mol) and NaOH (212.24 mg, 5.31 mmol) were dissolved in THF (12.5 mL), MeOH (12.5 mL), and H₂O (2.5 mL). The reaction was stirred at 25°C for 2 hours. The reaction was monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 3N HCl, resulting in the precipitation of a large amount of solid. The filter cake was collected by filtration and dried to afford 380 mg of compound C-07-3.

[0436] Step 3:

[0437] 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 solution was purified by preparative HPLC and freeze-dried to yield 40 mg of compound C-07-3.

[0438] The HPLC preparation method is as follows:

[0439] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0440] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0441] Step 4:

[0442] Compound C-07-4 (40 mg, 64.96 μmol) was dissolved in DCM (3 mL) and TFA (1.5 mL), reacted at 25° C. for 1.5 hours, monitored by LC-MS, and the reaction system was concentrated to dryness to obtain 36 mg of compound C-07-5.

[0443] Step 5:

[0444] 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 the mixture was reacted at 25°C for 40 minutes. The reaction was monitored by LC-MS, and water and ethyl acetate were added for extraction. The ethyl acetate layer was concentrated to obtain 28 mg of compound C-07-6.

[0445] Step 6:

[0446] Isotecan 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-tetraazaheptane-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 solution, resulting in the precipitation of a large amount of solid. The solid was collected by filtration, dissolved in DCM, and concentrated to obtain the crude product, which was purified by column chromatography (DCM / MeOH = 0-10%) to afford 660 mg of compound C-07-7.

[0447] Step 7:

[0448] 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. The mixture was allowed to react at room temperature for 1 hour. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 325 mg of compound C-07-8.

[0449] The HPLC preparation method is as follows:

[0450] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0451] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0452] Step 8:

[0453] Compound 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 allowed to react at 25°C for 2 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative HPLC and freeze-dried to yield 7 mg of compound C-07.

[0454] The structural characterization data are as follows:

[0455] ESI-MS (m / z): 1342.4 [M+H] + .

[0456] The HPLC preparation method is as follows:

[0457] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0458] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0459] Example 8 N-((7S,10S,13S)-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]indolizin[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxo-5,8,11,14-tetraazapentacan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-10)

[0460] Step 1:

[0461] The raw 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 H2O (4 mL). The reaction system was stirred at 90°C under a nitrogen atmosphere for 3 hours. The reaction was monitored by LC-MS, filtered through celite, and water and ethyl acetate were added to the filtrate. The mixture was extracted and concentrated to obtain a crude product, which was purified by column chromatography (EA / PE = 0-25%) to obtain 710 mg of C-10-1 compound.

[0462] Step 2:

[0463] 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 H2O (2 mL). The reaction was stirred at 25°C for 2 hours. The reaction was monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 1N HCl. A large amount of solid precipitated. The filter cake was collected by filtration and dried to obtain 560 mg of compound C-10-2.

[0464] Step 3:

[0465] 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 system. The reaction was allowed to proceed at 25°C for 12 hours, monitored by LC-MS. The solvent was evaporated under a stream of nitrogen to afford a crude product, which was purified by preparative HPLC and freeze-dried to afford 153 mg of compound C-10-3.

[0466] The HPLC preparation method is as follows:

[0467] Chromatographic column: Phenomenex Luna C18 200*40mm*10um.

[0468] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% hydrochloric acid)

[0469] Mobile phase: [water (HCl) -ACN]; B%: 13% - 43%, 10 min).

[0470] Step 4:

[0471] 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 HPLC and freeze-dried to obtain 51 mg of compound C-10-4.

[0472] The HPLC preparation method is as follows:

[0473] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0474] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0475] Step 5:

[0476] Compound C-10-4 (50 mg, 73.56 μmol) was added to DCM (2 mL) and TFA (1 mL), reacted at 25°C for 1 h, monitored by LC-MS, and the reaction system was concentrated to dryness to obtain 45 mg of compound C-10-5.

[0477] Step 6:

[0478] 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 solution was purified by preparative HPLC and freeze-dried to yield 13 mg of compound C-10.

[0479] The structural characterization data are as follows:

[0480] ESI-MS (m / z): 1342.5 [M+H] +.

[0481] The HPLC preparation method is as follows:

[0482] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0483] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0484] Example 9: N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-chloro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxo-5,8,11,14-tetraazapentacan-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-17)

[0485] Step 1:

[0486] 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). 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 sequentially. The temperature was raised to 60°C and the reaction mixture was reacted for 2 hours. Water (50 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to afford crude C-17-1 (3.8 g, 4.75 mmol), which was used directly in the next step without purification.

[0487] Step 2:

[0488] Dissolve C-17-1 (3.40 g, 5.40 mmol) in dichloromethane (30 mL) and add trifluoroacetic acid (10.8 g, 94.2 mmol, 7 mL). Stir the reaction mixture at 25°C for 2 hours. The reaction mixture is then concentrated, purified by preparative HPLC, and freeze-dried to afford C-17-2 (2.09 g, 3.64 mmol).

[0489] The HPLC preparation method is as follows:

[0490] Chromatographic column: Phenomenex luna C18 (250mm*70mm*10μm)

[0491] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0492] Step 3:

[0493] 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 trichloride hydrate (8.10 mg, 39.05 μmol) were added to the reaction system. The reaction was stirred at 25 ° C for 30 minutes. The reaction was monitored by LC-MS. Water and ethyl acetate were added for extraction and concentrated to obtain C-17-3 (60 mg).

[0494] Step 4:

[0495] 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 sequentially to DMF (3 mL). The reaction system was reacted at 25°C for 1 hour. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain C-17 (16 mg).

[0496] The structural characterization data are as follows:

[0497] ESI-MS (m / z): 1371.4 [M+H] + .

[0498] The HPLC preparation method is as follows:

[0499] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0500] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0501] 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-tetraazahexadecane-26-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-19)

[0502] Step 1:

[0503] 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 system was purged with nitrogen three times and then reacted at 100°C for 5 hours. After the reaction system was cooled to room temperature, water (50.0 mL) was added to the reaction solution, which was filtered and concentrated to obtain the crude product. The product was slurried with petroleum ether and filtered again. The filter cake was dried under vacuum to obtain C-19-2 (5.65 g).

[0504] Step 2:

[0505] C-19-2 (5.26 g, 13.7 mmol) was dissolved in THF (30.0 mL), MeOH (30.0 mL), and water (30.0 mL). LiOH·H2O (1.72 g, 40.9 mmol) was added and stirred at 25°C for 2 hours. The reaction mixture was adjusted to pH 3 with 1N aqueous hydrochloric acid. A solid precipitated and was filtered. The filter cake was dried under vacuum to afford C-19-3 (4.20 g).

[0506] Step 3:

[0507] 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 (20.0 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 sequentially. The mixture was heated to 60°C and stirred for 2 hours. After the reaction system was cooled to room temperature, water (10.0 mL) and ethyl acetate (20.0 mL) were added to the reaction solution. The aqueous phase was extracted twice with ethyl acetate (25.0 mL*2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude C-19-4 (2.50 g), which was used directly in the next step without purification.

[0508] Step 4:

[0509] 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 system was stirred at 25°C for 12 hours. The reaction solution was directly concentrated, purified by preparative HPLC, and freeze-dried to obtain C-19-5 (1.20 g).

[0510] The HPLC preparation method is as follows:

[0511] Chromatographic column: Phenomenex luna C18 (150mm*25mm*10μm)

[0512] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0513] Step 5:

[0514] 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 trichloride hydrate (39.70 mg, 0.19 mmol) and sodium periodate (4.09 g, 19.14 mmol) were added. The reaction system was reacted at 25 ° C for 1 hour, extracted with water (50 mL) and ethyl acetate (80 mL), and the organic phase was concentrated to obtain a crude product. The crude product was purified by column chromatography (MeOH / DCM = 10-20%) and concentrated to obtain C-19-6 (130 mg).

[0515] Step 6:

[0516] 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 stirred to dissolve. HATU (19.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were added and reacted at room temperature for 2 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain C-19 (20.4 mg).

[0517] The structural characterization data are as follows:

[0518] ESI-MS (m / z): 1372.4 [M+H] + .

[0519] The HPLC preparation method is as follows:

[0520] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0521] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0522] 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-tetraazacyclotetradecane-41-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-5-yl)benzamide (C-21)

[0523] Step 1:

[0524] C-10-2 (3.00 g, 8.10 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-ate (4.03 g, 8.10 mmol) were added to DMF (40 mL). 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 sequentially. The reaction system was stirred at 60°C for 2 hours. Water (100 mL) and ethyl acetate (60 mL x 3) were added to the reaction solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to afford C-21-1 (4.20 g, 4.14 mmol), which was used directly in the next step without purification.

[0525] Step 2:

[0526] C-21-1 (3.60 g, 4.24 mmol) was dissolved in dichloromethane (30 mL), and TFA (15.3 g, 134 mmol, 10 mL) was added. The reaction system was stirred at 25°C for 6 hours. Water (60 mL) and ethyl acetate (40 mL x 3) were added to the reaction solution, and the mixture was extracted. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by preparative HPLC and freeze-dried to obtain C-21-2 (2.93 g, 3.63 mmol).

[0527] The HPLC preparation method is as follows:

[0528] Chromatographic column: Phenomenex luna C18 (250mm*70mm*10μm)

[0529] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0530] Step 3:

[0531] 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 trichloride hydrate (15.47 mg, 74.56 μmol) were added to the reaction system. The reaction was stirred at 25°C for 30 minutes. The reaction system was extracted with water and ethyl acetate and concentrated to obtain C-21-3 (155 mg).

[0532] Step 4:

[0533] 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 system was reacted at 25°C for 1 hour. The reaction solution was purified by HPLC and freeze-dried to obtain C-21 (15 mg).

[0534] The structural characterization data are as follows:

[0535] ESI-MS (m / z): 1591.7 [M+H] + .

[0536] The HPLC preparation method is as follows:

[0537] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0538] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0539] 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-tetraazatetracan-4-yl)-2,6-bis(2-(methylsulfonyl)pyrimidin-5-yl)isonicotinamide (C-23)

[0540] Step 1:

[0541] C-19-3 (1.50 g, 4.04 mmol) and tert-butyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptane-27-ate (2.01 g, 4.04 mmol) were added to DMF (20.0 mL), followed by HOBt (1.64 g, 12.1 mmol), EDCI (2.32 g, 12.1 mmol), and DIEA (2.09 g, 16.2 mmol). The mixture was heated to 60°C and stirred for 2 hours. After cooling to room temperature, water (10.0 mL) and ethyl acetate (20.0 mL) were added to the reaction solution for separation. The aqueous phase was extracted twice with ethyl acetate (25.0 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product C-23-1 (3.00 g), which was used directly in the next step.

[0542] Step 2:

[0543] 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 to obtain a crude product, which was purified by preparative HPLC and freeze-dried to afford C-23-2 (1.20 g).

[0544] The HPLC preparation method is as follows:

[0545] Chromatographic column: Welch Ultimate C18 (150mm*25mm*5μm)

[0546] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0547] Step 3:

[0548] C-23-2 (500 mg, 0.63 mmol) was added to acetonitrile (10 mL) and water (5 mL), and ruthenium trichloride hydrate (13.0 mg, 0.063 mmol) and sodium periodate (1.35 g, 6.29 mmol) were added. The system 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 obtain a crude product, which was purified by column chromatography (MeOH / DCM = 10-20%) and concentrated to obtain C-23-3 (350 mg).

[0549] Step 4:

[0550] 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 reacted at room temperature for 2 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain C-23 (17.0 mg).

[0551] The structural characterization data are as follows:

[0552] ESI-MS (m / z): 1592.6 [M+H] + .

[0553] The HPLC preparation method is as follows:

[0554] Chromatographic column: Waters XBridge Prep C18OBD (5μm*19mm*150mm)

[0555] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0556] Example 13 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-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)

[0557] Step 1: Preparation of 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 (B-03-1)

[0558] Dissolve (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) in DMF (50 mL). Add DIPEA (1.18 g, 9.12 mmol, 1.59 mL) dropwise. Add acetoxyacetyl chloride (548.12 mg, 4.01 mmol, 431.59 μL) dropwise with ice-cooling and stirring. Continue stirring for 1 hour. Add the reaction solution to 0.1 M dilute hydrochloric acid to precipitate a solid, which is then filtered. The filter cake was dissolved in dichloromethane and methanol, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (methanol / dichloromethane = 0% to 5%) and concentrated again to obtain the title compound (1.7 g, 3.077 mmol).

[0559] Its structural characterization data are as follows:

[0560] ESI-MS (m / z): 552.2 [M+1] + .

[0561] Step 2: Preparation of ethyl 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]indazolidin-1-yl)amino)-2-oxoacetate (B-03-2)

[0562] 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), cooled to 0 ° C under nitrogen protection, and a dichloromethane solution (5 mL) of triphosgene (268.81 mg, 0.905 mmol) was added dropwise, and the reaction was stirred for 0.5 hour. A solution of (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatrinitroamino)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (1.44 g, 1.36 mmol) in dichloromethane was slowly added dropwise and allowed to react at room temperature for 4 hours. The reaction was quenched with water and extracted three times with dichloromethane (100 ml x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification on a silica gel column (MeOH / DCM = 0% to 5%) afforded the title compound (498 mg, 0.304 mmol).

[0563] Its structural characterization data are as follows:

[0564] ESI-MS (m / z): 1352.8 [M+1] + .

[0565] 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)

[0566] 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-2,3,9,1 Ethyl 2-oxoacetate (200 mg, 0.122 mmol) (0,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidin[1,2-b]quinolin-1-yl)amino) was dissolved in THF (3 mL) and MeOH (3 mL). A 1 mL aqueous solution of sodium carbonate (25.88 mg, 0.224 mmol) was added dropwise with stirring. Stirring was continued for 1 hour after the addition was complete. Dilute hydrochloric acid was added dropwise to the reaction mixture to neutralize the reaction. After concentration under reduced pressure, the mixture was directly transferred to the next step.

[0567] Its structural characterization data are as follows:

[0568] ESI-MS (m / z): 1596.7 [M+1] + .

[0569] 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)

[0570] 4-((S)-35-Azido-2-(4-((4-methoxyphenyl)benzhydryl)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 (190 mg, 119.04 μmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (0.5 mL) was added and the reaction was continued for 1 hour. Saturated sodium bicarbonate aqueous solution was added dropwise to the reaction solution for neutralization, and the organic phase was concentrated to obtain a crude product, which was purified by reverse phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0% to 50%) and freeze-dried to obtain the title compound (95 mg, 69.35 μmol).

[0571] Its structural characterization data are as follows:

[0572] ESI-MS (m / z): 1323.6 [M+1] + .

[0573] Step 5: Preparation of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-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)

[0574] 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 (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). Cuprous bromide (9.42 mg, 0.066 mmol) was added and stirring continued for 2 hours. The reaction mixture was directly filtered and the concentrated crude product was purified by preparative HPLC and freeze-dried to obtain the title compound (42.2 mg, 24.69 μmol).

[0575] The structural characterization data are as follows:

[0576] ESI-MS (m / z): 1628.7 [M+1] + .

[0577] The preparative HPLC method is as follows:

[0578] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0579] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0580] 2. Antibody Preparation

[0581] Fully humanized mice were immunized with human B7-H3-4Ig-His protein, and serum titers were monitored by ELISA and flow cytometry. The optimal mice were selected based on the titer results, and spleen cells were fused, screened, and subcloned. The binding activities of different monoclonal clones to human and monkey proteins and cells were tested, and the preferred clone 20G11G6 / 2# was obtained. The antibody sequence was modified by removing PTM sites, reducing PI, and removing ADCC activity in the heavy chain constant region. Finally, the fully human antibody 2#8890 (heavy chain variable region, SEQ ID NO: 3; light chain variable region, SEQ ID NO: 4) was obtained, along with the sequence of the human IgG1 heavy chain constant region with ADCC activity removed (SEQ ID NO: 31) and the human kappa light chain constant region (SEQ ID NO: 32), forming a complete humanized antibody (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 simultaneously transfected into CHO-S cells. Protein A purifies the expressed antibody in the supernatant to obtain the corresponding antibody protein 2#8890. The heavy chain amino acid sequence and light chain amino acid sequence of 2#8890 are shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

[0582] hIgG1 is an anti-chicken lysozyme antibody, the heavy chain variable region is fused to a mutant human IgG1 heavy chain constant region (SEQ ID NO: 31), and the light chain variable region is fused to a wild-type human kappa light chain constant region (SEQ ID NO: 32). The antibody hIgG1 was expressed and purified according to the above method.

[0583] The B7-H3 control antibody DS7300 was obtained from patent CN 103687945A. After codon optimization, the antibody heavy chain variable region nucleotide sequence was synthetically cloned into a human IgG1 heavy chain constant region containing a mutant (SEQ ID NO: 31), and the light chain variable region nucleotide sequence was synthesized into a pTT5 vector containing a wild-type kappa light chain constant region (SEQ ID NO: 32). The antibody DS7300 was expressed and purified as described above.

[0584] Table 1: Variable region and CDR amino acid sequences of 2#8890

[0585] 3. Conjugation of Compounds Containing Cellular Bioactive Molecules and Linkers to Antibodies

[0586] The antibodies 2#8890, DS7300, and hIgG1 involved in the antibody-drug conjugates prepared in the following examples are the corresponding antibodies described in the second part above.

[0587] 1. Preparation of ADC 1 (DS7300-M-01, DAR 4)

[0588] Take 38.041 ml of homemade DS7300 antibody (26.287 mg / mL), adjust the pH to 7.4 with 1 M Na2HPO4 solution, dilute the antibody to 3 mg / mL with 20 mM PB, and add 4 mM ZnCl2 (3.413 mL) and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 4.096 mL, pH 7.4) solution in an ice bath, mix well, and let it stand at 4°C overnight. A 6-fold amount of M-01 (4.18 mL, 10 mM) dissolved in dimethyl sulfoxide was added and mixed. The mixture was reacted at 4°C for 4 h. A cysteine ​​solution (10 mM, 6.826 mL) was then added. After 1.5 h of reaction, the reaction solution was brought to room temperature and an EDTA solution (10 mM, 6.826 mL) was added. After 30 minutes, a DHAA solution (10 mM, 6.826 mL) was added and the reaction continued for 30 minutes. After completion, the buffer was exchanged with a 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 was determined by mass spectrometry to be 3.8.

[0589] 2. Preparation of ADC 2 (hIgG1-M-01, DAR 4)

[0590] Take 1.2245 ml of hIgG1 antibody (24.5 mg / mL), adjust the pH to 7.3 with 1 M Na2HPO4 solution, dilute the antibody to 3 mg / mL with 20 mM PB, and add 4 mM ZnCl2 (52.04 uL) and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 124.89 uL, pH 7.3) solution in an ice bath, mix well, and incubate at 4°C overnight. A 6-fold amount of 171 (127.44 μL, 10 mM) dissolved in dimethyl sulfoxide was added and mixed thoroughly. The mixture was reacted at 4°C for 4 h. A cysteine ​​solution (10 mM, 208.15 μL) was then added. After 1.5 h of reaction, the reaction solution was brought to room temperature and an EDTA solution (10 mM, 208.15 μL) was added. After 30 minutes, a DHAA solution (10 mM, 208.15 μL) was added and the reaction continued for 30 minutes. After completion, the buffer was exchanged 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 was determined by mass spectrometry to be 4.46.

[0591] 3. Preparation of ADC 3 (2#8890-M-01, DAR 4)

[0592] Take 9.36 ml of 2#8890 antibody (3.205 mg / mL), adjust the pH to 7.4 with 1 M Na2HPO4 solution, dilute the antibody to 3 mg / mL with 20 mM PB, and add 4 mM ZnCl2 (57.31 uL) and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 124.9 uL, pH 7.4) solution in an ice bath, mix well, and let it stand at 4°C overnight. A 6-fold amount of M-01 (124.9 μL, 10 mM) dissolved in dimethyl sulfoxide was added and mixed. The mixture was allowed to react at 4°C for 4 h. A cysteine ​​solution (10 mM, 208.2 μL) was then added. After 1.5 h of reaction, the reaction solution was brought to room temperature and an EDTA solution (10 mM, 208.2 μL) was added. After 30 minutes, a DHAA solution (10 mM, 208.2 μL) was added and the reaction continued for 30 minutes. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC3 (2#8890-M-01). The DAR value was determined by mass spectrometry to be 3.89.

[0593] 4. Preparation of ADC 4 (hIgG1-A-05, DAR 8)

[0594] 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 then adjusted to 7.60 with 1 M Na₂HPO₄ solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 57 μL) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-05 dissolved in dimethyl sulfoxide (103 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 4 (hIgG1-A-05). The DAR value, determined by mass spectrometry, was 8.03.

[0595] 5. Preparation of ADC 5 (2#8890-A-05, DAR 8)

[0596] 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 then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 114.5 μL, pH 7.60) was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-05 dissolved in dimethyl sulfoxide (219.1 μL, 10 mM) was then added and mixed thoroughly. The mixture was allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 5 (2#8890-A-05). The DAR value was 7.90 as determined by mass spectrometry.

[0597] 6. Preparation of ADC 6 (hIgG1-A-07, DAR 8)

[0598] 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 then adjusted to 7.60 with 1 M Na₂HPO₄ solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 38.1 μL, pH 7.60) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-07 dissolved in dimethyl sulfoxide (69.2 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 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 value, determined by mass spectrometry, was 8.04.

[0599] 7. Preparation of ADC 7 (2#8890-A-07, DAR 8)

[0600] 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 then adjusted to 7.60 with 1 M Na2HPO4 solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 38.2 μL, pH 7.60) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 12-fold amount of A-07 dissolved in dimethyl sulfoxide (87.6 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 7 (2#8890-A-07). The DAR value, determined by mass spectrometry, was 7.76.

[0601] 8. Preparation of ADC 8 (hIgG1-A-14, DAR 8)

[0602] 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 then adjusted to 7.60 with 1 M Na₂HPO₄ solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 66.86 μL, pH 7.60) solution was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-14 dissolved in dimethyl sulfoxide (260.53 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. The buffer was then exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 8 (hIgG1-A-14). The DAR value, as determined by mass spectrometry, was 8.0.

[0603] 9. Preparation of ADC 9 (2#8890-A-14, DAR 8)

[0604] 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 then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 152 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-14 dissolved in dimethyl sulfoxide (292.26 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at 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 was 7.22 as determined by mass spectrometry.

[0605] 10. Preparation of ADC 10 (hIgG1-B-01, DAR 8)

[0606] 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 then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 114.5 μL, pH 7.60) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of B-01 dissolved in dimethyl sulfoxide (212.4 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 10 (hIgG1-B-01). The DAR value, determined by mass spectrometry, was 8.03.

[0607] 11. Preparation of ADC 11 (2#8890-B-01, DAR 8)

[0608] 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 then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 114.5 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of B-01 dissolved in dimethyl sulfoxide (212.4 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at 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.

[0609] The ADC samples after coupling were subjected to LC-MS molecular weight analysis.

[0610] Chromatographic determination conditions:

[0611] Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

[0612] Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN;

[0613] Flow rate: 0.25 ml / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 2 μl;

[0614] Mass spectrometry conditions:

[0615] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0616] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 200; CE 10; Accumulation time 0.5s;

[0617] m / z 600-4000; Time bins to sum 40.

[0618] 12. Preparation of ADC 12 (2#8890-C-07, DAR 4)

[0619] 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 then adjusted to 7.60 with 1 M Na2HPO4 solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine, 9.5 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. An 8-fold amount of C-07 dissolved in dimethyl sulfoxide (14.6 μL, 10 mM) was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at 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 was 4.12 as determined by mass spectrometry.

[0620] 13. Preparation of ADC 13 (2#8890-C-10, DAR 4)

[0621] 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 then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 9.5 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. An 8-fold amount of C-10 dissolved in dimethyl sulfoxide (14 μL, 10 mM) was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate, ADC 13 (2#8890-C-10). The DAR value was 4.17 as determined by mass spectrometry.

[0622] The ADC samples after coupling were subjected to LC-MS molecular weight analysis.

[0623] Chromatographic determination conditions:

[0624] Liquid chromatography column: ACQUITY UPLC MAbPac BEH SEC;

[0625] Mobile phase A: 20 mM NH4Ac;

[0626] Flow rate: 0.1 ml / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 2 μl;

[0627] Mass spectrometry conditions:

[0628] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0629] GS1 55; GS2 55; CUR 30; TEM 450; ISVF 5500; DP 75; CE 5; Accumulation time 0.5s; m / z 900-7000; Time bins to sum 40.

[0630] 14. Preparation of ADC 14 (2#8890-C-17, DAR 4)

[0631] 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 then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 4.77 μL of 20 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A 5-fold amount of C-17 dissolved in dimethyl sulfoxide (8.76 μL, 10 mM) was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at 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 was 3.86 as determined by mass spectrometry.

[0632] 15. Preparation of ADC 15 (2#8890-C-19, DAR 4)

[0633] 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 then adjusted to 7.60 with 1 M Na₂HPO₄ solution. 20 mM TCEP (tris(2-carboxyethyl)phosphine, 9.54 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A 5.5-fold amount of C-19 dissolved in dimethyl sulfoxide (17.9 μL, 10 mM) was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at 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 was 4.05 as determined by mass spectrometry.

[0634] 16. Preparation of ADC 16 (2#8890-C-21, DAR 4)

[0635] 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 then adjusted to 7.60 with 1 M Na₂HPO₄ solution. A 20 mM TCEP (tris(2-carboxyethyl)phosphine, 9.54 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A 5.5-fold amount of C-21 dissolved in dimethyl sulfoxide (17.9 μL, 10 mM) was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at 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 was 3.92 as determined by mass spectrometry.

[0636] 17. Preparation of ADC 17 (2#8890-B-03, DAR 8)

[0637] 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 then adjusted to 7.60 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 112.65 μL, pH 7.60) was added, mixed, and allowed to stand at room temperature for 1.5 hours. An 11-fold amount of B-03 dissolved in dimethyl sulfoxide (227.58 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at 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.

[0638] 18. Preparation of ADC 18 (2#8890-B-03, DAR 8)

[0639] 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 then adjusted to 7.60 with 1 M Na2HPO4 solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 112.65 μL, pH 7.60) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of B-03 dissolved in dimethyl sulfoxide (211.1 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer at 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.

[0640] IV. Detecting the activity of antibody-drug conjugates

[0641] 1. Dynamic affinity testing of anti-human B7-H3 antibodies and their conjugates

[0642] The dynamic affinity of anti-human B7-H3 antibodies and their conjugates to human B7-H3-4Ig-his, human B7-H3-2Ig-his, rat B7-H3-his, and monkey B7-H3-his proteins was tested using ForteBio (Pall Life Sciences). The specific method is as follows: the test antibody and its conjugate 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 gradiently diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, and 0 nM. The test antibody and its conjugate were then captured in PBST (0.02% Tween-20) solution using a Protein A Sensor (Pall Life Sciences) for 60 s. The antibodies were then bound to the four proteins for 60 s, followed by dissociation for 180 s. The results were analyzed in Data Analysis 11.0 software using a 1:1 global fitting mode to obtain affinity constants. The results are shown in Table 2-1 and Table 2-2, which show that antibody 2#8890 and its conjugate bind to monkey B7-H3 but not to rat B7-H3.

[0643] Table 2-1: Dynamic affinity test results of anti-human B7-H3 antibodies and their conjugates

[0644] Table 2-2: Dynamic affinity test results of anti-human B7-H3 antibodies and their conjugates II

[0645] 2. Cellular affinity testing of anti-human B7-H3 antibodies and their conjugates

[0646] The affinity of fully human anti-human B7-H3 antibodies and their conjugates to human colon cancer cells HT29 (Cell Bank, Chinese Academy of Sciences), human gastric cancer cells NCI-N87 (ATCC), human breast squamous carcinoma cells HCC1806 (ATCC), and human non-small cell lung cancer cells HCC827 (ATCC) was determined using flow cytometry (Beckman, Cytoflex). The affinity of fully human anti-human B7-H3 antibodies and their conjugates to CHOS-human B7-H3-4Ig and CHOS-human B7-H3-2Ig was also determined. The cross-species affinity of fully human anti-human B7-H3 antibodies and their conjugates to CHOS-rat B7-H3 and CHOS-monkey B7-H3 was also determined. Adherent cells were digested with Trypsin-EDTA (0.25%) (Thermo) solution, counted, and the cell density adjusted to 4.0 × 10 6 / ml, washed twice with 1% BSA, resuspended in 1% BSA solution, and 50 μl of cell suspension was added to each well of a 96-well pointed bottom plate (cell number 2×10 5 1% BSA was used to dilute the antibody and its conjugate (starting at a final concentration of 10 μg / ml, with a 3-fold serial dilution for a total of 11 concentration points). hIgG1 antibody and its conjugate served as a control (final concentration of 10 μg / ml). 50 μl of the diluted antibody was added to the conical bottom plate containing cells and incubated at 4°C for 60 min. The cells were washed twice with 1% BSA, and 50 μl of the diluted secondary antibody was added to each well, mixed, and incubated at 4°C for 30 min. The cells were washed twice with 1% BSA, resuspended in 200 μl of 1% BSA, and analyzed by flow cytometry. Data processing: Median PE values ​​were exported and then imported into GraphPad Prism 6 software to calculate EC values. 50 .

[0647] The affinity results of anti-human B7-H3 antibodies and their conjugates for HT29, NCI-N87, HCC1806, and HCC827 tumor cells are shown in Figures 1A1, 1A2, 1B, 1C, 1D1, and 1D2, and Tables 3-1 and 3-2, respectively. 2#8890 exhibited stronger tumor cell affinity than DS7300, and the ADC exhibited consistent affinities with the corresponding antibodies. The affinity results of fully human anti-human B7-H3 antibodies and their conjugates for CHOS-human B7-H3-4Ig and CHOS-human B7-H3-2IG cells are shown in Figures 1E and 1F, and Table 4. 2#8890 and DS7300 exhibited comparable affinities for these two overexpressing cell lines, and the ADC exhibited consistent affinities with the corresponding antibodies. The affinity results of anti-human B7-H3 antibodies and their conjugates for CHOS-rat B7-H3 and CHOS-monkey B7-H3 cells are shown in Figure 2A, Figure 2B and Table 5. The results showed that 2#8890 and its conjugates bound to monkey B7-H3-overexpressing cells but not to rat B7-H3-overexpressing cells.

[0648] Table 3-1: Anti-human B7-H3 antibody and ADC tumor cell affinity test results

[0649] Table 3-2: Anti-human B7-H3 antibody and ADC tumor cell affinity test results II

[0650] Table 4: Affinity determination results of anti-human B7-H3 antibodies and ADCs with CHOS-human B7-H3-4Ig and CHOS-human B7-H3-2Ig cells

[0651] Table 5: Affinity determination results of anti-human B7-H3 antibodies and ADCs with CHOS-rat B7-H3 and CHOS-monkey B7-H3 cells

[0652] 3. Detection of endocytic activity of anti-human B7-H3 antibodies and their conjugates

[0653] The endocytic activity of anti-human B7-H3 antibodies and their conjugates in human gastric cancer cells NCI-N87 (ATCC), human breast squamous 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 and counted. The cell density was adjusted to 1×10 5 100 μl of cell suspension was added to each well of a 96-well plate (the number of cells was 1×10 4 / well), place the 96-well plate in a 37°C, CO2 constant temperature incubator and incubate for 24 hours. Remove the 96-well plate, aspirate the culture medium, and add 50 μl of fresh complete culture medium to each well; dilute the test antibody and its conjugate with complete culture medium to a total of 6 concentration points, and use a single concentration for hIgG1 antibody and its conjugate; dilute 300 μg / ml pHrodo reagent (Thermo, Cat#Z25612) to 12 μg / ml in complete culture medium (the final concentration of pHrodo is 3 μg / ml); mix the serially diluted test antibody and the diluted pHrodo reagent in a 1:1 ratio (30 μl:30 μl) and incubate at room temperature in the dark for 30 minutes; take 50 μl of the test antibody and pHrodo reagent mixture and add it to the 96-well plate, incubate at 37°C, 5% CO2 for 24 hours; remove the 96-well plate, aspirate the culture medium, wash once with sterile PBS, and add 100 μl of Trypsin-EDTA to each well. (0.25%) digested cells, then added 100 μl complete medium to neutralize; after pipetting and dispersing the cells in the wells, the cells were detected by FACS. Data processing: Median YL-1H values ​​were exported and then imported into GraphPad Prism 6 software to calculate EC 50 The results are shown in Figures 3A, 3B, and 3C and Table 6. The endocytic activity of 2#8890 is stronger than that of DS7300, and the endocytic activity of ADC is consistent with that of the corresponding antibody.

[0654] Table 6: Results of endocytic activity assays of anti-human B7-H3 antibodies and ADCs

[0655] 4. In vitro cell killing assay of anti-human B7-H3 antibody-drug conjugates

[0656] Adherent A375, Calu6-B7-H3, and U87MG-B7-H3 cells were digested with Trypsin-EDTA (0.25%) (Thermo) solution and counted. The cell density was adjusted to 1×10 4 , 5×10 4 , 1×10 4 Cells / ml, add 100 μl of cell suspension to each well of a 96-well plate (cell numbers are 1000, 5000, and 1000 / well, respectively), and place the 96-well plate in a 37°C, CO2 constant temperature incubator for 24 hours. Use complete culture medium to dilute the ADC to be tested, starting with a final concentration of 3333.3nM, and 4-fold serial dilution for a total of 12 concentration points; take 100 μl of diluted ADC and add it to a 96-well plate, and incubate at 37°C, 5% CO2 for 4 to 7 days. Remove the 96-well plate, add 20 μl of CCK8 reagent to each well, incubate at 37°C for 2 to 3 hours, and use a microplate reader to detect OD 450nmThe signal values ​​were then imported into GraphPad Prism 6 software to calculate the IC 50 The results are shown in Table 7 and Figures 4A-C. The killing activity of the conjugate was significantly stronger than that of the negative antibody conjugate, indicating that the killing effect was target-mediated.

[0657] Table 7 ADC in vitro cell killing results

[0658] 5. In vivo efficacy testing of different antibody-drug conjugates in the HT29 model

[0659] Human colon cancer cells HT29 (Cell Bank, Chinese Academy of Sciences) were cultured in monolayers in McCoy's 5a medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The cells were digested and passaged 2-3 times per week using trypsin-EDTA. When the cells reached the exponential growth phase, the culture medium was collected for mycoplasma testing, and the cells were collected and counted. Each mouse was inoculated subcutaneously with 5×10 cells at the right scapula. 6 HT29 cells were suspended in 0.05 ml PBS + 0.05 ml Matrigel. When the average tumor volume grew to 100-200 mm 3 At the same time, mice with irregular, too small, or too large tumor volumes were eliminated, and the remaining mice were randomly divided into 4 groups according to tumor volume and animal body weight, with 6 mice in each group. The drug was injected into the tail vein (iv) once a week (QW) and twice a week (10 mg / kg). After administration, the tumor was measured with a vernier caliper twice a week, and the tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. The antitumor drug efficacy was evaluated by tumor growth inhibition rate TGI (%), calculated as follows: TGI (%) (tumor volume) = [1-(T Vt -T V0 ) / (C Vt -C V0 )]×100%; when the tumor regressed, TGI (%) (tumor volume) = 100% - (T Vt -T V0 ) / T V0 × 100%. T V0 T is the average tumor volume of the test drug group at the time of group administration; Vt C is the average tumor volume of the test drug group t days after administration; V0 is the average tumor volume of the vehicle group when the drug was administered; C Vt =V = the average tumor volume of the vehicle group on day t after administration. If the tumor is smaller than the initial volume, V tWhen <V0, it is defined as partial tumor regression (PR); if the tumor completely disappears, it is defined as complete tumor regression (CR). The death of animals was observed and recorded every day. The specific results are shown in Table 8 and Figures 5A - B.

[0660] Table 8 Analysis of the efficacy of different antibody - conjugated drugs on the HT29 cell - bearing mouse model

[0661] Note: * P < 0.05, ** P < 0.01, *** P < 0.001 indicates a significant difference compared with the solvent group.

[0662] The results showed that: Except for the negative control ADC 2 group, the treatment groups all showed obvious pharmacodynamic effects. TGI (%) : The pharmacodynamic effect of ADC 3 group was better than that of the positive control ADC 1 group. The animals in each group had good tolerance, showing the good efficacy and safety of the 2#8890 antibody.

[0663] 6. Detection of the in - vivo pharmacodynamic effects of different antibody - conjugated drugs on the HCC1806 model

[0664] Human breast squamous carcinoma cells HCC1806 (ATCC) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum in an incubator at 37°C with 5% CO2 air. When the cells were in the exponential growth phase, after detecting mycoplasma in the culture medium, the cells were collected and counted. 2 × 10 6 HCC1806 cells were subcutaneously inoculated at the right scapular region of each mouse, suspended in 0.1 ml PBS. When the average tumor volume grew to about 100 - 200 mm 3 , mice with too small or too large tumor volumes were excluded. The remaining mice were randomly divided into 4 groups with 6 mice in each group, and a single intravenous injection (10 mg / kg) was given. After administration, the tumor volume and body weight were measured twice a week. The specific results are shown in Table 9 and Figures 6A - B.

[0665] Table 9 Analysis of the efficacy of different antibody - conjugated drugs on the HCC1806 cell - bearing mouse model

[0666] Note: * P < 0.05, ** P < 0.01, *** P < 0.001 indicates a significant difference compared with the solvent group.

[0667] The results showed that: TGI (%) : The pharmacodynamic effect of ADC 3 group was better than that of the positive control ADC 1 group. The animals in each group had good tolerance, showing the good efficacy and safety of the 2#8890 antibody.

[0668] 7.2#8890 Different Conjugated Drugs in HCC1806 Model In Vivo Efficacy Testing

[0669] 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 collected for mycoplasma testing, and the cells were collected and counted. Each mouse was inoculated subcutaneously at the right scapula with 2×10 6 HCC1806 cells were suspended in 0.1 ml PBS. When the average tumor volume grew to about 100-200 mm 3 At the same time, mice with small or large tumors were removed. The remaining mice were randomly divided into 6 groups of 6 mice based on tumor volume and body weight. They were given a single dose 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. Detailed results are shown in Table 10 and Figures 7A-B.

[0670] Table 10 Analysis of the efficacy of different conjugated drugs of 2#8890 on HCC1806 cell tumor-bearing mouse model

[0671] Note: * P<0.05, ** P<0.01, *** P<0.001 indicated a significant difference compared with the vehicle group.

[0672] Results showed that the weight of mice in the treatment groups remained stable and the drug was well tolerated. At the same toxin dose, the efficacy of ADC 3 was comparable to that of ADC 11, but weaker than that of ADC 5. Ninety-five days after a single dose, tumors in mice in the ADC 5 group completely regressed.

[0673] 8. Efficacy testing of different doses of antibody-drug conjugates in the HCC1806 model

[0674] 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 collected for mycoplasma testing, and the cells were collected and counted. Each mouse was inoculated subcutaneously at the right scapula with 2×10 6 HCC1806 cells were suspended in 0.1 ml PBS. When the average tumor volume grew to about 100-200 mm 3At the same time, mice with small or large tumors were removed. The remaining mice were randomly divided into 12 groups of 6 mice each 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. Detailed results are shown in Table 11 and Figures 8A-B.

[0675] Table 11 Analysis of the efficacy of different doses of antibody-drug conjugates in HCC1806 cell tumor-bearing mouse models

[0676] Note: * P<0.05, ** P<0.01, *** P<0.001 indicated a significant difference compared with the vehicle group.

[0677] Results showed that ADC 5 and ADC 9 were equally effective across all dose groups, achieving partial remission (PR) at a 5 mg / kg dose. The 3 mg / kg dose of ADC 1 was less effective than the 1.5 mg / kg doses of ADC 5 and ADC 9 (P < 0.001). Two mice in the 1.5 mg / kg ADC 1 group developed hypothermia and decreased activity, subsequently dying. One mouse each died in the ADC 4 and ADC 8 groups. The remaining groups maintained stable body weights and demonstrated good tolerance.

[0678] 9. Efficacy testing of different antibody-drug conjugates in the NCI-N87 model

[0679] Human gastric cancer cells NCI-N87 (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 collected for mycoplasma testing, and the cells were collected and counted. Each mouse was inoculated subcutaneously at the right shoulder blade with 5×10 6 NCI-N87 cells were suspended in 0.1 ml of PBS matrix gel. When the average tumor volume grew to about 100-200 mm 3 At the same time, mice with small or large tumors were removed. The remaining mice were randomly divided into 8 groups of 6 mice each based on tumor volume and body weight. Drugs were administered intravenously in the tail vein twice a week. Tumor volume and body weight were measured twice a week after administration. Detailed results are shown in Table 12 and Figures 9A-B.

[0680] Table 12 Analysis of the efficacy of different antibody-drug conjugates in the NCI-N87 cell tumor-bearing mouse model

[0681] Note: * P<0.05, ** P<0.01, *** P<0.001 indicated a significant difference compared with the vehicle group.

[0682] Results showed that the body weight of mice in each treatment group was stable and well-tolerated. ADC 3 was comparable in efficacy to ADC 1. ADC 11, ADC 5, and ADC 9 were comparable in efficacy, but significantly superior to ADC 1.

[0683] 10. Hydrophilicity detection of anti-human B7-H3 antibodies and conjugates

[0684] The hydrophilicity of antibodies and conjugates was determined using an Agilent 1260 using a TSKgel Butyl-NPR analytical column. An appropriate amount of the test sample was diluted with a diluent (0.75 mol / L (NH₄)₂SO₄) to a 1.0 mg / ml solution, serving as the test solution. A 1 mg / ml solution of each control antibody (hydrophilic control, teterizumab; hydrophobic control, sacituzumab, both produced by Sichuan Kelun Botai Biopharmaceutical Co., Ltd.) was prepared with the diluent to serve as the system suitability solution. Approximately 40 μg of the sample was injected, and gradient elution was performed. After testing, the hydrophobicity value of the test sample was calculated based on the control sample using the formula: (test sample retention time - hydrophilic control retention time) / (hydrophobic control retention time - hydrophilic control retention time). Lower retention times and hydrophobicity values ​​indicate greater antibody hydrophilicity. The results are shown in Tables 13-15. Antibody 2#8890 exhibits good hydrophilicity, even higher than the control antibody DS7300. 2#8890 antibody conjugate hydrophilicity: ADC9>ADC 11>ADC 5, especially ADC 9 has good hydrophilicity.

[0685] Table 13: Hydrophilicity determination of anti-human B7-H3 antibodies

[0686] Table 14: Hydrophilicity determination of anti-human B7-H3 antibody conjugates (DAR4)

[0687] Table 15: Hydrophilicity determination of anti-human B7-H3 antibody conjugates (DAR8)

[0688] 11. Detection of Anti-Human B7-H3 Antibody-Drug Conjugate Binding Activity to Fc Receptors

[0689] ForteBio (Pall Life Sciences) was used to test the dynamic affinity of antibodies DS7300, 2#8890, and DS7300 and 2#8890 antibody-drug conjugates for human Fc receptor proteins CD16a, CD32a, CD32b, C1q, and FcRn. The specific method is as follows: The biotinylated proteins to be tested were captured using an SA Sensor (Pall Life Sciences) in PBST. The test antibodies and conjugates were diluted to a starting concentration of 5000 nM in PBST and then diluted two-fold at seven concentration points. Binding and dissociation were performed, and the results were analyzed in Data Analysis 11.0 software using a 1:1 mode and global fitting. The association rates, dissociation rates, and affinity constants were calculated, as shown in Table 16.

[0690] Table 16 Antibody and conjugate binding to Fc receptor activity detection

[0691] The results showed that the mutated 2#8890 and its conjugates did not bind to Fc receptors CD16a, CD32a, CD32b, and C1q proteins, which could reduce Fc receptor-mediated nonspecific killing and improve drug safety. At the same time, 2#8890 and its conjugates retained the FcRn protein binding activity and did not affect the half-life of the drug.

[0692] 12. Pharmacokinetic study of total antibody (Tab), ADC, and payload in serum of cynomolgus monkeys after multiple intravenous injections of anti-human B7-H3 antibody-drug conjugates

[0693] ADCs (ADC 5, ADC 9), total antibodies (TAb), and payload were quantitatively detected in cynomolgus macaque serum using ELISA and LC-MS / MS. The standard curves for both the ADCs (ADC 5, ADC 9) and total antibodies (TAb) assays had a quantification range of 11.72 to 3000 ng / mL, and the payload assay had a linear range of 0.1 to 40 ng / mL. Both the ADCs (ADC 5, ADC 9) and total antibodies (TAb) assays used B7-H3 protein as the capture protein, coated in a 96-well microtiter plate. Total antibodies (TAb) were then detected using goat anti-human IgG-HRP. For the ADCs (ADC 5, ADC 9), anti-toxin mouse antibodies and goat anti-mouse IgG were used as secondary and detection antibodies, respectively. Color development was achieved through the interaction of the enzyme and substrate, and the results were read on a SpectraMax i3x (Molecular Devices) microplate reader. The 4-P parameter method was used to fit a standard curve and calculate the concentration of each sample. ADC (ADC 5, ADC 9) and total antibody (TAb) concentrations were positively correlated with the color intensity. LC-MS / MS was performed on a Shimadzu LC 30-AD liquid chromatography system coupled to a SCIEX QTRAP 5500+ (SCIEX) mass spectrometer, using (+)ESI ionization and multiple reaction monitoring (MRM) mode. The column was an Xbridge C18 50*4.6mm, 5μm. The ion pairs for compounds 1-10 were 510.2 / 435.2. Sample pretreatment involved protein precipitation with acetonitrile. Results: Cynomolgus monkeys administered multiple intravenous injections of ADC5 and ADC9 demonstrated favorable pharmacokinetic properties for the ADC molecules described herein (e.g., ADC5 and ADC9), with relatively stable systemic circulation and minimal free toxin release. ADC9 exhibited a longer half-life in cynomolgus monkeys and more rapid clearance of free toxins.

[0694] Table 17 Pharmacokinetic parameters of TAb in serum after the last intravenous injection of ADC 50 mg / kg

[0695] Table 18 Pharmacokinetic parameters of ADC in serum after the last intravenous injection of 50 mg / kg

[0696] Table 19 Pharmacokinetic parameters of Payload in serum after the last intravenous injection of ADC at 50 mg / kg

[0697] 13. Repeated dose toxicity test

[0698] The repeated-dose toxicity study included a repeated toxicity study in which ADC5 and ADC 9 were intravenously injected four times into cynomolgus monkeys.

[0699] This study included three groups, one animal per group of each sex, each receiving 30 mg / kg of ADC5 or ADC9 intravenously in addition to normal saline (volume: 10 mL / kg) once weekly for two doses. The dose was then increased to 50 mg / kg of ADC5 or ADC9 in addition to normal saline, administered once weekly for two doses. No test article-related deaths or near-deaths were observed during the study. During the dosing period, animals in the ADC5 group experienced decreased appetite, hair loss, skin pigmentation, and weight loss. Female monkeys showed decreased white blood cell count (WBC), neutrophil count (NEUT), lysozyme (LYM), and mononuclear cell count (MONO). Male monkeys showed decreased red blood cell count (RBC), hematocrit (HGB), and hematocrit (HCT), and increased fibroblast growth factor (FGB), with a trend toward recovery during the recovery period. During the dosing period, animals in the ADC9 group experienced decreased appetite. Female monkeys showed decreased white blood cell count (WBC), neutrophil count (NEUT), lysozyme (LYM), and hematocrit (HCT), and increased fibroblast growth factor (FGB). Male monkeys showed increased fibroblast growth factor (FBG), decreased RBC, HGB, and HCT. During the recovery period, hyperpigmentation and mild hair loss at the site of administration were also observed, with all other changes showing a trend toward recovery. In this study, cynomolgus monkeys received intravenous injections of 30 mg / kg of ADC 5 and ADC 9 once weekly for two consecutive weeks. The dose was then increased to 50 mg / kg of ADC 5 and ADC 9 once weekly for two consecutive weeks. All animals tolerated the treatment, with the highest no-serious-toxicity dose (HNSTD) being 50 mg / kg.

[0700] 14. Antibody-drug conjugates inhibit tumor growth in a mouse subcutaneous transplant tumor model

[0701] The preparation containing the ADC of the present invention was administered via tail vein injection to a CDX mouse model subcutaneously transplanted with human breast squamous cell carcinoma cell HCC1806. The tumor volume and animal body weight changes were measured twice a week to calculate the tumor inhibition efficacy of the ADC of the present invention on tumor-bearing mice.

[0702] Experimental animals: Balb / c Nude mice (Chengdu Yaokang Biotechnology Co., Ltd.)

[0703] Cell line: Human breast squamous carcinoma cell line HCC1806 (ATCC)

[0704] Experimental methods:

[0705] HCC1806 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. HCC1806 cells were collected during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into female Balb / c-nude mice to establish a breast squamous cell carcinoma model. The average tumor volume was approximately 200 mm.3 Around 30 days, the mice were randomly divided into groups according to the size of the tumor and given drugs separately. The groups and their dosages were as follows: vehicle control group (i.e., negative control, Vehicle group): given 0.9% NaCl injection; ADC 5: dosage of 3 mg / kg; ADC 9: dosage of 3 mg / kg; ADC 11: dosage of 3.16 mg / kg; ADC 17: dosage of 3.32 mg / kg. Each group was injected with tail vein (iv) and given on Day 0, for a total of 1 dose. After administration, the body weight of the mice was measured twice a week, and the long and short diameters of the tumors were measured with a vernier caliper. The tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. Animal deaths were observed and recorded every day.

[0706] The tumor growth inhibition rate (TGI) was calculated using the following formula: 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%.

[0707] Where V T末 : Mean tumor volume of treatment group at the end of the experiment

[0708] V T0 : Mean tumor volume at the start of drug administration in the treatment group

[0709] V C末 : Mean tumor volume of negative control group at the end of the experiment

[0710] V C0 : Mean tumor volume of negative control group at the beginning of drug administration

[0711] The relative tumor proliferation rate T / C (%) was calculated using the following formula: T / C = (V T末 / V T0 ) / (V C末 / V C0 ).

[0712] The ADCs of the present invention demonstrated significant tumor growth inhibition in an HCC1806 breast squamous cell carcinoma xenograft model. On day 14, compared to the vehicle group, the tumor growth inhibition rates (TGI) of ADCs 5, 9, 11, and 17 were 96.68%, 98.50%, 82.61%, and 86.69%, respectively, demonstrating significant differences compared to the control group. During treatment, there were no animal deaths or significant weight loss in any of the treatment groups, and no significant drug toxicity was observed. The ADCs of the present invention were well tolerated by mice. Specific results are shown in Table 20.

[0713] Table 20 Analysis of the efficacy of different antibody-drug conjugates in HCC1806 cell tumor-bearing mouse models

[0714] Note: TGI is tumor growth inhibition rate, T / C is relative tumor proliferation rate.

[0715] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are all within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody drug conjugate having the formula Ab-[MLED] x The structure shown, wherein: Ab is an antibody or antigen-binding fragment thereof that specifically binds to the B7-H3 antigen; M is a linker connected to the antibody or antigen-binding fragment thereof; L is a linker between the linkers M and E; E is a structural fragment connecting L and D; D is the cytotoxic drug fragment; x is selected from 1 to 10.

2. The antibody-drug conjugate according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): (a) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 1; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 2; (b) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 3; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) set forth in SEQ ID NO: 4; or (c) CDR-H1, CDR-H2 and CDR-H3 contained in the following heavy chain variable region (VH), and / or CDR-L1, CDR-L2 and CDR-L3 contained in the following light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation compared to the heavy chain variable region and / or light chain variable region described in any one of (a) or (b), and the mutation is a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitution is a conservative substitution; Preferably, the CDRs are defined according to the IMGT, Kabat, Chothia or AbM numbering systems.

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

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein: The antibody or antigen-binding fragment thereof 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; wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

5. The antibody-drug conjugate according to 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-drug conjugate according to any one of claims 1 to 5, wherein: The antibody or antigen-binding fragment thereof further comprises a constant region from or derived from a human immunoglobulin; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region from or derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4); preferably, the antibody or antigen-binding fragment thereof comprises a wild-type Fc region, or comprises a mutated or chemically modified Fc region having altered effector function compared to the wild-type Fc region; Preferably, the antibody or antigen-binding fragment thereof comprises a variant of the human IgG1 heavy chain constant region having the following substitutions compared to the wild-type sequence from which it is derived: Leu234Ala, Leu235Ala and Gly237Ala (positions according to the EU numbering system); Preferably, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region from or derived from a human immunoglobulin (eg, κ or λ); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 30, or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions compared to SEQ ID NO: 30 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 31 or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions compared to SEQ ID NO: 31 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions); Preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as set forth in SEQ ID NO: 32, or a variant thereof having up to 20 conservative amino acid substitutions compared to SEQ ID NO: 32 (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; for example, 1, 2, 3, 4 or 5 conservative amino acid substitutions); More preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 30 and a light chain constant region (CL) as shown in SEQ ID NO: 32; or, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 31 and a light chain constant region (CL) as shown in SEQ ID NO:

32.

7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein: The antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising the VH region represented by SEQ ID NO: 1 and the heavy chain constant region (CH) represented by SEQ ID NO: 30, and a light chain comprising the VL region represented by SEQ ID NO: 2 and the light chain constant region (CL) represented by SEQ ID NO: 32; (2) a heavy chain comprising the VH region of SEQ ID NO: 3 and the heavy chain constant region (CH) of SEQ ID NO: 30, and a light chain comprising the VL region of SEQ ID NO: 4 and the light chain constant region (CL) of SEQ ID NO: 32; (3) a heavy chain comprising the VH region of SEQ ID NO: 1 and the heavy chain constant region (CH) of SEQ ID NO: 31, and a light chain comprising the VL region of SEQ ID NO: 2 and the light chain constant region (CL) of SEQ ID NO: 32; or, (4) a heavy chain comprising the VH region of SEQ ID NO: 3 and the heavy chain constant region (CH) of SEQ ID NO: 31, and a light chain comprising the VL region of SEQ ID NO: 4 and the light chain constant region (CL) of SEQ ID NO: 32; Preferably, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain having the sequence shown in SEQ ID NO: 40 and a light chain having the sequence shown in SEQ ID NO: 41; or, (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.

8. The antibody-drug conjugate according to 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), diabody, bispecific antibody and multispecific antibody.

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

10. The antibody drug conjugate according to any one of claims 1 to 9, wherein M is in, Ring A is a 5-6 membered alicyclic heterocyclic ring or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl group substituted; M1 is selected from single bond and C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 an alkynylene or amine group; Preferably, M is wherein Ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more (e.g., 2) 6-membered heteroaromatic rings to a benzene ring or a 6-membered heteroaromatic ring via a single bond, wherein the alicyclic heterocycle is optionally substituted by one or more selected from oxy (=O), halogen and C 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 Alkenylene, C 2-20 an alkynylene or amine group; Preferably, M is wherein ring A is selected from M1 is selected from a single bond and C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 an alkynylene or amine group; Preferably, M is selected from Preferably, M is selected from Preferably, M is selected from Preferably, M is selected from 11. The antibody drug conjugate according to any one of claims 1 to 10, wherein L is selected from one or more of the following structures: C 1-6 Alkylene, -N(R')-, carbonyl, -O-, natural or unnatural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys (COCH2CH2 (OCH2CH2) r OCH3)), and short peptides composed of amino acids (such as 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, Val- Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly), Where R' represents hydrogen, C 1-6 Alkyl or polyethylene glycol fragment containing 1-10 EO units; s is an integer selected from 1-20; Preferably, L is selected from the group consisting of one or more of the following structures: C 1-6 Alkylene, carbonyl, -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、 wherein s is an integer selected from 1 to 20; Preferably, L is selected from the group consisting of one or more of the following: Preferably, L is selected from the following structures: Preferably, L is selected from the following structures: Preferably, L is selected from the following structures: Preferably, L is selected from the following structures:

12. The antibody drug conjugate according to any one of claims 1 to 11, wherein E is a single bond, -NHCH2-, or is selected from the following structures: Preferably, E is a single bond, -NHCH2-, Preferably, E is -NHCH2- or Preferably, E is -NHCH2-; Preferably, E is a single bond; Preferably, E is 13. The antibody-drug conjugate according to any one of claims 1 to 12, Selected from the following structures: Preferably, Selected from the following structures:

14. The antibody-drug conjugate of any one of claims 1 to 13, wherein the cytotoxic drug is selected from a microtubule inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor; preferably, the microtubule 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, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, 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 cytotoxic drug is selected from the compounds represented by Formula I or Formula II, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds represented by Formula I or Formula II: in, R1, R2 are each independently selected from C 1-6 Alkyl and halogen; R3 is selected from H and -CO-CH2OH; R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring; R6 is selected from hydrogen or -C 1-4 Alkylene-NR a R b ; R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ; where R a 、R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6 alkyl; Preferably, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds: The corresponding fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is D in the general formula; preferably, D is a monovalent structure obtained by losing one H from the -OH, -NH2 or secondary amine group on the cytotoxic drug; Preferably, the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:

15. The antibody-drug conjugate according to any one of claims 1 to 14, selected from the group consisting of ADC A-01 to ADC A-34, ADC B-01 to ADC B-07, or ADC C-01 to ADC C-28 shown below; wherein the thiol group on the antibody and the drug linker compound form a thioether bond through an addition reaction or a substitution reaction to obtain the complete antibody-drug conjugate, and x represents the drug loading amount: Preferably, Ab in each antibody-drug conjugate represents an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO: 3 and VL as shown in SEQ ID NO: 4, for example, an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO: 3 and CH as shown in SEQ ID NO: 31, and VL as shown in SEQ ID NO: 4 and VL as shown in SEQ ID NO: 51; in, Indicates the specific connection method between the thiol group in the antibody or its antigen-binding fragment and the linker.

16. The antibody drug conjugate according to any one of claims 1 to 15, wherein the DAR value (drug-antibody conjugate ratio) is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-9, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3.5-6.0, 3.5-6.5, 3.5-7.0, 3.5-7.5, 3.5-8.0, 4.0~4.5,4.0~5.0,4.0~5.5,4.0~6.0,4.0~6.5,4.0~7.0,4.0~7.5,4.0~8.0,4.5~5.0,4.5~5.5,4.5~6.0,4.5~6.5,4.5~7.0,4.5~7.5,4.5~8.0,5.0~5.5,5.0~6.0,5.0~6.5,5.0~ 7.0, 5.0-7.5, 5.0-8.0, 5.5-6.0, 5.5-6.5, 5.5-7.0, 5.5-7.5, 5.5-8.0, 6.0-6.5, 6.0-7.0, 6.0-7.5, 6.0-8.5, 6.5-7.0, 6.5-7.5, 6.5-8.5, 7.0-7.5, 7.0-9.0 or 7.5-9.0; more preferably 4-8.

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

18. Use of the antibody-drug conjugate according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 19 in the preparation of a medicament for treating B7-H3-positive tumors; Preferably, the B7-H3-positive tumor includes solid tumors or hematological malignancies, 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.

Images