TW202421204A - Antibody-drug conjugate and method for preparation and use thereof - Google Patents

Abstract

The present application relates to an antibody-drug conjugate and a method for preparation and use thereof, and specifically relates to an antibody-drug conjugate for treating PTK7-positive cancer, including a PTK7 antibody, and drug-linker molecules conjugated to the antibody. In some embodiments, the antibody is humanized, has excellent binding activity to PTK7-positive cells, and can efficiently deliver drugs to PTK7-positive cells. In some embodiments, the drug includes a DNA topoisomerase inhibitor. The antibody-drug conjugate of the present application has a better drug-antibody conjugation ratio, and has a good targeted killing effect on lung cancer, breast cancer, epidermal cancer, ovarian cancer, esophageal cancer, and the like. Therefore, the present application further provides a method for preparation of the antibody-drug conjugate and use thereof in treatment of PTK7-positive cancer.

Description

Antibody-drug conjugate and preparation method and use thereof

This application relates to the field of targeted therapy, and in particular to an antibody-drug conjugate for treating PTK7-positive cancers. In particular, the antibody-drug conjugate comprises a PTK7 antibody and a drug-linker molecule bound to the antibody. In some embodiments, the antibody is humanized, has excellent activity in binding to PTK7-positive cells, and can effectively deliver drugs to PTK7-positive cells. In some embodiments, the drug comprises a DNA topoisomerase inhibitor. The antibody-drug conjugate of this application has an excellent drug-antibody binding ratio and has a good targeted killing effect on lung cancer, breast cancer, epithelial cancer, ovarian cancer and esophageal cancer. Therefore, the present application further provides methods for preparing antibody-drug conjugates and their use in treating PTK7-positive cancers.

Cancer is one of the leading causes of death in modern times. It is a type of disease caused by the malignant transformation of healthy cells and the malignant proliferation of cells due to genetic changes such as chromosomal translocation, tumor suppressor genes and growth factor receptor mutations. Defective apoptosis or programmed cell death further promotes the malignant transformation of cells, leading to cancer. According to the latest assessment of the International Agency for Research on Cancer (IARC) under the World Health Organization (WHO), there will be 19.29 million new cancer cases worldwide in 2020, including 10.06 million men and 9.23 million women. In 2020, there will be 9.96 million cancer deaths worldwide, including 5.53 million men and 4.43 million women. During this century, cancer is predicted to surpass cardiovascular disease as the leading cause of premature death in most countries.

PTK7 (protein tyrosine kinase 7) belongs to the receptor tyrosine kinase family and may lack kinase activity due to mutations in the kinase domain. The PTK7 protein is composed of 7 extracellular immunoglobulin domains, a transmembrane domain, and an intracellular tyrosine kinase domain, and its ligand is unknown. The extracellular segment of PTK7 can be cleaved by ADAM and MT1-MMP proteases to produce soluble fragments, and the concentration of free PTK7 in healthy human serum is about 12.4 ± 3.3 ng/ml; and the concentration in tumor patients is higher, about 24.6 ± 3.8 ng/ml.

PTK7 is expressed in a variety of solid tumors: it is reported to be highly expressed in 47.4% of NSCLC, 45.1% of ovarian cancer, 28.6% of TNBC, and 60% of esophageal cancer, and studies have shown that PTK7 expression promotes tumor cell growth and that knockout of the PTK7 gene reduces tumor burden in mice. High expression of PTK7 is positively correlated with disease stage and lymph node metastasis, and negatively correlated with survival. Currently, PTK7-targeted antibody-drug conjugates, CAR-T, and other treatments are being studied. In preclinical studies, tumor cell lines with high PTK7 expression will be effectively killed. These studies have shown strong tumor inhibitory activity in human-derived tumor cell-derived xenograft (CDX) and human-derived tumor xenograft (PDX) models. Cofetuzumab Pelidotin, an ADC drug targeting PTK7 and developed by Pfizer, has shown good safety and preliminary efficacy in Phase I clinical trials.

ADC drugs are composed of antibodies, bioactive molecules and linkers. The bioactive molecules are covalently bound to the antibodies via linkers; the antibodies (e.g. monoclonal antibodies) can specifically recognize specific targets on the surface of tumor cells, and then guide the ADC to the surface of cancer cells, allowing the ADC to enter the cancer cells via endocytosis; and then the bioactive molecules are released in the cancer cells to kill the cancer cells while minimizing damage to normal tissue cells. Developing antibodies with better targeting, hydrophilicity and endocytosis activity for binding to cytotoxic small molecules to prepare ADC drugs with a larger therapeutic window will provide patients with better treatment options.

The present application relates to an antibody-drug conjugate for treating PTK7-positive cancers, and exemplarily discloses an antibody-drug conjugate using humanized antibodies 101A6HZ and 64A10HZ as targeting moieties and having a structure represented by the general formula Ab-[MLED] _x . The results show that the conjugate has a better drug-antibody binding ratio, and the conjugate has excellent activity in binding to PTK7-positive cells, and has a good target killing effect on PTK7-positive cells (e.g., lung cancer, breast cancer, epithelial cancer, ovarian cancer, and esophageal cancer cells). Therefore, the present application provides an antibody-drug conjugate for treating cancers with high PTK7 expression, a pharmaceutical composition comprising the antibody-drug conjugate, and its use for treating cancers with high PTK7 expression. Antibody-drug conjugates

In one embodiment, the present application provides an antibody-drug conjugate having a structure shown as formula Ab-[MLED] _x , wherein: Ab is an antibody or an antigen-binding fragment thereof that specifically binds to human tyrosine kinase 7 (PTK7); M is a linker site linked to the antibody or its antigen-binding fragment; L is a linker between M and E; E is a structural fragment connecting L and D; D is a cytotoxic drug fragment; and x is selected from 1 to 10.

In some embodiments, 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 Chothia numbering system: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having a sequence shown as SEQ ID NO: 11, CDR-H2 or a variant thereof having a sequence shown as SEQ ID NO: 12, and CDR-H3 or a variant thereof having a sequence shown as SEQ ID NO: 13; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having a sequence shown as SEQ ID NO: 14, CDR-L2 or a variant thereof having a sequence shown as SEQ ID NO: 15 or a variant thereof and a CDR-L3 or a variant thereof having a sequence shown as SEQ ID NO: 16; or (1b) a heavy chain variable region (VH) comprising the following three CDRs: a CDR-H1 or a variant thereof having a sequence shown as SEQ ID NO: 27, a CDR-H2 or a variant thereof having a sequence shown as SEQ ID NO: 28, a CDR-H3 or a variant thereof having a sequence shown as SEQ ID NO: 29; and/or a light chain variable region (VL) comprising the following three CDRs: a CDR-L1 or a variant thereof having a sequence shown as SEQ ID NO: 30, a CDR-L2 or a variant thereof having a sequence shown as SEQ ID NO: 31, and a CDR-L3 or a variant thereof having a sequence shown as SEQ ID NO: 32 or a variant thereof; wherein the variant of any one of (1a) and (1b) has at least 70%, 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 the variant is derived, or has undergone one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which the variant is derived; preferably, the substitutions are conservative substitutions; or (2) 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: (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence shown as SEQ ID NO: 17 or a variant thereof, CDR-H2 having a sequence shown as SEQ ID NO: 18 or 19 or a variant thereof, CDR-H3 having a sequence shown as SEQ ID NO: 13 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence shown as SEQ ID NO: 14 or a variant thereof, CDR-L2 having a sequence shown as SEQ ID NO: 15 or a variant thereof, and CDR-L3 having a sequence shown as SEQ ID NO: 16 or a variant thereof; or (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence shown as SEQ ID NO: 17 or a variant thereof, CDR-H2 having a sequence shown as SEQ ID NO: 18 or 19 or a variant thereof, and CDR-H3 having a sequence shown as SEQ ID NO: 13 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence shown as SEQ ID NO: 14 or a variant thereof, CDR-L2 having a sequence shown as SEQ ID NO: 15 or a variant thereof, and CDR-L3 having a sequence shown as SEQ ID NO: 16 or a variant thereof; or 33 or its variant, CDR-H1 having a sequence shown as SEQ ID NO: 34 or 35 or its variant, and CDR-H3 having a sequence shown as SEQ ID NO: 29 or its variant; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence shown as SEQ ID NO: 30 or its variant, CDR-L2 having a sequence shown as SEQ ID NO: 31 or its variant, and CDR-L3 having a sequence shown as SEQ ID NO: 32 or its variant; wherein the variant of any of (2a) and (2b) has at least 70%, 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 the variant is derived, or undergoes one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which the variant is derived; preferably, the substitutions are conservative substitutions; or (3) 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: (3a) a heavy chain variable region (VH) comprising the following 3 CDRs: having a sequence shown as SEQ ID NO: 20 or its variant, CDR-H2 or its variant having a sequence shown as SEQ ID NO: 21 and CDR-H3 or its variant having a sequence shown as SEQ ID NO: 22; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or its variant having a sequence shown as SEQ ID NO: 23, CDR-L2 or its variant having a sequence shown as SEQ ID NO: 24 and CDR-L3 or its variant having a sequence shown as SEQ ID NO: 16; or (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or its variant having a sequence shown as SEQ ID NO: 36, CDR-H2 or its variant having a sequence shown as SEQ ID NO: 37 or its variants and CDR-H2 or its variants having the sequence shown as SEQ ID NO: 38 and CDR-H3 or its variants having the sequence shown as SEQ ID NO: 38; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or its variants having the sequence shown as SEQ ID NO: 39, CDR-L2 or its variants having the sequence shown as SEQ ID NO: 40 and CDR-L3 or its variants having the sequence shown as SEQ ID NO: 32; wherein the variant of any of (3a) and (3b) has at least 70%, 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 the variant is derived, or undergoes one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which the variant is derived; preferably, the substitutions are conservative substitutions; 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 3 CDRs: having a sequence shown as SEQ ID NO: 25 or its variant, CDR-H2 or its variant having a sequence shown as SEQ ID NO: 26 and CDR-H3 or its variant having a sequence shown as SEQ ID NO: 13; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or its variant having a sequence shown as SEQ ID NO: 14, CDR-L2 or its variant having a sequence shown as SEQ ID NO: 15 and CDR-L3 or its variant having a sequence shown as SEQ ID NO: 16; or (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or its variant having a sequence shown as SEQ ID NO: 41, CDR-H2 or its variant having a sequence shown as SEQ ID NO: 42 or its variants and CDR-H2 or its variants having the sequence shown as SEQ ID NO: 29 and CDR-H3 or its variants; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or its variants having the sequence shown as SEQ ID NO: 30, CDR-L2 or its variants having the sequence shown as SEQ ID NO: 31 and CDR-L3 or its variants having the sequence shown as SEQ ID NO: 32; The variant of any one of (4a) and (4b) has at least 70%, 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 the variant is derived, or has undergone substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared to the sequence from which the variant is derived; preferably, the substitution is a conservative substitution.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: (a) VH or a variant thereof shown as SEQ ID NO: 1, and/or VL or a variant thereof shown as SEQ ID NO: 2; (b) VH or a variant thereof shown as SEQ ID NO: 3, and/or VL or a variant thereof shown as SEQ ID NO: 4; or (c) VH or a variant thereof shown as SEQ ID NO: 5, and/or VL or a variant thereof shown as SEQ ID NO: 6; (d) VH or a variant thereof shown as SEQ ID NO: 7, and/or VL or a variant thereof shown as SEQ ID NO: 8; (e) VH or a variant thereof shown as SEQ ID NO: 9, and/or VL or a variant thereof shown as SEQ ID NO: 10 VL or its variant; wherein the variant has at least 70%, 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 the variant is derived, or undergoes substitution, deletion or addition of one or more amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) compared to the sequence from which the variant is derived; and preferably, the substitution is a conservative substitution.

In some embodiments, the antibody or antigen-binding fragment thereof further comprises: (a) a human immunoglobulin heavy chain constant region (CH) or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which the variant is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; for example, substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids); and (b) A human immunoglobulin light chain constant region (CL) or variant, wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids) compared to the wild-type sequence from which the variant is derived.

In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, such as a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region. In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) shown as SEQ ID NO: 43 or a variant thereof, and compared with SEQ ID NO: 43, the variant has a conservative substitution of up to 20 amino acids (e.g., a conservative substitution of up to 15, up to 10 or up to 5 amino acids; for example, a conservative substitution of 1, 2, 3, 4 or 5 amino acids); In some embodiments, the light chain constant region is a kappa light chain constant region. In some embodiments, the antibody or its antigen-binding region comprises a light chain constant region (CL) shown as SEQ ID NO: 44 or a variant thereof, and the variant has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 44 (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 some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) shown as SEQ ID NO: 43 or 45, and a light chain constant region (CL) shown as SEQ ID NO: 44.

In some embodiments, the antibody or its antigen-binding fragment comprises: (1) a heavy chain comprising a VH sequence shown as SEQ ID NO: 1 and a heavy chain constant region (CH) shown as SEQ ID NO: 43, and a light chain comprising a VL sequence shown as SEQ ID NO: 2 and a light chain constant region (CL) shown as SEQ ID NO: 44; (2) a heavy chain comprising a VH sequence shown as SEQ ID NO: 3 and a heavy chain constant region (CH) shown as SEQ ID NO: 43 or 45, and a light chain comprising a VL sequence shown as SEQ ID NO: 4 and a light chain constant region (CL) shown as SEQ ID NO: 44; (3) a VH sequence shown as SEQ ID NO: 5 and a light chain constant region (CL) shown as SEQ ID NO: 43, and a light chain comprising a VL sequence shown as SEQ ID NO: 6 and a light chain constant region (CL) shown as SEQ ID NO: 44; (4) a heavy chain comprising a VH sequence shown as SEQ ID NO: 7 and a heavy chain constant region (CH) shown as SEQ ID NO: 43, and a light chain comprising a VL sequence shown as SEQ ID NO: 8 and a light chain constant region (CL) shown as SEQ ID NO: 44; or (5) a heavy chain comprising a VH sequence shown as SEQ ID NO: 9 and a heavy chain constant region (CH) shown as SEQ ID NO: 43, and a VL sequence shown as SEQ ID NO: 10 and a light chain constant region (CL) shown as SEQ ID NO: The light chain of the light chain constant region (CL) of 44.

In the antibody-drug conjugate, the cytotoxic drug can be linked to the antibody or antigen-binding fragment via a linker (such as the "M-L-E" fragment shown in this application).

In some embodiments, M is , wherein Ring A is a 5-6 membered aliphatic heterocyclic ring or a 5-20 membered aromatic ring system, and the aliphatic heterocyclic ring and the aromatic ring system are optionally substituted with one or more members selected from the group consisting of a oxo group (=O), a halogen, a cyano group, an amine group, a carboxyl group, a hydroxyl group, and a C _1-6 alkyl group; and M ₁ is selected from a single bond, a C _1-20 alkylene group, a C _2-20 alkenylene group, and a C _2-20 alkynylene group.

In some embodiments, M is , wherein Ring A is a 5-membered aliphatic heterocyclic ring, a 6-membered aromatic heterocyclic ring, or a polycyclic ring formed by connecting one or more 6-membered aromatic heterocyclic rings and a benzene ring via a single bond, and the aliphatic heterocyclic ring is optionally substituted with one or more members selected from the group consisting of a pendooxy group (=O), a halogen, and a C _1-4 alkyl group; and M ₁ is selected from a single bond, a C _3-10 alkylene group, a C _3-10 alkenylene group, and a C _3-10 alkynylene group.

In some embodiments, M is , wherein ring A is selected from , , and ; and M ₁ is selected from a single bond, a C _5-8 alkylene group, a C _5-8 alkenyl group and a C _5-8 alkynyl group.

In some embodiments, M is selected from the following structures: and .

In some embodiments, M is selected from the following structures: .

In some embodiments, L is selected from a structure comprising one or more of the following: C _1-6 alkylene, -N(R')-, carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH ₂ CH ₂ (OCH ₂ CH ₂ ) _s OCH ₃ ), D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly (SEQ ID NO: 48), Gly-Phe-Leu-Gly (SEQ ID NO: 49), Gly-Gly-Val-Ala (SEQ ID NO: 50), Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 51), , , , , , , , , , , and , wherein R' represents hydrogen, C _1-6 alkyl or an alkyl group containing -(CH ₂ CH ₂ O)r-; r is selected from an integer of 1-10; and s is selected from an integer of 1-20.

In some embodiments, L is selected from a structure comprising one or more of the following: C _1-6 alkylene, -N(R')-, carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH ₂ CH ₂ (OCH ₂ CH ₂ ) _s OCH ₃ ), D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly (SEQ ID NO: 48), Gly-Phe-Leu-Gly (SEQ ID NO: 49), Gly-Gly-Val-Ala (SEQ ID NO: 50), Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 51), , , , , , , , , , and , wherein R' represents hydrogen, C _1-6 alkyl or an alkyl group containing -(CH ₂ CH ₂ O)r-; r is selected from an integer of 1-10; and s is selected from an integer of 1-20.

In some embodiments, L is a structure selected from one or more of the following: C _1-6 alkylene, -NH-, Val, Cit, Phe, Lys, Lys(COCH ₂ CH ₂ (OCH ₂ CH ₂ ) _s OCH ₃ ), Gly, Val-Cit, Gly-Gly-Phe-Gly (SEQ ID NO: 48), , , , , , , and , where s is an integer selected from 1-20.

In some embodiments, L is selected from a structure comprising one or more of the following: C _1-6 alkylene, -NH-, Val, Cit, Phe, Lys, Lys(COCH ₂ CH ₂ (OCH ₂ CH ₂ ) _s OCH ₃ ), Gly, Val-Cit, Gly-Gly-Phe-Gly (SEQ ID NO: 48), , , , , , and ; where s is an integer from 1 to 20.

In some embodiments, L is selected from the following structures: , , , , , , , , , , and ; Where s is an integer selected from 1 to 20.

In some embodiments, L is selected from the following structures: , , , , , , , , , , and ; Where s is an integer selected from 1-20.

In some embodiments, L is selected from the following structures: , , , , and .

In some embodiments, L is selected from the following structures: , , , and .

In some embodiments, L is selected from the following structures: , , and .

In some embodiments, L is selected from the following structures: , and .

In some embodiments, E is a single bond, -NH-CH ₂ -, -NH-CH ₂ -O-CH ₂ -CO-, , , , or .

In some embodiments, E is a single bond, -NH-CH ₂ -, -NH-CH ₂ -O-CH ₂ -CO-, , or .

In some embodiments, E is -NH- _CH2- , or .

In some embodiments, E is -NH- _CH2- or .

In some embodiments, M is selected from the following structures: and ; L is selected from the following structures: , , , and ; E is -NH-CH ₂ , or .

In some embodiments, M is selected from the following structures: and ; L is selected from the following structures: , , and ; E is -NH-CH ₂ , or In some embodiments, Select from the following structures: , , , , , , , , , , , , and .

In some embodiments, Select from the following structures: , , , , , , , , , , and .

In some embodiments, Select from the following structures: , , , , , , , and .

In some embodiments, Select from the following structures: , , , , , and .

In some embodiments, the cytotoxic drug is selected from tubulin inhibitors, DNA intercalators, DNA topoisomerase inhibitors, and RNA polymerase inhibitors. In some embodiments, the tubulin inhibitor is an auristatin compound or a maytansinoid 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, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester or analog thereof.

The cytotoxic drugs disclosed in the present application generally contain multiple functional groups, such as hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH), primary amine (-NH ₂ ), diamine (-NR _A H) or tertiary amine (-NR _{B RC} ), and _RA , _RB and _RC herein represent only non-hydrogen substituents on N; and the cytotoxic drugs can be linked to the linker in the conjugate via these functional groups.

In some embodiments, the cytotoxic drug is linked to E in the antibody-drug conjugate via a -OH, -SH, primary amine, secondary amine, or tertiary amine group thereon.

In some embodiments, the cytotoxic drug is .

In some embodiments, the cytotoxic drug is selected from the following Formula I and Formula II: wherein R ₁ and R ₂ are each independently selected from the group consisting of C _1-6 alkyl and halogen; R ₃ is selected from the group consisting of H and -CO-CH ₂ OH; R ₄ and R ₅ are each independently selected from the group consisting of H, halogen and hydroxyl; or R ₄ and R ₅ together with the carbon atom to which they are attached form a 5-6 membered oxygen-containing heterocyclic ring; R ₆ is selected from the group consisting of hydrogen and -C _1-4 alkylene-NR _a R _b ; R ₇ is selected from the group consisting of H, C _1-6 alkyl and -C _1-4 alkylene-NR _a R _b ; wherein, at each occurrence, R _a and R _b are each independently selected from the group consisting of: H, C _1-6 alkyl, -SO ₂ -C _1-6 alkyl and -CO-C _1-6 alkyl.

In some embodiments, the cytotoxic drug is selected from the following Formula I and Formula II: R ₁ and R ₂ are independently selected from C _1-6 alkyl and halogen; R ₃ is selected from H and -CO-CH ₂ OH; R ₄ and R ₅ are independently selected from H, halogen and hydroxyl; or R ₄ and R ₅ are connected to the relevant carbon atoms to form a 5-6 membered oxygen-containing heterocyclic ring; R ₆ is selected from hydrogen or -C _1-4 alkylene-NR _a R _b ; R ₇ is selected from C _1-6 alkyl and -C _1-4 alkylene-NR _a R _b ; wherein R _a and R _b are independently selected from H, C _1-6 alkyl, -SO ₂ -C _1-6 alkyl and -CO-C _1-6 alkyl at each occurrence.

In some embodiments, the cytotoxic drug is selected from the following compounds: and .

In some embodiments, the cytotoxic drug is selected from the following compounds: and .

According to the present application, the corresponding fragment of the cytotoxic drug obtained after the cytotoxic drug is linked to the linker is D in the formula Ab-[MLED] _x . In some embodiments, D is a monovalent structure obtained by losing one H from -OH, -NH ₂ or a diamine group on the cytotoxic drug.

In some embodiments, D is selected from the following structures: and .

In some embodiments, the antibody-drug conjugate is selected from ADC A-01 to ADC A-26, ADC B-01 to ADC B-06, and ADC C-01, as shown below: ADC A-01 、ADC A-02 、ADC A-03 、ADC A-04 、ADC A-05 、ADC A-06 、ADC A-07 、ADC A-08 、ADC A-09 、ADC A-10 、ADC A-11 、ADC A-12 、ADC A-13 、ADC A-14 、ADC A-15 、ADC A-16 、ADC A-17 、ADC A-18 、ADC A-19 、ADC A-20 、ADC A-21 、ADC A-22 、ADC A-23 、ADC A-24 、ADC A-25 、ADC A-26 ADC B-01 、ADC B-02 、ADC B-03 、ADC B-04 、ADC B-05 、ADC B-06 and ADC C-01 .

The HA in each antibody-drug conjugate represents an antibody or antigen-binding fragment having a VH represented by SEQ ID NO: 1, 3, 5, 7 or 9 and a VL represented by SEQ ID NO: 2, 4, 6, 8 or 10, for example, an antibody or antigen-binding fragment having a VH represented by SEQ ID NO: 3 and a CH represented by SEQ ID NO: 43 or 45, a VL represented by SEQ ID NO: 4 and a CL represented by SEQ ID NO: 44; and Represents the specific bonding pattern of sulfhydryl groups and linkers in an antibody or antigen-binding fragment.

In some embodiments, the antibody-drug conjugate is selected from ADC A-01 to ADC A-25, ADC B-01 to ADC B-06, and ADC C-01, as shown below: ADC A-01 、ADC A-02 、ADC A-03 、ADC A-04 、ADC A-05 、ADC A-06 、ADC A-07 、ADC A-08 、ADC A-09 、ADC A-10 、ADC A-11 、ADC A-12 、ADC A-13 、ADC A-14 、ADC A-15 、ADC A-16 、ADC A-17 、ADC A-18 、ADC A-19 、ADC A-20 、ADC A-21 、ADC A-22 、ADC A-23 、ADC A-24 、ADC A-25 、ADC B-01 、ADC B-02 、ADC B-03 、ADC B-04 、ADC B-05 、ADC B-06 、ADC C-01 .

The HA in each antibody-drug conjugate represents an antibody or antigen-binding fragment having a VH represented by SEQ ID NO: 1, 3, 5, 7 or 9 and a VL represented by SEQ ID NO: 2, 4, 6, 8 or 10, for example, an antibody or antigen-binding fragment having a VH represented by SEQ ID NO: 3 and a CH represented by SEQ ID NO: 43 or 45, a VL represented by SEQ ID NO: 4 and a CL represented by SEQ ID NO: 44; and Represents the specific bonding pattern of sulfhydryl groups and linkers in an antibody or antigen-binding fragment.

In certain embodiments of the antibodies or antibody-drug conjugates disclosed herein, the heavy chain constant domain may comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In certain embodiments of the antibodies or antibody-drug conjugates disclosed herein, the N-terminal amino acid of the antibody variable domain may be cyclized to pyroglutamate. In certain embodiments of the antibodies or antibody-drug conjugates disclosed herein, the N-terminal amino acid of the antibody variable domain may be cyclized to pyroglutamate.

In certain embodiments, the antibodies or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to an antigen and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage, glutamic acid or conversion of glutamic acid to pyroglutamic acid salt or pyroglutamic acid) that may occur during recombinant expression in a host cell (e.g., CHO cells) or during purification/storage.

Thus, a composition may comprise a population of antibody-drug conjugate species, wherein each species may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and/or comprise an N-terminal glutamate or glutamine or an N-terminal amino acid cyclized to pyroglutamate. In certain embodiments, a composition may comprise a population of antibody-drug conjugate species, wherein each species may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and/or comprise an N-terminal glutamate or glutamine or an N-terminal amino acid cyclized to pyroglutamate.

Therefore, in specific embodiments, the present invention further provides a composition comprising the ADC disclosed herein, wherein the major ADC species in the composition comprises (i) an antibody lacking a lysine residue at the heavy chain C-terminus; (ii) an antibody having glutamic acid, glutamine, pyroglutamic acid salt or pyroglutamic acid at the heavy chain N-terminus; or (iii) an antibody lacking a lysine residue at the heavy chain C-terminus and having glutamic acid, glutamine, pyroglutamic acid salt or pyroglutamic acid at the heavy chain N-terminus.

In a specific embodiment, the present invention further provides a composition comprising the ADC disclosed herein, wherein the major ADC species in the composition comprises an antibody lacking a lysine residue at the heavy chain C-terminus and having pyroglutamate or pyroglutamine at the heavy chain N-terminus.

In some embodiments, the antibodies disclosed herein are genetically engineered to comprise one or more cysteine or lysine residues or atypical amino acid substitutions at defined amino acid positions within the antibody.

In some embodiments, the antibodies disclosed herein are genetically engineered to include one or more cysteine or atypical amino acid substitutions of amino acids at defined positions within the antibody. The cysteine residues or atypical amino acid residues can then be conjugated to a drug-linker via the sulfhydryl group of the cysteine residue or the reactive group of the atypical amino acid. Thus, the antibody-drug conjugates of the present invention can include one or more substitutions of cysteine residues or atypical amino acid residues for amino acids in the heavy or light chain of the antibody, which cysteine residues or atypical amino acid residues are then conjugated to a drug-linker disclosed herein. In a specific embodiment, the amino acid position that can be substituted is selected from positions 152, 153, 171, 172, 173 and 375 of the heavy chain homeostasis domain (numbered according to the Eu numbering scheme) and positions 165 and 168 of the light chain homeostasis domain (numbered starting from the amino acid 1 of the N-terminus). In a specific embodiment, cysteine can replace the amino acid at one or more of positions 152, 153, 171, 172, 173 and 375 of the heavy chain homeostasis domain (numbered according to the Eu numbering scheme) and positions 165 and 168 of the light chain homeostasis domain (numbered starting from the amino acid 1 of the N-terminus). In specific embodiments, the antibody-drug conjugate comprises an S375C amino acid substitution that is bound to a drug-linker disclosed herein. In specific embodiments, the antibody comprises an S375C amino acid substitution and an E152C amino acid substitution that are each bound to a drug-linker disclosed herein. In specific embodiments, the antibody comprises an S375C amino acid substitution and an S168C amino acid substitution that are each bound to a drug-linker disclosed herein. Compositions

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. Thus, the composition may be characterized by a "drug to antibody" ratio (DAR) ranging from about 1 to about 10. Methods for determining the DAR are well known to those skilled in the art and include methods using reverse phase chromatography or HPLC-MS.

In some embodiments, the DAR value (drug-antibody binding ratio) of the drug-antibody 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, 4-1 0, 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 1-8, 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. Drug-conjugate

Those skilled in the art will appreciate that the antibody-drug conjugate described in the present application can be prepared in a modular manner. For example, a "drug-linker" in free form (which can be understood as M'-LED, wherein M' is the structural form of M before covalently linking to the antibody or its antigen-binding fragment) is obtained, and then the "drug-linker" in free form is covalently linked to the antibody or its antigen-binding fragment to obtain the antibody-drug conjugate of the present application. Accordingly, M' in the "drug-linker" in free form is linked to one or more sulfhydryl (-SH), amine (-NH ₂ ) or carboxyl (-COOH) groups on the antibody or its antigen-binding fragment via a substitution reaction (e.g., removal of -SO ₂ Me or -Br and similar structures thereon) or via an addition reaction and other means.

In another aspect, the present invention provides a drug-linker having a structure shown as the formula M'-LED, wherein M' is , and Lg is a leaving group for nucleophilic substitution reaction (e.g., halogen, mesyl, fluorophenol or ) or a hydroxyl group (-OH), a sulfhydryl group (-SH) or an amine group (-NH ₂ ); or Lg forms an unsaturated double bond with an adjacent atom on ring A; ring A is a 5-6 membered aliphatic heterocyclic ring or a 5-20 membered aromatic ring system, and the aliphatic heterocyclic ring and the aromatic ring system are optionally substituted by one or more groups selected from the following: a pendoxy group (=O), a halogen, a cyano group, an amine group, a carboxyl group, a sulfhydryl group and a C _1-6 alkyl group; and M ₁ is selected from a single bond, a C _1-20 alkylene group, a C _2-20 alkenyl group and a C _2-20 alkynyl group; and the L, E and D structures are defined according to any one of the above-mentioned antibody-drug conjugates.

In some embodiments, M' is , Lg is a methanesulfonyl group, or Lg and adjacent atoms on ring A form a carbon-carbon double bond; ring A is a 5-membered alicyclic heterocyclic ring, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more 6-membered heteroaromatic rings and a benzene ring via a single bond, and the alicyclic heterocyclic ring is optionally substituted by one or more groups selected from the following: a pendoxy group (=O), a halogen, and a C _1-4 alkyl group; and M ₁ is selected from a single bond, a C _3-10 alkylene group, a C _3-10 alkenylene group, and a C _3-10 alkynylene group.

In some embodiments, M' is , Selected from , , and ; and M ₁ is selected from a single bond, a C _5-8 alkylene group, a C _5-8 alkenyl group and a C _5-8 alkynyl group.

In some embodiments, M' is selected from and .

In some embodiments, M' is .

In some embodiments, the drug-linker in free form is selected from A-01 to A-26, B-01 to B-06 and C-01, as shown below: A-01: , A-02: , A-03: , A-04: , A-05: , A-06: , A-07: , A-08: , A-09: , A-10: , A-11: , A-12: , A-13: , A-14: , A-15: , A-16: , A-17: , A-18: , A-19: , A-20: , A-21: , A-22: , A-23: , A-24: , A-25: , A-26: , B-01: , B-02: , B-03: , B-04: , B-05: , B-06: , C-01: .

In some embodiments, the drug-linker in free form is selected from A-01 to A-25, B-01 to B-06 and C-01, as shown below: A-01: , A-02: , A-03: , A-04: , A-05: , A-06: , A-07: , A-08: , A-09: , A-10: , A-11: , A-12: , A-13: , A-14: , A-15: , A-16: , A-17: , A-18: , A-19: , A-20: , A-21: , A-22: , A-23: , A-24: , A-25: , B-01: , B-02: , B-03: , B-04: , B-05: , B-06: , C-01: . Pharmaceutical composition

In another aspect, the present application provides a pharmaceutical composition comprising any of the aforementioned antibody-drug conjugates, optionally any of the aforementioned drug-linkers, and one or more pharmaceutical adjuvants.

The antibody-drug conjugates described herein are generally formulated in a unit injectable form together with a pharmaceutically acceptable parenteral vehicle for parenteral use (e.g., concentrated injection, intravenous injection, intratumoral injection, and the like). 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 substance or solution (Remington’s Pharmaceutical Sciences (1980), 16th edition, Osol, A. ed.). The antibody-drug conjugates described herein or pharmaceutical compositions comprising the antibody-drug conjugates can be administered by any route suitable for the subject to be treated. Uses

The antibody-drug conjugates or pharmaceutical compositions thereof described herein can be used to treat a variety of diseases or disorders, such as cancers with high expression of PTK7, including solid tumors or hematological malignancies, such as lung cancer, breast cancer, epithelial cancers (e.g., skin squamous cell carcinoma and oral squamous cell carcinoma), ovarian cancer, esophageal cancer (e.g., esophageal squamous cell carcinoma), and the like.

Therefore, the present application provides the use of an antibody-drug conjugate, a drug-linker, or a pharmaceutical composition comprising the same for preparing a drug for treating a cancer with high PTK7 expression.

At the same time, the present application also provides a method for treating cancer with high PTK7 expression, the method comprising the following steps: administering an effective amount of any of the above-mentioned antibody-drug conjugates, drug linkers, or pharmaceutical compositions containing the same to an individual in need. Definition

Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Reference to the techniques used herein is intended to refer to the techniques commonly understood in the art, including those variations of techniques that are obvious to those skilled in the art or replacement of equivalent techniques. In addition, the laboratory procedures used herein (e.g., genomics, nucleic acid chemistry, and molecular biology) are all routine procedures widely used in their respective fields. Although it is believed that those skilled in the art fully understand the following terms, the following definitions are set forth to better explain the present invention.

The term "antibody" generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). The light chain of an antibody can be classified as kappa (κ) and lambda (λ) light chains. The heavy chain can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. In the light chain and the heavy chain, the variable region and the constant region are connected by a "J" region having about 12 or more amino acids, and the heavy chain further includes a "D" region having about 3 or more amino acids. Each heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region (CH). The constant region of the heavy chain is composed of three domains (CH1, CH2 and CH3). Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). The constant region of the light chain is composed of one domain, CL. The constant domain is not directly involved in the binding between the antibody and the antigen, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can also be subdivided into regions with high variability, called complementation determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and each VL is composed of 3 CDRs and 4 FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions (VH and VL) of each heavy chain/light chain pair form an antigen binding site. The arrangement of amino acids in each region or domain is based on various numbering systems known in the art.

The term "complementary determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The heavy and light chain variable regions each contain three CDRs, named CDR1, CDR2, and CDR3. The exact boundaries of the CDRs may be defined according to various numbering systems known in the art, such as, for example, the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia and 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, one skilled in the art will readily identify the CDRs defined according to each numbering system. In addition, the correspondence between different numbering systems is well known to one skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

In the present invention, the CDR in an antibody or antigen-binding fragment can be defined according to various numbering systems known in the art, such as, for example, according to the Kabat, Chothia, IMGT or AbM numbering systems. In certain embodiments, the CDR contained in an antibody or antigen-binding fragment is defined according to the Chothia numbering system.

The following general rules disclosed in www.bioinf.org.uk: Prof. Andrew CR Martin's group and reproduced below can be used to define CDRs in antibody sequences that include those amino acids that specifically interact with amino acids that comprise an antigenic determinant in the antigen to which the antibody binds. There are rare instances where these generally constant features do not occur; however, Cys residues are the most conserved feature. Ring Kabat AbM Chothia1 Contact2 IMGT L1 L24--L34 L24--L34 L24--L34 L30--L36 L27--L32 L2 L50--L56 L50--L56 L50--L56 L46--L55 L50--L52 L3 L89--L97 L89--L97 L89--L97 L89--L96 L89--L97 H1 H31--H35B (Kabat number)3 H26--H35B H26--H32..34 H30--H35B H26--H35B H1 H31--H35 (Chothia number) H26--H35 H26--H32 H30--H35 H26--H33 H2 H50--H65 H50--H58 H52--H56 H47--H58 H51--H56 H3 H95--H102 H95--H102 H95--H102 H93--H101 H93--H102 1 Some of these numbering schemes, especially for Chothia loops, vary depending on the individual publication examined. 2 Either numbering scheme can be used for these CDR definitions, except that the Contact numbering scheme uses either the Chothia or Martin (enhanced Chothia) definition. 3 When numbering using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies in length between H32 and H34. (This is because the Kabat numbering scheme places insertions at H35A and H35B.) If both H35A and H35B are absent, the loop ends at H32 If only H35A is present, the loop ends at H33 If both H35A and H35B are present, the loop ends at H34

The entire amino acid sequence of VH is usually numbered according to Kabat, and the three CDRs in the variable region can be defined according to any of the numbering schemes mentioned above. In a specific embodiment, the numbering of the amino acid positions in VH can be continuous, starting from amino acid position 1 and continuing to the end of the sequence or according to Kabat. Unless otherwise specified, the amino acid positions in VH and VL herein are defined according to continuous numbering.

The numbering of amino acid positions in the heavy chain constant domain can be consecutive, starting at amino acid position 1 and continuing to the end of the sequence or according to Eu numbering. The IgG1 heavy chain constant domain amino acid sequence has 330 amino acids numbered consecutively from 1 to 330. The corresponding sequence according to Eu numbering starts at position number 118 and ends at position number 447. Unless otherwise stated, amino acid positions in the heavy chain and light chain herein are defined according to consecutive numbering.

The term "framework region" or "FR" residues refer to those amino acid residues in the variable regions of an antibody other than the CDR residues as defined above.

The term "antigen-binding fragment" of an antibody refers to a polypeptide that is a fragment of an antibody, such as a polypeptide that is a fragment of a full-length antibody, which 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 antigen binding, which is also referred to as an "antigen-binding fragment". Generally, reference is made to Fundamental Immunology, Chapter 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 technology 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 (sdAb, nanobodies) and similar polypeptides, which contain at least a portion of an antibody that is sufficient to achieve antigen-specific binding ability to a polypeptide. Engineered variants of antibodies are summarized in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

The term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of VH domain (Ward et al., Nature 341: 544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of 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 on the hinge region; the term "Fab'fragment" means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in the F(ab') ₂ fragment, and the obtained fragment consists of the complete Fd fragment (consisting of VH and CH1 domains) of the light chain and heavy chain.

The term "Fv" means 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 capable of forming a complete antigen binding site. It is generally believed that the six CDRs achieve antigen binding specificity for an antibody. However, one variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) can also be used to recognize and bind an antigen, although its affinity may be lower than that of a complete binding site.

The term "Fc" refers to an antibody fragment formed by binding the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc fragment of an antibody has a variety of different functions but is not involved in antigen binding.

The term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein 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 linkers in the prior art include repeating GGGGS (SEQ ID NO: 55) amino acid sequences or variants thereof. For example, a linker having an amino acid sequence (GGGGS) ₄ (SEQ ID NO: 56) can be used, and variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers suitable for use in the present invention are described in Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31: 94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56; and Roovers et al. (2001), Cancer Immunol. In some instances, a disulfide bond may exist between the VL and VH of the scFv. In certain embodiments, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, a scFv comprising _NH2 -VH-VH-COOH and NH2 _- VL-VL-COOH.

The term "single domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, which refers to an antibody fragment composed of a single monomeric variable antibody domain (such as a single heavy chain variable region), and such antibody fragments retain the ability to bind to the same antigen-specific binding as the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single domain antibodies are also called nanobodies.

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.

Herein, unless otherwise specified, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.

Antigen-binding fragments of antibodies (such as the above-mentioned antibody fragments) can be obtained from a given antibody (such as the antibodies provided by the present invention) using conventional techniques known to those skilled in the art (such as 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 intact antibodies.

The term "mouse antibody" refers to antibodies obtained by fusing B cells and myeloma cells of immunized mice, screening for immortalized and antibody-secreting mouse hybrid fusion cells, and then performing screening, antibody preparation, and antibody purification; or refers to antibodies secreted by plasma cells formed by differentiation and proliferation of B cells after antigens invade the mouse body.

The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody. Typically, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies typically retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, and the like. The donor antibody may be a mouse, rat, rabbit or non-human primate (such as cynomolgus monkey) antibody having the desired properties (eg, antigen specificity, affinity, reactivity, ability to enhance immune cell activity and/or ability to enhance immune response).

As used herein, the term "identity" refers to the sequence match between two polypeptides or between two nucleic acids. When a position in the two sequences to be 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), the molecules are identical at that position. The "percent identity" between two sequences refers to the function obtained by the following formula: number of matching positions shared by the two sequences/number of positions to be compared × 100. For example, if there are 6 matches out of 10 positions in the two sequences, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have a total of 50% identity (there are 3 matches out of a total of 6 positions). Typically, comparison is performed when two sequences are aligned for maximum consistency. This alignment can be achieved using, for example, the method proposed by Needleman et al. (1970) J. Mol. Biol. 48: 443-453, and is typically performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined by using the E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) algorithm integrated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined by the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm integrated into the GAP program in the GCG software package (available at www.gcg.com), using either the Blossum 62 matrix or the 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.

The term "conservative substitution" means an amino acid substitution that does not adversely affect or alter 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 (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include replacing an amino acid residue with an amino acid residue with a similar side chain, such as a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). 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), amino acids with acidic side chains (e.g., aspartic acid and glutamine), and amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan). , amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine and methionine), amino acids with β-branched side chains (e.g., threonine, valine and isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan and histidine). Therefore, the corresponding amino acid residue is preferably replaced by 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).

The compilation of the 20 common amino acids involved in this article follows the common 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 shown as single-letter or three-letter abbreviations known in the art. For example, alanine can be shown as A or Ala.

The terms "include", "comprising", "having", "containing" or "involving" and other variations thereof herein are inclusive or open and do not exclude other unlisted elements or method steps.

The term "alkyl" means a group obtained by losing a hydrogen atom from a straight or branched hydrocarbon group, for example, "C _1-20 alkyl", "C _1-10 alkyl", "C _1-6 alkyl", "C _1-4 alkyl" and "C _1-3 alkyl". Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-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, and the like.

The term "alkylene" means a group obtained by losing two hydrogen atoms from a straight or branched alkyl group, such as "C _1-20 alkylene", "C _1-10 alkylene", "C _3-10 alkylene", "C _5-8 alkylene", "C _1-6 alkylene", "C _1-4 alkylene" and "C _1-3 alkylene". Specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene, 1,6-hexylene or the like.

The term "alkenyl" refers to a divalent group obtained by losing two hydrogen atoms from a straight or branched alkyl group containing at least one carbon-carbon double bond, including, for example, " _C2-20 alkenyl", " _C3-10 alkenyl", " _C5-8 alkenyl", etc. Examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, and the like.

The term "alkynylene" refers to a divalent group obtained by losing two hydrogen atoms from a straight or branched alkyl group containing at least one carbon-carbon triple bond, including, for example, " _C2-20 alkynylene", " _C3-10 alkynylene" and " _C5-8 alkynylene". Examples 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, etc.

The term "heteroaliphatic cyclocyclic ring" refers to a saturated or partially saturated cyclic structure containing at least one ring member selected from N, O and S. Specific examples include, but are not limited to, 5-6 membered aliphatic heterocyclic rings, 5-6 membered nitrogen-containing aliphatic heterocyclic rings, 5-6 membered oxygen-containing aliphatic heterocyclic rings, and the like, such as tetrahydrofuran, pyrrolidine, hexahydropyridine, tetrahydropyran, and the like.

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.

The term "aromatic ring system" refers to a monocyclic or polycyclic ring system comprising at least one aromatic ring (e.g., benzene ring) or heteroaromatic ring (e.g., pyrimidine ring). Two or more aromatic rings and/or heteroaromatic aromatic rings may form a fused ring or may be connected by a single bond (e.g., bipyrimidinylphenyl, etc.), and the aromatic ring system may be divalent or higher valent (e.g., trivalent or tetravalent), for example, a 5-20 membered aromatic ring system.

Cross-reference to related applications

This application claims the rights of Chinese Application No. 202311284321.4 filed on September 28, 2023 and Chinese Application No. 202211263034.0 filed on October 14, 2022, the disclosures of each of which are incorporated herein by reference in their entirety. Sequence Listing

This application contains a computer-readable sequence listing, which has been submitted with this application in XML file format, and the entire contents of the sequence listing are incorporated herein by reference. The sequence listing XML file submitted with this application is named "14463-051-228_SEQ_LISTING.xml", created on September 14, 2023, and is 63,238 bytes in size.

The following description of specific embodiments will further illustrate the present invention, but does not 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 concept and scope of the present invention.

The information about the sequences involved in the present invention is described in the following table: Serial Number Sequence Name Sequence information 1 101A6 VH amino acid sequence EVKLVESGGGLVQPGGSLSLSCAASGFTFTDYYLSWVRQPPGKALEWLALIRNKANGYTTEYSASVKGRFTISRDSSQSILYLQMDALRAEDSATYYCARDTLAAYWGQGTSVTVSS 2 101A6 VL amino acid sequence DIVMTQSHKFMSTSVGDRVSFTCKASQDVGSAVIWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPLTFGAGTKLEIK 3 101A6HZ/101A6HZm VH amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYLSWVRQAPGKGLEWLALIRNKANGYTTEYAASVKGRFTISRDSSKNSLYLQMNSLKTEDTAVYYCARDTLAAYWGQGTLVTVSS 4 101A6HZ/101A6HZm VL amino acid sequence DIVMTQSPDSLAVSLGERATINCKASQDVGSAVIWYQQKPGQSPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSSYPLTFGQGTKLEIK 5 64A10 VH amino acid sequence QVQLKESGPVLVAPSQSLSITCTVSGFSLTSYGVYWVRQPPGKGPEWLGVIWTSGNTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCVKHDGGNYVGVMNYWGQGTSVTVSS 6 64A10 VL amino acid sequence DIQMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSQESPRLLIKYASQSISGIPSRFSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPPTFGGGTKLEIK 7 64A10HZ VH amino acid sequence QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVYWVRQPPGKGPEWLGVIWTSGNTNYNPSLKSRLTISKDNSKNQVSLKLSSVTAADTAVYYCVKHDGGNYVGVMNYWGQGTLVTVSS 8 64A10HZ VL amino acid sequence EIVMTQSPDFQSVTPKEKVTITCRASQSISDYLHWYQQKPDQSPKLLIKYASQSISGVPSRFSGSGSGSDFTLTINSLEAEDAATYYCQNGHSFPPTFGQGTKLEIK 9 64A10HZ05 VH amino acid sequence QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVYWVRQPPGKGLEWLGVIWTSGNTNYNPSLKSRLTISKDNSKNQVSLKLSSVTAADTAVYYCVKHDGGNYVGVMNYWGQGTLVTVSS 10 64A10HZ05 VL amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISDYLHWYQQKPGKAPKLLIKYASQSISGVPSRFSGSGSDFTLTISSLQPEDFATYYCQNGHSFPPTFGGGTKVEIK 11 101A6/101A6HZ/101A6HZm Chothia CDR H1 GFTFTDY 12 101A6/101A6HZ/101A6HZm Chothia CDR H2 RNKANGYT 13 101A6/101A6HZ/101A6HZm Chothia/kabat/AbM CDR H3 DTLAAY 14 101A6/101A6HZ/101A6HZm Chothia/Kabat/AbM CDR L1 KASQDVGSAVI 15 101A6/101A6HZ/101A6HZm Chothia/Kabat/AbM CDR L2 WASTRHT 16 101A6/101A6HZ/101A6HZm Chothia/Kabat/IMGT/AbM CDR L3 QQYSSYPLT 17 101A6/101A6HZ/101A6HZm Kabat CDR H1 DYYLS 18 101A6HZ/101A6HZm Kabat CDR H2 LIRNKANGYTTEYAASVKG 19 101A6 Kabat CDR H2 LIRNKANGYTTEYSASVKG 20 101A6/101A6HZ/101A6HZm IMGT CDR H1 GFTFTDYY twenty one 101A6/101A6HZ/101A6HZm IMGT CDR H2 IRNKANGYTT twenty two 101A6/101A6HZ/101A6HZm IMGT CDR H3 ARDTLAAY twenty three 101A6/101A6HZ/101A6HZm IMGT CDR L1 QDV twenty four 101A6/101A6HZ/101A6HZm IMGT CDR L2 WAS 25 101A6/101A6HZ/101A6HZm AbM CDR H1 GFTFTDYYLS 26 101A6/101A6HZ/101A6HZm AbM CDR H2 LIRNKANGYTTE 27 64A10/64A10HZ/64A10HZ05 Chothia CDR H1 GFSLTSY 28 64A10/64A10HZ/64A10HZ05 Chothia CDR H2 WTSGN 29 64A10/64A10HZ/64A10HZ05 Chothia/Kabat/AbM CDR H3 HDGGNYVGVMNY 30 64A10/64A10HZ/64A10HZ05 Chothia/Kabat/AbM CDR L1 RASQSISDYLH 31 64A10/64A10HZ/64A10HZ05 Chothia/Kabat/AbM CDR L2 YASQSIS 32 64A10/64A10HZ/64A10HZ05 Chothia/Kabat/IMGT/AbM CDR L3 QNGHSFPPT 33 64A10/64A10HZ/64A10HZ05 Kabat CDR H1 SYGVY 34 64A10HZ/64A10HZ05 Kabat CDR H2 VIWTSGNTNYNPSLKS 35 64A10 Kabat CDR H2 VIWTSGNTNYNSALMS 36 64A10/64A10HZ/64A10HZ05 IMGT CDR H1 GFSLTSYG 37 64A10/64A10HZ/64A10HZ05 IMGT CDR H2 IWTSGNT 38 64A10/64A10HZ/64A10HZ05 IMGT CDR H3 VKHDGGNYVGVMNY 39 64A10/64A10HZ/64A10HZ05 IMGT CDR L1 QSISDY 40 64A10/64A10HZ/64A10HZ05 IMGT CDR L2 YAS 41 64A10/64A10HZ/64A10HZ05 AbM CDR H1 GFSLTSYGVY 42 64A10/64A10HZ/64A10HZ05 AbM CDR H2 VIWTSGNTN 43 Human heavy chain constant region IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 44 Human light chain constant region CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 45 Mutant human heavy chain constant region IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 46 101A6HZm HC EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYLSWVRQAPGKGLEWLALIRNKANGYTTEYAASVKGRFTISRDSSKNSLYLQMNSLKTEDTAVYYCARDTLAAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 47 101A6HZ/101A6HZm LC DIVMTQSPDSLAVSLGERATINCKASQDVGSAVIWYQQKPGQSPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 48 Connector GGFG 49 Connector GFLG 51 Connector GGGGG 52 101A6HZm HC Heavy chain amino acid sequence (N-terminal pyroglutamic acid or pyroglutamine; no C-terminal lysine) XVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYLSWVRQAPGKGLEWLALIRNKANGYTTEYAASVKGRFTISRDSSKNSLYLQMNSLKTEDTAVYYCARDTLAAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 53 101A6HZm HC Heavy chain amino acid sequence (without C-terminal lysine) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYLSWVRQAPGKGLEWLALIRNKANGYTTEYAASVKGRFTISRDSSKNSLYLQMNSLKTEDTAVYYCARDTLAAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 54 101A6HZm HC Heavy chain amino acid sequence (N-terminal pyroglutamic acid salt or pyroglutamic acid) XVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYLSWVRQAPGKGLEWLALIRNKANGYTTEYAASVKGRFTISRDSSKNSLYLQMNSLKTEDTAVYYCARDTLAAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK X is pyroglutamine salt or pyroglutamine 55 Connector GGGGS 56 Connector GGGGSGGGGSGGGGSGGGGS

The abbreviations used in this article have the following meanings: Abbreviation Meaning Abbreviation Meaning HATU N,N,N',N'-Tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate NBS N-Bromosuccinimide DIPEA Diisopropylethylamine THF Tetrahydrofuran HPLC HPLC DMSO Dimethyl sulfoxide TFA Trifluoroacetate DMF N,N-Dimethylformamide Pd/C Carbon supported palladium PyroE Pyroglutamate

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

Nuclear magnetic resonance ( ¹ H 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).

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

s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad peak, J: coupling constant, Hz: Hertz, and DMSO-d6: deuterated dimethyl sulfoxide. δ values are expressed in ppm.

Mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B. 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 (A-01)

Compound A-01-1 (0.40 g, 640.59 μmol, its synthesis reference patent CN 111936169A) and exatecan 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 to react at 25°C for 4 h. DIPEA was removed under reduced pressure, and most of DMF was removed by freeze drying with water to obtain a crude product, and the crude product was purified by preparative high performance liquid chromatography (conditions as follows) to obtain 273 mg of the title compound. Chromatographic column: Waters XBridge C18 OBD 45 mm×450 mm×8.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0.00 30 70 70 5.00 30 70 70 60.00 70 30 70

The structural characterization data are as follows: ESI-MS (m/z): 1034.4 [M+H] ⁺ . Example 2 Synthesis of N-((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)-2-hydroxyacetamide and N-((1 R,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)-2-hydroxyacetamide (1-8-A and 1-8-B) Step 1: Synthesis of 1-chloro-3-bromo-2-methyl-5-nitrobenzene (1-8-2)

Compound 1-8-1 (5.00 g, 29.14 mmol) was dissolved in n-heptane (25 mL) at a temperature below 25°C; concentrated sulfuric acid (25 mL) was added and heated to 50°C; NBS (6.22 g, 34.97 mmol) was added in batches at 50°C, and the temperature was maintained at 50°C and reacted for 2 h; and the reaction was monitored by thin layer chromatography (ethyl acetate: petroleum ether = 1:10). The reaction solution was cooled to room temperature, then added dropwise to ice water, and extracted with toluene; the organic phases were combined, washed with sodium sulfite solution, water and saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure; and the crude product was purified by preparative high performance liquid chromatography (conditions as shown below), and the resulting solution was freeze-dried to obtain 4.88 g of the title compound. Chromatographic column: C18 ODS 45 mm×450 mm×8.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 60 40 60 10 60 40 60 40 100 0 60 Step 2: Synthesis of 3-chloro-5-bromo-4-methylaniline (1-8-3)

Compound 1-8-2 (4.88 g, 19.48 mmol) was dissolved in ethyl acetate (100 mL) at 25°C, and carbon-supported platinum (2.00 g, 19.48 mmol, 5% content) was added. After hydrogen substitution, the reaction was carried out at 60°C under hydrogen protection for 4 h, and the reaction was monitored by HPLC-MS. The reaction solution was filtered and concentrated to obtain 3.68 g of the crude product of the title compound, which was directly used in the next reaction without further purification. Step 3: Synthesis of N-(3-chloro-5-bromo-4-methylphenyl)acetamide (1-8-4)

Compound 1-8-3 (3.63 g, 14.82 mmol) was dissolved in ethyl acetate (70 mL) at 20°C; triethylamine (4.50 g, 44.45 mmol) and acetic anhydride (2.27 g, 22.23 mmol) were added, the reaction was kept at 20°C for 20 h, and monitored by HPLC-MS. Water was added to the reaction liquid; extraction was performed with ethyl acetate; the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was slurried in a mixture of ethyl acetate: petroleum ether = 1:5 to obtain 2.86 g of the title compound. Step 4: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid (1-8-5)

Compound 1-8-4 (1.80 g, 6.86 mmol) was dissolved in THF (20 mL) and water (5 mL) at 20°C; vinylacetic acid (708.31 mg, 8.23 mmol), DIPEA (1.95 g, 15.08 mmol) and tris(o-methylphenyl)phosphine (62.60 mg, 0.20 mmol) were added; the reaction system was flushed with nitrogen and then heated to 70°C and maintained for 5 h; and the reaction was monitored by HPLC-MS. 1 N sodium hydroxide solution was added to the reaction solution to adjust pH=8; ethyl acetate was added for extraction; the aqueous phase was adjusted with 1 N hydrochloric acid until pH=3 was reached, and then extracted with ethyl acetate; and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 0.82 g of the title compound, which was directly used in the next reaction. Step 5: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)butanoic acid (1-8-6)

Compound 1-8-5 (2.60 g, 9.71 mmol) was dissolved in THF (50 mL) at 20°C; Pd/C (0.52 g, content 10%) was added; the system was placed under positive hydrogen pressure using a balloon filled with hydrogen, and reacted at 40°C for 2 h; and the reaction was monitored by HPLC-MS. The reaction solution was filtered, and the filtrate was concentrated to obtain 2.43 g of the title compound, which was directly used in the next reaction without further purification. Step 6: Synthesis of N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-7)

Compound 1-8-6 (2.43 g, 9.01 mmol) was dissolved in trifluoroacetic acid (10 mL) and cooled to 5°C; trifluoroacetic anhydride (3.78 g, 18.02 mmol, 2.50 mL) was added dropwise; the temperature was maintained at 5°C and reacted for 4 h; and the reaction was monitored by HPLC-MS. The reaction solution was added to water and dissolved in 10 N sodium hydroxide to adjust pH = 9; ethyl acetate was added for extraction; the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column (ethyl acetate: petroleum ether = 0-20%) to obtain 1.53 g of the title compound. Step 7: Synthesis of (Z)-N-(3-chloro-7-(hydroxyimino)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-8)

Potassium tert-butoxide (1.50 g, 13.37 mmol) was dissolved in THF (16 mL) and tert-butanol (4 mL) at 5°C; compound 1-8-7 (1.53 g, 6.08 mmol) in the THF solution (16 mL) was added dropwise; after 10 min, amyl nitrite (1.14 g, 9.73 mmol) was added dropwise; the reaction was kept at 5°C for 1 h and monitored by HPLC-MS. The reaction solution was adjusted to pH=5 with 1 N hydrochloric acid and extracted with ethyl acetate; the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure; and the crude product was slurried with methyl tert-butyl ether to obtain 1.20 g of the title compound. Step 8: Synthesis of N-(7-amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-9)

Compound 1-8-8 (0.50 g, 1.78 mmol) was dissolved in methanol (8 mL) and 2 N hydrochloric acid (8 mL) at 20°C; Pd/C (0.15 g, content 10%) was added; the system was placed under positive hydrogen pressure using a balloon filled with hydrogen, and reacted at 5°C for 2 h; and the reaction was monitored by HPLC-MS. The reaction solution was filtered and concentrated to obtain 0.52 g of the title compound, which was directly used in the next reaction without further purification. Step 9: Synthesis of N,N'-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (1-8-10)

Compound 1-8-9 (0.52 g, 1.70 mmol) was dissolved in pyridine (5 mL) at 20°C; acetic anhydride (2 mL) was added; and the reaction was maintained at 20°C for 2 h and monitored by HPLC-MS. The reaction solution was added to water; ethyl acetate was added for extraction; the organic phase was washed with water, combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column (ethyl acetate: petroleum ether = 0-30%) to obtain 0.22 g of the title compound. Step 10: Synthesis of N-(8-amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-8-11)

Compound 1-8-10 (450.97 mg, 1.46 mmol) was dissolved in methanol (16 mL) at 20°C; 2 N hydrochloric acid (16 mL) was added, and the mixture was heated to 60°C and maintained for 2 h; and the reaction was monitored by HPLC-MS. Saturated sodium bicarbonate solution was added to the cooled reaction solution to adjust pH=8; ethyl acetate was added for extraction; the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 230.00 mg of the title compound, which was directly used in the next step without further purification. Step 11: Synthesis of N-((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-8-12)

Compound 1-8-11 (230.00 mg, 0.78 mmol) was dissolved in toluene (10 mL); (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (230.00 mg, 0.87 mmol) and p-toluenesulfonic acid (26.73 mg, 0.16 mmol) were added, and the mixture was heated to 140°C and reacted for 5 h; the reaction was monitored by HPLC-MS. The reaction solution was concentrated, and the crude product was purified by silica gel column (methanol: dichloromethane = 0-10%) to obtain 150.00 mg of the title compound. Step 12: Synthesis of (9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1-2)

Compound 1-8-12 (40.00 mg, 0.081 mmol) was added to concentrated hydrochloric acid (1 mL), and heated to 100°C for 5 h; and the reaction was monitored by HPLC-MS. The reaction solution was filtered, the filtrate was purified by preparative HPLC (conditions as shown below), and the resulting solution was freeze-dried to obtain the title compound 1-2 hydrochloride. Compound 1-2 hydrochloride was separated under the following purification conditions to obtain two isomers, named 1-2-A (5.00 mg trifluoroacetic acid, retention time 9.85 min) and 1-2-B (7.00 mg trifluoroacetic acid, retention time 10.62 min) according to the retention time. Chromatographic column: SunFire preparative C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 5 95 28 2 5 95 28 18 50 50 28

The structural characterization data are as follows: 1-2-A: ¹ H NMR (400 MHz, DMSO-d6) δ 8.42 (s, 3H), 8.27 (s, 1H), 7.36 (s, 1H), 6.59 (s, 1H), 5.78-5.63 (m, 1H), 5.50-5.36 (m, 3H), 5.10-5.06 (m, 1H), 3.20-3.04 (m, 2H), 2.56 (s, 3H), 2.26-2.13 (m, 2H), 1.93-1.79 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H).

ESI-MS (m/z): 452.1 [M+H] ⁺ .

1-2-B: ¹ H NMR (400 MHz, DMSO-d6) δ 8.42 (s, 3H), 8.27 (s, 1H), 7.36 (s, 1H), 6.58 (s, 1H), 5.78-5.63 (m, 1H), 5.50-5.36 (m, 3H), 5.10-5.06 (m, 1H), 3.20-3.04 (m, 2H), 2.55 (s, 3H), 2.26-2.13 (m, 2H), 1.93-1.79 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H).

ESI-MS (m/z): 452.0 [M+H] ⁺ . Step 13: Synthesis of 2-((tert-butyldiphenylsilyl)oxy)-N-((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)acetamide and 2-((tert-butyldiphenylsilyl)oxy)-N-((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)acetamide N-((1R,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)acetamide (1-8-13-A and 1-8-13-B)

The hydrochloride of compound 1-2 (40.00 mg, 81.91 μmol) was dissolved in N,N-dimethylformamide (1 mL) at 25°C; 2-((tert-butyldiphenylsilyl)oxy)acetic acid (30.91 mg, 98.29 μmol), HATU (62.25 mg, 163.81 μmol) and N,N-diisopropylethylamine (42.34 mg, 327.63 μmol) were added; the reaction was maintained at 25°C for 0.5 h and monitored by HPLC-MS. After the reaction was completed, water was added to the reaction solution; dichloromethane/methanol (v/v=10/1) was added for extraction; the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure; and the crude product was purified and separated by preparative thin layer chromatography (dichloromethane:methanol=20:1) to obtain two isomers; and according to the Rf value, the two isomers were named 1-8-13-A (15.00 mg, Rf value is 0.3) and 1-8-13-B (12.00 mg, Rf value is 0.35). Step 14: Synthesis of N-((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)-2-hydroxyacetamide and N-( (1R,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)-2-hydroxyacetamide (1-8-A and 1-8-B)

1-8-13-A (15.00 mg) and 1-8-13-B (12.00 mg) were dissolved in tetrahydrofuran (1 mL) in two reaction flasks at 25°C; tetrabutylammonium fluoride (1M in tetrahydrofuran)/glacial acetic acid mixture (v/v=13/1) (50 uL) was added dropwise; and the reaction was maintained at 25°C for 0.5 h and monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative HPLC, and the obtained solution was freeze-dried to obtain the respective title compounds 1-8-A (ie, 1-10) (6.94 mg) and 1-8-B (ie, 1-9) (4.00 mg). Chromatographic column: SunFire prepared C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 20 80 28 3 20 80 28 18 90 10 28

The structural characteristics of 1-8-A (i.e. 1-10) are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.43 (d, J = 8.8 Hz, 1H), 8.16 (s, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.65-5.36 (m, 4H), 5.21 (q, J = 19.0 Hz, 2H), 3.95 (d, J = 5.7 Hz, 2H), 3.26-3.11 (m, 2H), 2.53 (s, 3H), 2.30-2.08 (m, 2H), 1.94-1.79 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

ESI-MS (m/z): 510.1 [M+H] ⁺ .

The structural characteristics of 1-8-B (i.e. 1-9) are as follows: 1H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 8.9 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.64-5.35 (m, 4H), 5.19 (q, J = 19.0 Hz, 2H), 3.97 (d, J = 5.2 Hz, 2H), 3.27-3.10 (m, 2H), 2.51 (s, 3H), 2.27-2.10 (m, 2H), 1.93-1.80 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H).

ESI-MS (m/z): 510.1 [M+H] ⁺ . Example 3 N-((10S)-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]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-(Methylsulfonyl)pyrimidin-5-yl)-hex-5-ynylamide and N-((10S)-10-benzyl-1-(((1R,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)-1,6,9,12,15-pentaoxo-3-oxo-5-nitropropane ,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-hex-5-ynylamide (A-07-A (A-05) and A-07-B (A-06)) Step 1: Synthesis of (S)-10-benzyl-23-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentaoxy-3-oxa-5,8,11,14,17-pentaazatricosane-22-ynecarboxylic acid (A-07-3)

Compound A-07-2 (30.00 mg, 0.07 mmol) was dissolved in DMF (0.2 mL) at 25°C; 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyn-5-ate (A-07-1, 28.00 mg, 0.08 mmol) was added and reacted at 30°C for 1 h; and the reaction was monitored by HPLC-MS. The reaction solution was directly purified by preparative HPLC (conditions as shown below), and the resulting solution was freeze-dried to obtain 20.00 mg of the title compound. Chromatographic column: SunFire prepared C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

The structural characterization data are as follows: ESI-MS (m/z): 691.0 [M+H ₂ O] ⁺ . Step 2: Synthesis of N-((10S)-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]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-(methylsulfonyl)pyrimidin-5-yl)-hexane-5-amide and N-(( 10S)-10-benzyl-1-(((1R,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)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-hexane-5-amide (A-07-A and A-07-B)

The hydrochloride of 1-2 (30.00 mg, 61.43 μmol) was dissolved in N,N-dimethylformamide (1 mL) at 25°C; A-07-3 (49.66 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol) and N,N-diisopropylethylamine (23.82 mg, 184.29 μmol) were added in sequence; the reaction was kept at 25°C for 0.5 h and monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative HPLC (conditions as shown below), and the resulting solution was freeze-dried to obtain the title compound A-07. A-07 was separated under the following purification conditions to obtain two isomers, named A-07-A (i.e. A-05) (11.04 mg, retention time 7.5 min) and A-07-B (i.e. A-06) (19.42 mg, retention time 8.0 min) according to the retention time. Chromatographic column: SunFire preparative C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 30 70 28 3 30 70 28 18 90 10 28

The structural characterization data are as follows: A-07-A (ie A-05): ESI-MS (m/z): 1107.3 [M+H] ⁺ .

¹ H NMR (400 MHz, DMSO) δ 9.10 (s, 2H), 8.66 - 8.63 (m, 1H), 8.51 (d, J = 8.8 Hz, 1H), 8.34 - 8.31 (m, 1H), 8.21 - 8.19 (m, 1H), 8.17 - 8.09 (m, 2H), 8.08 - 8.04 (m, 1H), 7.30 (s, 1H), 7.26 - 7.15 (m, 5H), 6.55 (s, 1H), 5.56 - 5.55 (m, 1H), 5.48 - 5.35 (m, 2H), 5.25 - 5.10 (m, 2H), 4.64 (d, J = 6.4 Hz, 2H), 4.45 - 4.44 (m, 1H), 4.06 - 3.98 (m, 2H), 3.77 -3.52 (m, 6H), 3.41 (s, 3H), 3.25 - 3.12 (m, 2H), 3.03 - 3.00 (m, 1H), 2.83 - 2.72 (m, 1H), 2.58 - 2.56 (m, 2H), 2.48 (s, 3H), 2.33 - 2.30 (m, 2H), 2.21 - 2.13 (m, 2H), 1.91 - 1.76 (m, 4H), 0.87 (t, J = 7.2 Hz, 3H).

A-07-B (ie A-06): ESI-MS (m/z): 1107.3 [M+H] ⁺ . Example 4 (S)-7-Ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxacyclopenta[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2) Step 1: Synthesis of 3-(isopropylamino)-1-(6-nitrobenzo[d][1,3]dioxacyclohexene-5-yl)propan-1-one (2-2-2)

Compound 2-2-1 (1.90 g, 8.08 mmol) was slowly added to nitric acid (8 mL) at 0°C, and the temperature was slowly raised to 25°C and reacted for 1 h. The reaction solution was directly purified by reverse phase column chromatography (acetonitrile/0.5% formic acid aqueous solution) to obtain 1.50 g of the formate salt of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 281.1 [M+H] ⁺ . Step 2: Synthesis of 1-(6-aminobenzo[d][1,3]dioxadien-5-yl)-3-(isopropylamino)propan-1-one (2-2-3)

The formate salt of compound 2-2-2 (1.25 g, 4.46 mmol) was added to tetrahydrofuran (20 mL); 10% palladium on carbon (125.00 mg) was added; hydrogen substitution was performed three times; and the reaction was then performed at 25° C. for 16 h. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to dryness under reduced pressure to obtain 895.00 mg of a crude product of the title compound, which was directly used in the next reaction without purification.

The structural characterization data are as follows: ESI-MS (m/z): 251.1 [M+H] ⁺ . Step 3: Synthesis of ((S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxacyclopenta[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)

Compound 2-2-3 (23.00 mg, 0.09 mmol) and (4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (21.00 mg, 0.09 mmol) were dissolved in toluene (4 mL); p-toluenesulfonic acid (1.40 mg, 0.009 mmol) was added, and the reaction was carried out at 140° C. for 12 h. The solvent was removed under reduced pressure to obtain a crude product of the title compound; the crude product was purified by HPLC (mobile phase A: acetonitrile, mobile phase B: 0.05% formic acid aqueous solution), and 3 drops of 3 M hydrochloric acid were added to the prepared solution, followed by freeze drying to obtain 8.70 mg of the hydrochloride salt of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 478.2 [M+H] ⁺ .

¹ H-NMR (400 MHz, DMSO-d6): δ 9.38 (brs, 2H), 7.85 (s, 1H), 7.52 (s, 1H), 7.25 (s, 1H), 6.30 (d, J = 2.0 Hz , 2H), 5.43 (s, 2H), 5.30 (s, 2H), 3.57 - 3.45 (m, 2H), 3.40 - 3.25 (m, 1H), 3.22 - 3.06 (m, 2H), 1.95 - 1.77 (m , 2H), 1.27 (d, J = 6.4 Hz, 6H), 0.87 (t, J = 7.6 Hz, 3H). Example 5 Synthesis of ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxy-3,4,12,14 -tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl) ester 4-((S)-2-(4- Aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)methyl)-1H-1,2,3 -triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazopenta Benzyl ester (B-01) Step 1: Synthesis of (4-((S)-2-(4-(((4-methyloxyphenyl)diphenylmethyl)amino)butyl)carbonate 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)methyl)-1H-1,2,3-triazol-1-yl)-4,8- Dioxy-6,12,15,18,21,24, 27,30,33-nonaoxo-3,9-diazopentazoylamino)benzyl)ester (S)-4-ethyl-11-(2-(N-isopropylmethyl) Sulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl ester (B-01-2)

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) at room temperature; 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, and the mixture was stirred for 1 h and filtered; the filtrate was purified by preparative high performance liquid chromatography (conditions as shown below) to obtain 30.00 mg of the title compound. Chromatographic column: SunFire prepared C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0.00 40 60 28 8.00 40 60 28 50.00 90 10 28

The structural characterization data are as follows: ESI-MS (m/z): 815.9 [(M-273)/2+H] ⁺ . Step 2: Synthesis of ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate 5-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazopentazoneamido)benzyl ester (B-01)

Compound B-01-3 (30.00 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL); trifluoroacetic acid (0.2 mL) was added to the reaction solution and reacted at room temperature for 30 min. The reaction solution was concentrated under reduced pressure and purified by preparative high performance liquid chromatography (conditions as follows) to obtain 20.00 mg of the trifluoroacetate salt of the title compound. Chromatographic column: SunFire preparative C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0.00 15 85 28 8.00 15 85 28 50.00 60 40 28

The structural characterization data are as follows: ESI-MS (m/z): 508.2 [M+H] ⁺ . Example 6 (2S,3S,4S,5R,6S)-6-(4-(((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolanecyclopentano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)aminomethyl)oxy)methyl)-2-(2-(2-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)ethoxy)ethoxy)acetamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (B-03) Step 1: Synthesis of (2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-3)

Compound (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetic acid triester (B-03-1, 12.32 g, 31.02 mmol) and 4-hydroxy-3-nitrobenzyl alcohol (compound B-03-2, 5.00 g, 29.56 mmol) were dissolved in acetonitrile (200 mL); silver oxide (27.40 g, 118.25 mmol) was added while stirring; and after nitrogen substitution, the reaction was carried out at room temperature in the dark for 12 h. The reaction was monitored by HPLC-MS; the reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure and then purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:3) to obtain 12.80 g of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 503 [M+18] ⁺ . Step 2: Synthesis of (2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-4)

Compound B-03-3 (2.20 g, 4.53 mmol) was dissolved in ethyl acetate and tetrahydrofuran (50 mL each); PtO ₂ (0.20 g) was added; and then the reaction system was replaced with a hydrogen balloon three times; and the reaction was carried out under a hydrogen atmosphere for 2 h. The reaction was monitored by HPLC-MS; the reaction liquid was directly filtered; and the filter cake was rinsed with ethyl acetate, and the filtrate was evaporated to dryness under reduced pressure to obtain 2.02 g of a crude product of the title compound, which was directly used in the next reaction.

The structural characterization data are as follows: ESI-MS (m/z): 456.1 [M+1] ⁺ . Step 3: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxy-4-azadodecyl-12-amino)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-5): Compound B-03-4 (456.00 mg, 1.00 mmol) and [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (385.91 mg, 1.00 mmol) were dissolved in dichloromethane (10 mL); 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (495.22 mg, 1.00 mmol) was added while stirring. 2.00 mmol); and the reactant was stirred for 2 h. The reaction was monitored by HPLC-MS; and the reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography (methanol: dichloromethane = 1:20) to obtain 507.00 mg of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 823.3 [M+1] ⁺ . Step 4: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3,11-dioxo-2,7,10-trioxa-4,12-diazododecanyl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-6)

Compound B-03-5 (507.00 mg, 616.18 μmol) and diisopropylethylamine (238.91 mg, 1.85 mmol) were dissolved in dichloromethane (20 mL); then p-nitrophenyl chloroformate (372.60 mg, 1.85 mmol) was dissolved in dichloromethane (1 mL) and slowly added dropwise to the reaction solution; and after the addition was completed, the reaction was carried out at room temperature for 15 h. The reaction was monitored by high performance liquid chromatography-mass spectrometry; and the reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography (methanol: dichloromethane = 1:20) to obtain 496.00 mg of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 988.5 [M+1] ⁺ . Step 5: Synthesis of 4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxy-7,8,11,13-tetrahydro-10H-[1,3]dioxolanecyclopentano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)aminoformyl )oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (2S,3R,4S,5S,6S)-2-(2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxy-4-azadodecanoylamido-12-amino) ester (B-03-7)

Compound B-03-6 (165.51 mg, 0.17 mmol), compound 2-2 (40.00 mg, 0.084 mmol) and 1-hydroxybenzotriazole (33.96 mg, 0.25 mmol) were dissolved in DMF (4 mL); diisopropylethylamine (32.48 mg, 0.25 mmol) was added dropwise, and reacted under stirring for 12; and the reaction was monitored by high performance liquid chromatography-mass spectrometry. Water and ethyl acetate were added and stirred, and then allowed to stand to separate the phases; and the organic phase was washed with saturated brine, dried, and concentrated under reduced pressure to obtain 100.00 mg of a crude product of the title compound, which was directly used in the next reaction.

The structural characterization data are as follows: ESI-MS (m/z): 1326.2 [M+1] ⁺ . Step 6: Synthesis of (2S,3S,4S,5R,6S)-6-(2-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxy-7,8,11,13-tetrahydro-10H-[1,3]dioxacyclopentano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)aminoformyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (B-03-8).

Compound B-03-7 (100.00 mg, 0.08 mmol) was dissolved in MeOH (5 mL); 1 drop of dichloromethane was added dropwise; an aqueous solution (1 mL) of lithium hydroxide monohydrate (15.82 mg, 0.377 mmol) was added dropwise, and the reactant was stirred for 2 h. The reaction was monitored by HPLC-MS; 3 N aqueous hydrochloric acid was added dropwise to adjust the reaction solution to pH = 4; the product was concentrated under reduced pressure and then purified by preparative HPLC (conditions as shown below); and the resulting solution was freeze-dried to obtain 27.00 mg of the title compound. Chromatographic column: SunFire prepared C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0.00 20 80 28 2.00 20 80 28 18.00 80 20 28

The structural characterization data are as follows: ESI-MS (m/z): 964.2 [M+1] ⁺ . Step 7: Synthesis of (2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolane-[4,5-g]pyrano[3',4':6,7]indolizino[1,2- b]quinolin-14-yl)ethyl) (isopropyl)aminomethyl)oxy)methyl)-2-(2-(2-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)ethoxy)ethoxy)acetylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (B-03).

Compound B-03-8 (27.00 mg, 0.03 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyn-5-acid 2,5-dioxypyrrolidin-1-yl ester (A-07-1) (11.26 mg, 0.03 mmol) were dissolved in DMF (1 mL); diisopropylethylamine (3.62 mg, 0.03 mmol) was added dropwise while stirring, and reacted at room temperature for 4 h; and the reaction was monitored by HPLC-MS. The reaction solution was purified by preparative HPLC (conditions as shown below), and the resulting solution was freeze-dried to obtain 11.70 mg of the title compound. Chromatographic column: SunFire prepared C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0.00 20 80 28 2.00 20 80 28 18.00 80 20 28

The structural characterization data are as follows: ESI-MS (m/z): 1214.4 [M+1] ⁺ . Example 7 Synthesis of N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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)-2-hydroxyacetamide and N-((1R ,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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)-2-hydroxyacetamide (1-11-A and 1-11-B) Step 1: Synthesis of 3-bromo-4-chloro-5-fluoroaniline (1-5-02)

Compound 1-5-01 (2.00 g, 10.53 mmol) was dissolved in N,N-dimethylformamide (30 mL); then N-chlorosuccinimide (1.69 g, 12.63 mmol) was slowly added; after the addition was completed, the reaction was carried out at room temperature for 16 h; and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure to obtain a crude product, and the crude product was purified by flash chromatography using a silica gel column (ethyl acetate: petroleum ether = 0-25%) to obtain 0.95 g of the title compound.

The structural characterization data are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ 6.77 (dd, J = 2.5, 1.4 Hz, 1H), 6.51 (dd, J = 11.7, 2.5 Hz, 1H), 5.84 (s, 2H). Step 2: Synthesis of N-(3-bromo-4-chloro-5-fluorophenyl)acetamide (1-5-03)

Compound 1-5-02 (0.95 g, 4.23 mmol) was dissolved in ethyl acetate (20 mL), and acetic anhydride (648.13 mg, 6.35 mmol) was added under nitrogen protection; after the addition was completed, the temperature was raised to 50°C and reacted for 15 h; and the reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was quenched with methanol (5 mL) and directly evaporated to dryness under reduced pressure to obtain a crude product, which was purified by flash chromatography using a silica gel column (ethyl acetate: petroleum ether = 0-40%) to obtain 1.01 g of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 265.9 [M+H] ⁺ . Step 3: Synthesis of (E)-4-(5-acetamido-2-chloro-3-fluorophenyl)-3-butenoic acid (1-5-04)

Compound 1-5-03 and 3-butenoic acid (387.65 mg, 4.50 mmol) were dissolved in a mixture of 1,4-dioxane (24 mL) and water (8 mL), and then N,N-diisopropylethylamine (1.45 g, 11.26 mmol), tris(o-methylphenyl)phosphine (114.21 mg, 375.24 μmol) and potassium acetate (42.12 mg, 187.62 μmol) were added; after the addition was completed, the atmosphere in the reaction system was replaced with nitrogen, and the temperature was raised to 100° C. under nitrogen atmosphere and reacted for 16 h; and the reaction was monitored by high performance liquid chromatography-mass spectrometry. After the reaction solution was cooled to room temperature, 1 N sodium hydroxide aqueous solution (60 mL) and ethyl acetate (50 mL) were added and shaken to separate the phases. After the lower aqueous phase was separated, the pH was adjusted to about 3 with a 4 mol/L hydrochloric acid aqueous solution; then ethyl acetate was added for extraction; the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered; and the filtrate was evaporated to dryness under reduced pressure to obtain 1.00 g of a crude product of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 272.0 [M+H] ⁺ . Step 4: Synthesis of 4-(5-acetamido-2-chloro-3-fluorophenyl)butanoic acid (1-5-05)

The crude product of compound 1-5-04 (1.00 g, 3.68 mmol) was dissolved in tetrahydrofuran (15 mL); then 10% palladium on carbon (0.10 g) was added; after the addition was completed, the reaction system was replaced with a hydrogen balloon three times, and the reaction was carried out under a hydrogen atmosphere for 4 h; and the reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.00 g of the crude product of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 274.0 [M+H] ⁺ . Step 5: Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-06)

The crude product of compound 1-5-05 (1.00 g, 3.65 mmol) was dissolved in trifluoroacetic acid (5 mL); after cooling to 5°C, trifluoroacetic anhydride (3.84 g, 18.27 mmol, 2.54 mL) was slowly added; after the addition was completed, the temperature was maintained at 5°C and reacted for 2 h; and the reaction was monitored by HPLC-MS. The reaction solution was slowly poured into water, and then extracted with ethyl acetate; the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered; and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by flash chromatography using a silica gel column to obtain 0.43 g of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 256.1 [M+H] ⁺ . Step 6: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-07)

Tetrahydrofuran (16 mL) and tert-butanol (4 mL) were added to the reaction solution bottle; and after cooling to 5°C in an ice bath, potassium tert-butoxide (415.18 mg, 3.70 mmol) was added; compound 1-5-06 (0.43 mg, 1.68 mmol) was dissolved in tetrahydrofuran (1 mL) and added dropwise to the reaction solution; after 10 min, amyl nitrite (315.24 mg, 2.69 mmol) was further added; after completion of the addition, the temperature was maintained at 5°C and the reaction was carried out for 1 h; and the reaction was monitored by HPLC-MS. The reaction solution was quenched with a saturated aqueous ammonium chloride solution, and extracted with ethyl acetate; the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered; and the filtrate was concentrated under reduced pressure to obtain 455.00 mg of a crude product of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 285.0 [M+H] ⁺ . Step 7: Synthesis of N-(7-amino-4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-08)

The crude product of compound 1-5-07 (0.40 g, 1.41 mmol) was dissolved in methanol (10 mL); then 3 mol/L aqueous hydrochloric acid solution (1 mL) and 10% palladium on carbon (40.00 mg) were added; after the addition was completed, the atmosphere in the reaction system was replaced with hydrogen; the reaction was carried out at room temperature under a hydrogen atmosphere for 1 h and monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered and the filtrate was concentrated under reduced pressure to obtain 0.43 mg of the crude hydrochloride of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 271.0 [M+H] ⁺ . Step 8: Synthesis of (8-acetamido-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (1-5-09)

The crude hydrochloride of compound 1-5-08 (0.43 g, 1.19 mmol) was dissolved in 1,4-dioxane (15 mL); then sodium bicarbonate (400.35 mg, 4.77 mmol), water (5 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (481.81 mg, 1.43 mmol) were added; after the addition was completed, the reaction was carried out at room temperature for 2 h while stirring; and the reaction was monitored by HPLC-MS. The reaction solution was poured into water and then extracted with ethyl acetate; the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered; and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by a C18 reverse phase column (acetonitrile: 0.05% formic acid in water = 20%-100%) to obtain 301.00 mg of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 493.2 [M+H] ⁺ . Step 9: Synthesis of (8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (1-5-10)

Compound 1-5-09 (300.00 mg, 608.61 μmol) was dissolved in dioxane (5 mL); 12 mol/L concentrated hydrochloric acid (1 mL) was added; after the addition was completed, the temperature was raised to 60°C and reacted for 2 h; and the reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was poured into water and then extracted with ethyl acetate; the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered; and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography using a silica gel column (ethyl acetate: petroleum ether = 0-50%) to obtain 198.00 mg of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 451.1 [M+H] ⁺ . Step 10: Synthesis of ((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)carbamic acid (9H-fluoren-9-yl)methyl ester (1-5-11)

(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (138.72 mg, 526.96 μmol) and compound 1-5-10 (198.00 mg, 439.13 μmol) were added to toluene (10 mL); p-toluenesulfonic acid (75.53 mg, 439.13 μmol) was then added; after the addition was completed, the temperature was raised to 140°C and reacted for 4 h; the reaction solution was directly evaporated to dryness at 140°C under reduced pressure to obtain a crude product, which was purified by flash chromatography using a silica gel column (methanol: dichloromethane = 0-5%) to obtain 256.00 mg of the title compound.

The structural characterization data are as follows: ESI-MS (m/z): 678.1 [M+H] ⁺ . Step 11: Synthesis of (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1-5-A and 1-5-B)

Compound 1-5-11 (201.18 mg, 296.67 μmol) was dissolved in N,N-dimethylformamide (4 mL); then diethylamine (108.49 mg, 1.48 mmol) was added; after the addition was completed, the reaction was carried out at room temperature for 0.5 h; and the reaction was monitored by high performance liquid chromatography-mass spectrometry. After ethylenediamine was distilled off from the reaction solution under reduced pressure, the pH was adjusted to 2-3 with 1 mol/L aqueous hydrochloric acid solution; and the reaction solution was directly purified by preparative high performance liquid chromatography to obtain the title compounds 1-5-A (44.00 mg) and 1-5-B (43.00 mg). Chromatographic column: SunFire prepared C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 10 90 28 3 10 90 28 18 70 30 28 1-5-A (first LCMS peak at 6 min, retention time: 1.276 min)

The structural characterization data are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ 8.00 (d, J = 10.3 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.62 (d, J = 19.3 Hz, 1H), 5.44 (s, 2H), 5.38 (d, J = 19.3 Hz, 1H), 4.43-4.38 (m, 1H), 3.28-3.10 (m, 2H), 2.22-2.12 (m, 1H), 2.12-2.02 (m, 1H), 1.93-1.80 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

ESI-MS (m/z): 456.1 [M+H] ⁺ .

The structural characteristics of 1-5-B (LCMS peak appeared 6 min later, retention time: 1.300 min) are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ7.98 (d, J = 10.3 Hz, 1H), 7.32 (s, 1H), 5.61 (d, J = 19.4 Hz, 1H), 5.44 (s, 2H), 5.32 (d, J = 19.4 Hz, 1H), 4.44-4.36 (m, 1H), 3.33-3.25 (m, 1H), 3.22-3.11 (m, 1H), 2.23 - 2.13 (m, 1H), 2.11-2.03 (m, 1H), 1.96 -1.82 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H).

ESI-MS (m/z): 456.1 [M+H] ⁺ .

6 min LCMS conditions: Chromatographic column: Waters SunFire C18 OBD 4.6 mm×50 mm×5.0 μm Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 90 10 2 4.2 10 90 2 5.7 10 90 2 5.71 90 10 2 6.70 90 10 2 Step 12: Synthesis of N-((10S)-10-benzyl-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamide and N-((1 0S)-10-benzyl-1-(((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (1-5-12-A and 1-5-12-B)

Compound 1-5-A (36.00 mg, 79.70 μmol) and compound A-07-3 (64.43 mg, 95.64 μmol) in a single structure were dissolved in N,N-dimethylformamide (2 mL); then 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added; after the addition was completed, the reaction was carried out at room temperature for 1 h; and the reaction was monitored by HPLC-MS. The reaction solution was directly purified by HPLC to obtain the title compound 1-5-12-A (51.00 mg, A-14) in a single structure. Chromatographic column: SunFire prepared C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 30 70 28 3 30 70 28 18 90 10 28

The structural characterization data are as follows: ESI-MS (m/z): 1111.0 [M+H] ⁺ .

Compound 1-5-B (36.00 mg, 79.70 μmol) and compound A-07-3 (64.43 mg, 95.64 μmol) in a single structure were dissolved in N,N-dimethylformamide (2 mL); then 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added; after the addition was completed, the reaction was carried out at room temperature for 1 h; and the reaction was monitored by HPLC-MS. The reaction solution was directly purified by HPLC to obtain the title compound 1-5-12-B (52.00 mg, A-15) in a single structure. Chromatographic column: SunFire prepared C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 30 70 28 3 30 70 28 18 90 10 28

The structural characterization data are as follows: ESI-MS (m/z): 1111.0 [M+H] ⁺ . Step 13: Synthesis of N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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)-2-hydroxyacetamide and N-( (1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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)-2-hydroxyacetamide (1-11-A and 1-11-B)

Compound 1-5-12-A (40.00 mg, 35.99 μmol) was weighed and dissolved in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL); then 4 mol/L ethyl acetate hydrochloride solution (1 mL) was added; after the addition was completed, the reaction was carried out at room temperature for 0.5 h; and the reaction was monitored by HPLC-MS. The reaction solution was directly concentrated to dryness under reduced pressure to obtain a crude product, which was purified by HPLC to obtain the title compound 1-11-A (4.75 mg) in a single configuration. Chromatographic column: SunFire prepared C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 15 85 28 3 15 85 28 18 90 10 28

The structural characterization data are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ 8.50 (d, J = 8.9 Hz, 1H), 8.05 (d, J = 10.3 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H), 5.67-5.60 (m, 1H), 5.49 (t, J = 5.8 Hz, 1H), 5.43 (s, 2H), 5.21 (s, 2H), 3.96 (d, J = 5.8 Hz, 2H), 3.32-3.22 (m, 2H), 2.28-2.15 (m, 2H), 1.93-1.80 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

ESI-MS (m/z): 514.0 [M+H] ⁺ .

Compound 1-5-12-B (40.00 mg, 35.99 μmol) was weighed and dissolved in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL); then 4 mol/L ethyl acetate hydrochloride (1 mL) was added; after the addition was completed, the reaction was carried out at room temperature for 0.5 h; and the reaction was monitored by HPLC-MS. The reaction solution was directly concentrated to dryness under reduced pressure to obtain a crude product, which was purified by HPLC to obtain the title compound 1-11-B (8.24 mg) of single configuration. Chromatographic column: SunFire prepared C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 15 85 28 3 15 85 28 18 90 10 28

The structural characterization data are as follows: ¹ H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 9.0 Hz, 1H), 8.05 (d, J = 10.3 Hz, 1H), 7.34 (s, 1H), 6.55 (s, 1H), 5.68-5.58 (m, 1H), 5.53 (t, J = 5.8 Hz, 1H), 5.43 (d, J = 2.9 Hz, 2H), 5.20 (d, J = 7.3 Hz, 2H), 3.97 (d, J = 5.7 Hz, 2H), 3.31-3.21 (m, 2H), 2.26-2.15 (m, 2H), 1.92-1.82 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

ESI-MS (m/z): 514.0 [M+H] ⁺ . Example 8 N-((10S,19S)-amino-10-benzyl-1-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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-amino)-1,6 ,9,12,15,18,25-heptadioxy-3-oxadiazine-5,8,11,14,17,24-hexaaza-30-(2-methylsulfonylpyrimidin-5-yl)-29-tricarbazone-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecacyclohexane-38-amide (A-17-A) Step 1: Synthesis of (2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaproic acid-38-amido)-6-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)hexanoic acid (A-17-03)

The hydrochloride of compound A-17-02 (389.68 mg, 962.45 μmol) was dissolved in dichloromethane (8 mL), and DIPEA (518.28 mg, 4.01 mmol, 713.88 μL) and compound A-17-01 (550.00 mg, 802.04 μmol) were added, and reacted at 25°C for 1.5 h. The pH of the reaction solution was adjusted to neutral with dilute hydrochloric acid, and the solvent was removed under reduced pressure. The concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-03 (450.00 mg). Chromatographic column: SunFire prepared C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 10 90 28 2 10 90 28 18 80 20 28

ESI-MS (m/z): 939.3 [M+H] ⁺ . Step 2: Synthesis of 2-({2-[(2S)-2-(2-{2-[(2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaproic acid-38-amido)-6-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)hexanamido]acetamido}acetamido)-3-phenylpropyl]acetamido}methoxy)acetic acid (A-17-04)

Compound A-17-03 (50.00 mg, 118.09 μmol) was dissolved in DMF (2.5 mL); HATU (49.39 mg, 129.89 μmol), compound A-07-2 (133.07 mg, 141.70 μmol) and DIPEA (45.78 mg, 354.26 μmol, 63.06 μL) were added and reacted at 25°C for 1 h. The solvent was removed under reduced pressure; and the concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-04 (40.00 mg). Chromatographic column: SunFire prepared C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 10 90 28 2 10 90 28 18 80 20 28

ESI-MS (m/z): 1344.4 [M+H] ⁺ . Step 3: Synthesis of ((40S)-40-((10S)-10-benzyl-1-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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)-1-amino (9H-fluoren-9-yl)methyl 1,6,9,12,15-pentaoxadi ...

Compound 1-17-04 (29.86 mg, 59.50 μmol) was dissolved in DMF (3 mL); HATU (27.15 mg, 71.40 μmol) and compound 1-7 (80.00 mg, 59.50 μmol) and DIPEA (38.45 mg, 297.51 μmol, 52.96 μL) were added and reacted at 25°C for 1 h. The solvent was removed under reduced pressure; and the concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-05 (50.00 mg). Chromatographic column: SunFire preparative C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 10 90 28 2 10 90 28 18 80 20 28

ESI-MS (m/z): 1781.6 [M+H] ⁺ . Step 4: Synthesis of N-((10S,19S)-23-amino-10-benzyl-1-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4]:6,7]indolizino[1,2-b] Quinolinophenyl-1-amino)-1,6,9,12,15,18,25-heptaoxy-3-oxo-5,8,11,14,17,24-hexaazatriacontria-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxytriacontria-38-amide (A-17-06)

Compound A-17-05 (20.00 mg, 11.22 μmol) was dissolved in DMF (2.5 mL) and diethylamine (0.5 mL) and reacted at 25°C for 2 h. The solvent was removed under reduced pressure; and the concentrate was purified by preparative HPLC to obtain the formate salt of the title compound A-17-06 (10.00 mg). Chromatographic column: SunFire preparative C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 15 85 28 2 15 85 28 18 80 20 28

ESI-MS (m/z): 1559.7 [M+H]+. Step 5: Synthesis of N-((10S,19S)-amino-10-benzyl-1-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4]:6,7]indolizino[1,2-b]quinoline-1-amino)-1 ,6,9,12,15,18,25-heptadioxy-3-oxadiazine-5,8,11,14,17,24-hexaaza-30-(2-methylsulfonylpyrimidin-5-yl)-29-tricarbazone-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecacyclohexane-38-amide (A-17-A)

The formate salt of compound A-17-06 (7.00 mg, 4.49 μmol) and compound A-07-1 (3.28 mg, 8.97 μmol) were dissolved in DMF (1 mL), and DIPEA (1.74 mg, 13.46 μmol, 2.40 μL) was added thereto, and reacted at 25°C for 2 h. The solvent was removed under reduced pressure; and the concentrate was purified by preparative HPLC to obtain the title compound A-17-A (5.60 mg). Chromatographic column: SunFire preparative C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0 20 80 28 2 20 80 28 18 80 20 28

ESI-MS (m/z): 1809.8 [M+H] ⁺ . Example 9 (1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)(methyl) 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl amino)-3-methyl-1-oxobutan-2-yl)amino)-2-methyl-1-oxopropyl-2-yl)carbamate (C-01)

According to the synthesis procedure in International Patent Publication No. WO 2015/168019 A2, 4-((S)-2-((S)-2-(6-(2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)hexylamino)-3-methylbutylamino)-5-ureidopentanamido)benzyl carbonate (4-nitrophenyl) (C-01-1) and (S)-2-(2-amino-2-methylpropionamido)-N-((3R,4 The title compound C-01 was obtained by the reaction of 3-((S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((S)-2-phenyl-1-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-1-yl)-5-methyl-1-oxohept-4-yl)-N,3-dimethylbutyramide (Aur0101).

ESI-MS (m/z): 1341.7 [M+1]+. 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-dimethyl-1,6,9,12-tetraoxide-3-oxy-5,8,11-triazol-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido (A-26). Step 1:

Compound A-26-1 (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 added, and the reaction was carried out at room temperature under stirring for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain 700 mg of compound A-26-2.

The preparation method is as follows: Waters SunFire C18 OBD (5 μm*19 mm*150 mm) Chromatographic column: Waters SunFire C18 OBD (5 μm*19 mm*150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] A [%] Phase shift A B [%] Shift phase B Flow rate [mL/min] 0.00 30 70 28 2.00 30 70 28 18.00 90 10 28 Step 2:

Compound A-26-2 (500 mg, 0.513 mmol) was dissolved in N,N-dimethylformamide (2 mL), diethylamine (75.05 mg, 1.03 mmol) was added, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain 307 mg of compound A-26-3.

The preparation method is as follows: Chromatographic column: Waters SunFire preparative C18 OBD (5 μm*19 mm*150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0.00 20 80 twenty four 2.00 20 80 twenty four 18.00 80 20 twenty four Step 3:

Compound A-26-3 (170 mg, 0.226 mmol) and compound A-07-1 (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 was carried out at room temperature with stirring for 16 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain 50.56 mg of compound A-26.

The structural characterization data are as follows: MS m/z (ESI): 1002.4 [M+H] ⁺ .

The separation and purification methods are as follows: Chromatographic column: Waters SunFire preparative C18 OBD (5 μm*19 mm*150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) [min] Time Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0.00 30 70 28 2.00 30 70 28 18.00 90 10 28 ¹ H NMR (400 MHz, DMSO) δ 9.11 (s, 2H), 8.68 (t, J = 6.4 Hz, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.16 (s, 1H), 8.10 (d, J = 7.2 Hz, 1H), 8.01 (d, J = 7.2 Hz, 1H), 7.91 (d, J = 6.8 Hz, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.65-5.55 (m, 1H), 5.43 (s, 2H), 5.21(s, 2H), 4.67-4.55 (m, 2H), 4.29-4.15 (m, 3H), 3.98 (s, 2H), 3.41 (s, 3H), 3.25-3.15 (m, 2H), 2.57-2.56 (m, 2H), 2.35-2.27 (m, 2H), 2.22-2.12 (m, 2H), 1.91-1.75 (m, 4H), 1.23-1.09 (m, 9H), 0.87 (t, J = 7.2 Hz, 3H). Example 11 Carbonate ((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) 4-((S)-2-(4- (4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamide)benzyl aminobutyl)ester (B-04) Step 1: Preparation of 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl acetate (B-04-1).

(1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2 g, 3.65 mmol) was dissolved in DMF (50 mL); DIPEA (1.18 g, 9.12 mmol, 1.59 mL) was added dropwise; acetoxyacetyl chloride (548.12 mg, 4.01 mmol, 431.59 μL) was added dropwise while stirring in an ice bath; and the stirring reaction was continued for 1 hour. The reaction solution was added to a 0.1 M dilute hydrochloric acid aqueous solution to precipitate a solid, which was 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 a silica gel column (methanol/dichloromethane = 0% to 5%) and concentrated again to obtain the title compound (1.7 g, 3.077 mmol).

The structural characterization data are as follows: ESI-MS (m/z): 552.2 [M+1] ⁺ . Step 2: Preparation of 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazopentazotriamide)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]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl acetate (B-04-2)

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]indeno[1,2-b]quinolin-1-yl)amino)-2-oxoethyl acetate (500 mg, 0.905 mmol) and DMAP (885.33 mg, 7.25 mmol) were dissolved in anhydrous 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 mixture was stirred and reacted for 0.5 hours. A dichloromethane 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) was slowly added dropwise, and the mixture was naturally returned to room temperature and reacted for 4 hours. The reaction solution was quenched with water, extracted with dichloromethane three times (100 ml × 3), the organic phases were combined, washed with saturated brine, dried, concentrated, and purified by silica gel column (MeOH/DCM = 0% ~ 5%) to obtain the title compound (498 mg, 0.304 mmol).

The structural characterization data are as follows: ESI-MS (m/z): 1352.8 [M+1] ⁺ . Step 3: Preparation of (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 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamide)benzyl) carbonate (B-04-3)

2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentaazatriamide)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]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethyl acetate (200 mg, 0.122 mmol) was dissolved in THF (3 mL) and MeOH (3 mL), and an aqueous solution of sodium carbonate (25.88 mg, 0.224 mmol) (1 mL) was added dropwise while stirring, and the stirring reaction was continued for 1 hour. The reaction solution was neutralized by dropwise addition of dilute hydrochloric acid solution, concentrated under reduced pressure, and then directly carried out to the next step.

The structural characterization data are as follows: ESI-MS (m/z): 1596.7 [M+1] ⁺ . Step 4: Preparation of (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 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentaazatriamide)benzyl) carbonate (B-04-4)

Carbonate ((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) 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamide)benzyl) (190 mg, 119.04 μmol) was dissolved in dichloromethane (5 mL), trifluoroacetate (0.5 mL) was added, and the reaction was continued for 1 hour. Saturated aqueous sodium bicarbonate solution was added dropwise to the reaction solution for neutralization, and then the liquid was separated and the organic phase was concentrated to obtain a crude product. After purification by a reverse phase chromatography column (acetonitrile/1% formic acid aqueous solution = 0%~50%) and freeze drying, the title compound (95 mg, 69.35 μmol) was obtained.

The structural characterization data are as follows: ESI-MS (m/z): 1323.6 [M+1] ⁺ . Step 5: Preparation of (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 4-((S)-2- (4-Aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamino)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamide)benzyl ester (B-04)

Carbonate ((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) 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentaazatriamide)benzyl) ester (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 then stirred for 2 hours. The reaction solution was directly filtered and the crude product was concentrated. The crude product was purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (42.2 mg, 24.69 μmol).

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

The method of preparative HPLC is as follows: Chromatographic column: SunFire preparative C18 OBD 19 mm×150 mm×5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid) Time [min] Phase shift A [%] Shift phase B [%] Flow rate [mL/min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28 Example 12 Preparation of antibodies

Human PTK7 protein was used to immunize wild-type mice; serum titers were monitored by ELISA and flow cytometry; the best mice were selected based on the titer results, and spleen cells were taken out from them for fusion, screening and sub-selection; the activities of different monoclonal lines binding to human and monkey proteins and cells, endocytosis and other activities were detected; monoclonal lines were selected for humanization; and finally 64A10 humanized antibody variable regions (heavy chain variable region SEQ ID NO: 7; light chain variable region SEQ ID NO: 8) and 101A6 humanized antibody variable regions (heavy chain variable region SEQ ID NO: 3; light chain variable region SEQ ID NO: 4) were obtained; the above heavy chain variable region sequences were fused to the human IgG1 heavy chain constant region (SEQ ID NO: :43), and the above light chain variable region sequences were fused to the human kappa light chain constant region (SEQ ID NO:44) to form two complete humanized antibodies (see Table 1); Nanjing GenScript Biotech Co., Ltd. was commissioned to perform codon optimization and gene synthesis on the antibodies, and the antibodies were constructed into pTT5 plasmids; and the heavy chain and light chain plasmids were simultaneously transfected into CHO-S cells, and the antibodies expressed in the protein A purified supernatant were used to obtain the corresponding antibodies 64A10HZ and 101A6HZ. At the same time, in order to reduce the off-target toxicity mediated by the antibody Fc receptor, the humanized 101A6 heavy chain variable region was fused to the mutant human IgG1 heavy chain constant region (SEQ ID NO: 45), and the expressed antibody was named 101A6HZm. hIgG1 is an anti-chicken lysozyme antibody fused to the human IgG1 heavy chain constant region, and hIgG1m is an anti-chicken lysozyme antibody fused to the mutant human IgG1 heavy chain constant region.

The PTK7 control antibody was obtained from Hu24 in patent CN201580030255. After codon optimization, the antibody heavy chain and light chain nucleotide sequences were synthesized and cloned into the pTT5 vector, and expressed and purified according to the above method. Example 13 Antibody Binding and Detection of Binding of Antibody to C-01

64A10HZ, 101A6HZ, Hu24 or hIgG1m antibody was taken and diluted with 20 mM PB+0.1 M EDTA (pH 7.60); then the pH was adjusted to 7.60 with 1 M Na ₂ HPO ₄ solution; and 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly and allowed to stand at room temperature for 1.5 h. A solution of C-01 (10 mM, 5 equivalents of antibody) in DMSO was added to the above mixture, and then the resulting mixture was mixed evenly and allowed to stand at room temperature for 2 h; then the buffer solution was replaced with a 20 mM histidine buffer solution (pH 6.0) using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate. The DAR values determined by mass spectrometry are shown in Table 2. Binding of 101A6HZ and A-05

1.938 mol 101A6HZ antibody (25.8 mg/mL) was taken and diluted with 96.9 uL 20 mM PB + 0.1 M EDTA (pH 7.60); then the pH was adjusted to 7.60 with 1 M Na ₂ HPO ₄ solution; and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 83.1 uL, pH 7.60) solution was added, mixed evenly and allowed to stand at room temperature for 1.5 h. A solution of A-05 (10 mM, 5 equivalents of antibody) in DMSO was added to the above mixture, and the resulting mixture was then mixed evenly and allowed to stand at room temperature for 2 h; the buffer solution was then replaced with a 20 mM histidine buffer solution (pH 6.0) using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., ADC 101A6HZ-A-05-4). The DAR value determined by mass spectrometry was 3.73. Binding of the antibody to A-05, B-01, A-26, and B-04

101A6HZ, 101A6HZm, 64A10HZ, Hu24, hIgG1 or _hIgG1m antibody was diluted with 20 mM PB + 0.1 M EDTA (pH 7.60); then the pH was adjusted to 7.60 with 1 M _Na2HPO4 solution; and 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly and allowed to stand at room temperature for 1.5 h. A solution of A-05 and B-01, A-26 or B-04 (10 mM, 10 equivalents of antibody) in DMSO was added to the above mixture, and the resulting mixture was then mixed uniformly and allowed to stand at room temperature for 2 h; and then the buffer solution was replaced with a 20 mM histidine buffer solution (pH 6.0) using a NAP-5 gel column (Cytiva) to obtain an antibody-drug conjugate.

After the binding reaction, the methylsulfonyl group of A-05, B-01, A-26 or B-04 is replaced and the cysteine sulfhydryl group in the reference antibody is directly bonded to the pyrimidine ring as shown in ADC A-05, ADC B-01, ADC A-26 or ADC B-04.

The DAR values determined by mass spectrometry are shown in Table 2.

The drug/antibody ratio (DAR) of the bound samples was determined as follows: LC-MS molecular weight analysis was performed on the bound ADC samples.

HPLC conditions: HPLC column: Thermo MAbPac RP 3.0*100 mm; Mobile phase A: 0.1% FA/H ₂ O; Mobile phase B: 0.1% FA/ACN; Flow rate: 0.25 ml/min; Sample chamber temperature: 8 ℃; Column temperature: 60 ℃; Injection volume: 2 μl; Time(min) 2 20 twenty two 25 26 30 Mobile phase A (volume %) 75 60 5 5 75 75 Mobile phase B (volume %) 25 40 95 95 25 25

Mass spectrometry conditions: Mass spectrometer model: AB Sciex Triple TOF 5600+; GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 200; CE 10; accumulation time 0.5 s; m/z 600-4000; time bins to sum 40. Table 2 ADC quality test results Drug-Conjugate Number ADC Number Purity SEC% DAR A-05 64A10HZ-A-05 95.9 8.00 A-05 101A6HZ-A-05 98.8 8.03 B-01 64A10HZ-B-01 92.7 8.01 B-01 101A6HZ-B-01 99.4 7.97 C-01 64A10HZ-C-01 90.0 3.4 C-01 101A6HZ-C-01 89.1 3.96 C-01 Hu24-C-01 92.5 4.08 A-05 101A6HZ-A-05-4 99.2 3.73 A-05 101A6HZm-A-05 97.6 8.05 A-05 hIgG1-A-05 98.5 8.02 A-05 hIgG1m-A-05 97.9 7.18 C-01 hIgG1m-C-01 98.1 2.73 A-26 101A6HZm-A-26 96.7 8.15 B-04 101A6HZm-B-04 93.8 8.47 Example 12 Detection of the activity of antibody-drug conjugates 1. In vivo efficacy detection of different conjugate drugs in the NCI-H358 model 1. In vivo efficacy detection of different antibody-drug conjugates in the NCI-H358 model

Human non-small cell lung cancer NCI-H358 cells (NANJING COBIOER BIOSCIENCES CO., LTD) were cultured in vitro as monolayers in RPMI1640 medium supplemented with 10% fetal bovine serum and cultured at 37°C in an incubator containing 5% CO _2. 5×10 ⁶ NCI-H358 cells were subcutaneously inoculated into the right armpit of each mouse and suspended in 0.05 ml PBS + 0.05 ml Matrigel. When the average tumor volume grew to 150-200 ^mm3 , mice with irregular tumor volume, too small or too large tumor volume were excluded, and the remaining mice were randomly divided into 8 groups according to tumor volume and animal weight, with 6 mice in each group. The drug was administered IV at a single dose (3 mg/kg); after administration, the tumor was measured twice a week using a vernier caliper, and the tumor volume was calculated according to the following formula: V = 0.5a×b ² , where a and b represent the long and short diameters of the tumor, respectively; the antitumor drug efficacy was evaluated by the tumor growth inhibition rate TGI (%), and the calculation formula was: TGI (%) (tumor volume) = [1-(T _Vt - T _V0 )/(C _Vt - C _V0 )]×100%; when the tumor was relieved, TGI(%) (tumor volume) = 100%-(T _Vt -T _V0 )/ T _V0 ×100%. T _V0 is the average tumor volume of the test drug group at the time of group dosing; T _Vt is the average tumor volume of the test drug group on the tth day after dosing; C _V0 is the average tumor volume of the vehicle group (normal saline) at the time of group dosing; and C _Vt is the average tumor volume of the vehicle group on the tth day after dosing. If the tumor shrinks compared with the initial volume, that is, V _t <V ₀ , it is defined as a partial tumor response (PR); and if the tumor disappears completely, it is defined as a complete tumor response (CR). Animal deaths were observed and recorded every day, and the specific results are shown in Table 3 and Figures 1A-1B. Table 3 Analysis of the efficacy of different antibody-drug conjugates against human non-small cell lung cancer cells in the NCI-H358 subcutaneous tumor-bearing mouse model (N=6) Drugs V _P0 (mm ³ , mean ± SEM) _VP21 (mm ³ , mean ± SEM) TGI (%) P -value Physiological saline 191.91±11.32 647.23±67.63 / / hIgG1-A-05 190.46±9.11 421.66±34.42 49.22* 0.01398 64A10HZ-A-05 191.59±9.35 268.77±26.92 83.05*** 0.00040 101A6HZ-A-05 190.85±9.52 281.42±24.19 80.11*** 0.00047 64A10HZ-B-01 190.68±7.73 331.38±36.46 69.10** 0.00211 64A10HZ-C-01 188.74±9.49 443.05±33.63 44.15* 0.02219 101A6HZ-C-01 192.06±9.41 465.40±40.01 39.97* 0.04320 Hu24-C-01 189.77±6.92 449.71±50.28 42.91* 0.04107

Note: T test ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 means significant difference compared with the saline group.

The results showed that: after single intravenous administration, different drug combinations had significant drug effects on human non-small cell lung cancer cells in the NCI-H358 mouse subcutaneous xenograft tumor animal model. TGI (%): A-05 combination was superior to C-01 combination, and animals in each group were better tolerated. 2. In vivo efficacy detection of different antibody-drug combinations in the A431 model

Human epithelial squamous cell carcinoma A431 cells (NANJING COBIOER BIOSCIENCES CO., LTD) were cultured under the following conditions: 10% fetal bovine serum was added to the DMEM medium, and the culture was performed at 37°C in an incubator containing air with 5% CO _2. 5×10 ⁶ A431 cells were subcutaneously inoculated into the right armpit of each mouse and suspended in 0.1 ml PBS. When the average tumor volume grew to about 100-150 mm ³ , mice with tumor volumes that were too small or too large were excluded, and the remaining mice were randomly divided into 3 groups of 5 mice each according to the tumor volume and animal weight; drugs were administered IV once a week for a total of two times. Tumor volume and body weight were measured twice a week after administration, and the specific results are shown in Table 4 and Figures 2A-2B. Table 4 Analysis of the efficacy of antibody-drug conjugates against human epithelial squamous cell carcinoma cells in the A431 subcutaneous tumor-bearing mouse model (N=5) Drugs Drug dosage (mg/kg) V _P0 (mm ³ , mean ± SEM) _VP22 (mm ³ , mean ± SEM) TGI (%) P -value Physiological saline / 128.36±9.62 1675.96±199.83 / / hIgG1-A-05 10 126.58±9.30 1749.78±145.64 -4.89 0.77291 101A6HZ-A-05 10 127.77±9.58 624.86±96.98 67.88** 0.00148

Note: T test ^** P < 0.01 means significant difference compared with the saline group.

The results showed that 101A6HZ-A-05 had significant efficacy at 10 mg/kg. 3. Detection of binding of anti-human PTK7 antibodies and their conjugates to PTK7-overexpressing cells of different genera and species

The ability of the antibody and its conjugate to bind to CHO-PTK7 cells was detected by flow cytometry (Beckman, model: Cytoflex). CHO-PTK7 cells (human: hPTK7, monkey: cPKT7, mouse: mPTK7, rat: rPTK7) cultured in suspension were counted; cells were collected at a density of 2×10 ⁵ cells/well, washed twice with 1×PBS, and resuspended in 1% BSA solution; cells were then transferred to 96-well deep plates, 50 μl/well; antibodies and their drug conjugates were diluted with 1% BSA, starting from 10 μg/ml, and diluted in a three-fold gradient; 50 μl of diluted antibodies or antibody-drug conjugates were then added to the deep plates containing cells and incubated at 4°C for 40 min; cells were washed twice with PBS, and 50 μl of the diluted antibodies or antibody-drug conjugates were then added to the deep plates containing cells and incubated at 4°C for 40 min. 100 μl of diluted secondary antibody was added to each well and mixed evenly, and the cells were incubated at 4°C for 30 min; and the cells were washed twice with PBS, then resuspended in 400 μl PBS and monitored by flow cytometry. Data processing: The median fluorescence signal value was input into GraphPad Prism 6 software to calculate EC ₅₀ , and the results are shown in Table 5. The results show that the antibody 101A6HZ and 101A6HZm conjugates of the present invention can bind to human and monkey PTK7 cells overexpressing, but not to mouse and rat PTK7 cells. Table 5 Affinity determination results of anti-human PTK7_ADC cells Drugs _EC50 (ng/ml) hPTK7 cPTK7 mPTK7 rPTK7 101A6HZm-A-05 418.70 551.20 Unbound Unbound 101A6HZ-C-01 147.50 114.70 Unbound Unbound 4. Detection of cell affinity of anti-human PTK7 antibodies and their conjugates

The affinity of anti-human PTK7 antibody and bound ADC to OVCAR3 (ATCC), NCI-H358, HCC1806 (ATCC), A431, NCI-H520 (Nanjing Cobioer Biosciences Co., LTD.), and DU4475 cells was detected by flow cytometry (Beckman, model: Cytoflex). Adherent cells OVCAR, NCI-H358, HCC1806, A431, and NCI-H520 were digested by trypsin, and the suspended cells DU4475 were mixed by pipetting, and the appropriate amount of cells were counted and collected, and the detection steps of flow cytometry were the same as above. The results are shown in Table 6 and Figures 3A-3F. The results show that after the antibodies 101A6HZ and 101A6HZm of the present invention are incorporated into ADC, they can still bind to tumor cells with high affinity. Table 6-1 Affinity test results of anti-human PTK7 ADC for cells Drugs _EC50 (ng/ml) OVCAR3 NCI-H358 HCC1806 A431 101A6HZm-A-05 127.20 123.60 152.0 154.10 101A6HZ-C-01 102.60 108.10 67.07 97.40 Table 6-2 Results of cell affinity detection of anti-human PTK7 ADC Drugs _EC50 (ng/ml) NCI-H520 DU4475 101A6HZm-A-05 157.0 173.0 101A6HZm-A-26 114.3 89.13 101A6HZm-B-04 97.29 74.81 5. Detection of cellular endocytosis of anti-human PTK7 antibodies and their conjugates 5.1 Endocytosis detection method I

HCC1806 and OVCAR3 cells were trypsinized, resuspended and then counted; cells were collected at a density of approximately 2×10 ⁵ cells/well; cells were suspended with 1% BSA at a volume of 50 μl/well and spread on a deep plate; antibodies and corresponding ADCs were added and incubated at 4°C for 60 min; cells were washed twice with 1% BSA, and then secondary antibodies (anti-human IgG Alexa Fluor 488) were added and incubated at 4°C for 30 min; endocytosis at different temperatures was detected according to the literature (PLoS ONE 10(4): e0124708); and after incubation, 150 μl 1% BSA was added to resuspend and tested on the machine. Data analysis: The fluorescence signal values after quenching at 37°C were used to make fitting curves at different concentration points, and the EC ₅₀ was calculated. The results are shown in Table 7. The candidate antibody conjugates have good endocytosis activity. Table 7 Detection of cellular endocytosis of ADC Drugs _EC50 (ng/ml) OVCAR3 NCI-H358 HCC1806 A431 101A6HZm-A-05 168.70 178.40 185.0 96.62 101A6HZ-C-01 65.29 96.20 106.3 68.02 5.2 Endocytosis Detection Method II

Adherent cells NCI-H358, HCC1806, OVCAR3, NCI-H520 were digested by trypsin, and suspended cells DU4475 were mixed by pipetting, then the cells were counted and placed in a 96-well plate, with 10,000 cells per well in 100 μl of medium. The cells were then cultured in an incubator at 37°C. The next day, the antibody or ADC sample was diluted with complete medium, starting from 4.8 μg/ml, 3-fold, and graded at 8 concentrations. The antibody or ADC sample was labeled with pHrodo red diluted to 12 μg/ml in complete medium. 30 μl of diluted antibody/ADC sample and 30 μl of diluted pHrodo red sample were mixed in a 96-well plate and incubated at room temperature in the dark for 30 minutes. Then 50 μl of medium/well was removed from the cells, 50 μl of labeled antibody or ADC was added to the cells, and the cells were incubated in an incubator at 37°C 5% CO2 for 24 hours. The next day, the culture supernatant was removed, the cells were washed once with PBS, and digested by adding 40 μl 0.25% trypsin/well, then neutralized with 100 μl complete medium, and mixed thoroughly by pipetting up and down. Flow cytometry was used to detect the fluorescent signal excited by an excitation wavelength of 561 nm.

Data processing: The median values were exported and imported into GraphPad Prism 6 software to calculate EC ₅₀ . The results are shown in Table 8 and Figures 4A-4E . Table 8 Detection of endocytosis of antibodies and antibody-drug conjugates Drugs _EC50 (ng/ml) NCI-H358 HCC1806 OVCAR3 NCI-H520 DU4475 101A6HZm 47.43 137.8 32.94 45.40 42.16 101A6HZm-A-05 58.64 138.0 32.53 45.78 47.50 101A6HZm-A-26 47.34 134.5 24.98 38.73 46.56 101A6HZm-B-04 47.36 176.8 21.20 44.90 44.91 6. In vitro cytotoxicity assay of anti-human PTK7 antibody-drug conjugates

FADU (NANJING COBIOER BIOSCIENCES CO., LTD), HCC1806, DU4475 (NANJING COBIOER BIOSCIENCES CO., LTD) and H358-hPTK7 cells were digested by trypsin; the optimal cell number was selected and plated according to the proliferation curve of each tumor cell, and then cultured overnight at 37°C. Each antibody and conjugate was diluted with culture medium, added to the cells, and cultured continuously for 5-6 days. CCK8 was added to the cells and cultured continuously for 2-4 h; the value at OD450 nm was read with a microplate reader; the killing activity was calculated; and the results are shown in Table 9 and Figures 5A-5C. The killing activity of the conjugate was significantly stronger than that of the negative antibody conjugate, indicating that the killing effect was mediated by the target. Table 9 In vitro cell killing results of anti-human PTK7 ADC Drugs IC ₅₀ (nM) FADU HCC1806 DU4475 H358-hPTK7 hIgGm-A-05 187.54 / 130.17 / 101A6HZm-A-05 22.51 21.59 1.96 1.03

Note: "/" means no killing activity. 7. Hydrophilicity detection of anti-human PTK7 antibodies and conjugates

Agilent 1260 and analytical column TSKgel Butyl-NPR were used to detect the hydrophilicity of antibodies and conjugates; an appropriate amount of test sample was taken and diluted with diluent (0.75 mol/L (NH ₄ ) ₂ SO ₄ ) to prepare a 1.0 mg/ml solution as the test sample solution; and control antibodies (hydrophilic control, tetelizumab; hydrophobic control, satuzumab. Both are produced by Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd.) were taken out and prepared into a 1 mg/ml solution with diluent as the system suitability solution. Inject about 40 μg of sample; analyze gradient elution; calculate the hydrophobicity of the test sample based on the control sample after detection, and the calculation formula is: (retention time of test sample - retention time of hydrophilic control) / (retention time of hydrophobic control - retention time of hydrophilic control). The smaller the retention time and hydrophobicity, the better the hydrophilicity of the antibody. The results are shown in Table 10. Antibody 101A6HZm conjugate has good hydrophilicity, which is equivalent to naked satuzumab antibody and can improve the metabolism of ADC drugs in vivo. Table 10 Hydrophilicity detection of anti-human PTK7 antibodies and conjugates antibody Detention time Hydrophobicity Hydrophilic comparison 9.097 0.00 Hydrophobic control 15.962 1.00 101A6HZm-A-05 15.451 0.9 8. Detection of the binding activity of anti-human PTK7 antibody-drug conjugates to Fc receptors

ForteBio (Pall life sciences) was used to detect the dynamic affinity of antibodies 101A6HZm and 101A6HZm-A-05 to human Fc receptor proteins CD16a, CD32a, CD32b, CD64 and FcRn. The specific method includes: capturing the biotinylated protein to be detected by using SA sensor (Pall life sciences) in PBST solution; diluting the antibody and conjugate to be detected by using PBST to reach an initial concentration of 5,000 nM, and performing double dilution until 7 concentration points are reached; binding, dissociation and open detection results in Data Analysis 11.0 software; and analyzing the results by selecting 1:1 mode and global fitting to obtain the binding rate, dissociation rate and affinity constant; and the results are shown in Table 11. Table 11 Detection of binding activity of antibodies and conjugates to Fc receptors antibody KD(M) CD16a CD32a CD32b CD64 FcRn 101A6HZm Not detected Not detected Not detected Not detected 1.66E-8 101A6HZm-A-05 Not detected Not detected Not detected Not detected 1.42E-8

The results showed that the mutant 101A6HZm and its conjugates did not bind to the Fc receptors CD16a, CD32a, CD32b and CD64 proteins, which may reduce nonspecific killing mediated by Fc receptors and improve drug safety. At the same time, 101A6HZm and its conjugates retained FcRn protein binding activity and did not affect the half-life of the drug. 9. In vivo efficacy detection of anti-human PTK7 antibody-drug conjugates in the HCC1806 model

Human breast cancer HCC1806 cells were cultured in monolayer in vitro, and the culture conditions included: 10% fetal bovine serum was added to RPMI 1640 medium, and the culture was carried out at 37°C in an incubator containing air with 5% CO _2. 2×10 ⁶ HCC1806 cells were subcutaneously inoculated into the right armpit of each mouse and suspended in 0.1 ml PBS. When the average tumor volume grew to about 100-150 mm ³ , mice with tumor volumes that were too small or too large were excluded, and the remaining mice were randomly divided into 5 groups of 5 mice each according to tumor volume and animal weight; drugs were administered IV once every two weeks for a total of two times (P0 and P18). Tumor volume and body weight were measured twice a week after administration, and the specific results are shown in Table 12 and Figures 6A-6B. Table 12 Analysis of the efficacy of antibody-drug conjugates against human breast cancer cells in the HCC1806 subcutaneous tumor-bearing mouse model (N=5) Drugs Dosage (mg/kg) V _P0 (mm ³ , mean ± SEM) _VP31 (mm ³ , mean ± SEM) TGI (%) P -value Physiological saline / 100.87±9.32 2086.11±202.17 / / hIgG1m-A-05 3 100.47±8.76 1470.03±187.50 31.01 0.0559 hIgG1m-A-05 10 102.18±9.99 979.31±147.87 55.82** 0.0040 101A6HZm-A-05 3 100.52±9.40 801.28±213.70 64.70** 0.0024 101A6HZm-A-05 10 102.08±9.06 489.36±83.95 80.49*** 0.0001

Note: T test ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 means significant difference compared with the saline group.

The results showed that the 101A6HZm-A-05 group had significant drug efficacy at 3 mg/kg and 10 mg/kg, and the anti-tumor effect was significantly better than the anti-tumor effect of the hIgG1m-A-05 group at the same dose. 10. In vivo efficacy detection of anti-human PTK7 antibody-drug conjugate in the OVCAR3 model

Human ovarian cancer OVCAR3 cells were cultured in monolayer in vitro, and the culture conditions included: 20% fetal bovine serum was added to RPMI1640 medium, and the culture was carried out at 37°C in an incubator containing air with 5% CO _2. 5×10 ⁶ OVCAR3 cells were subcutaneously inoculated into the right armpit of each mouse and suspended in 0.05 ml PBS + 0.05 ml matrigel. When the average tumor volume grew to about 100-150 mm ³ , mice with tumor volumes that were too small or too large were excluded, and the remaining mice were randomly divided into 5 groups of 5 mice each according to tumor volume and animal weight; and the drug was given IV at a single dose. Tumor volume and body weight were measured twice a week after administration, and the specific results are shown in Table 13 and Figures 7A-B. Table 13 Analysis of the efficacy of antibody-drug conjugates against human ovarian cancer cells in the OVCAR3 subcutaneous tumor-bearing mouse model (N=5) Drugs Dosage (mg/kg) V _P0 (mm ³ , mean ± SEM) _VP36 (mm ³ , mean ± SEM) TGI (%) P -value Tumor remission Physiological saline / 131.40±9.19 2081.67±316.61 / / without hIgG1m-A-05 3 130.66±11.60 1550.40±281.78 27.20 0.2454 without hIgG1m-A-05 10 132.09±9.09 256.56±21.21 93.62*** 0.0004 without 101A6HZm-A-05 3 129.39±11.41 39.57±10.89 169.41*** 0.0002 1/5CR, 4/5PR 101A6HZm-A-05 10 129.93±8.95 45.53±3.11 164.96*** 0.0002 5/5PR

Note: T test ^** P < 0.01, ^*** P < 0.001 means significant difference compared with the saline group.

The results showed that the 101A6HZm-A-05 group had significant drug efficacy at 3 mg/kg and 10 mg/kg, and the anti-tumor effect was significantly better than the anti-tumor effect of the hIgG1m-A-05 group at the same dose. According to tumor regression, under the condition of 3 mg/kg 101A6HZm-A-05, 1 of 5 mice had a complete response (CR) and 4 had a partial response (PR); and under the condition of 101A6HZm-A-05 at 10 mg/kg, all 5 mice achieved a partial response (PR). Example 13 Antibody binding and detection Antibody binding to C-01

64A10HZ, 101A6HZ, Hu24 or hIgG1m antibody was taken and diluted with 20 mM PB+0.1 M EDTA (pH 7.60); then the pH was adjusted to 7.60 with 1 M Na ₂ HPO ₄ solution; and 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly and allowed to stand at room temperature for 1.5 h. A solution of C-01 (10 mM, 5 equivalents of antibody) in DMSO was added to the above mixture, and then the resulting mixture was mixed evenly and allowed to stand at room temperature for 2 h; then the buffer solution was replaced with a 20 mM histidine buffer solution (pH 6.0) using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate. The DAR values determined by mass spectrometry are shown in Table 2. Binding of 101A6HZ and A-05

1.938 mol 101A6HZ antibody (25.8 mg/mL) was taken and diluted with 96.9 uL 20 mM PB + 0.1 M EDTA (pH 7.60); then the pH was adjusted to 7.60 with 1 M Na ₂ HPO ₄ solution; and 10 mM TCEP (tris(2-carboxyethyl)phosphine, 83.1 uL, pH 7.60) solution was added, mixed evenly and allowed to stand at room temperature for 1.5 h. A solution of A-05 (10 mM, 5 equivalents of antibody) in DMSO was added to the above mixture, and the resulting mixture was then mixed evenly and allowed to stand at room temperature for 2 h; the buffer solution was then replaced with a 20 mM histidine buffer solution (pH 6.0) using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., ADC 101A6HZ-A-05-4). The DAR value determined by mass spectrometry was 3.73. Binding of A-05, B-01, A-26, and B-04

101A6HZ, 101A6HZm, 64A10HZ, Hu24, hIgG1 or _hIgG1m antibody was diluted with 20 mM PB + 0.1 M EDTA (pH 7.60); then the pH was adjusted to 7.60 with 1 M _Na2HPO4 solution; and 10 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60) solution was added, mixed evenly and allowed to stand at room temperature for 1.5 h. A solution of A-05, B-01, A-26 or B-04 (10 mM, 10 equivalents of antibody) in DMSO was added to the above mixture, and the resulting mixture was then mixed uniformly and allowed to stand at room temperature for 2 h; and then the buffer solution was replaced with a 20 mM histidine buffer solution (pH 6.0) using a NAP-5 gel column (Cytiva) to obtain an antibody-drug conjugate.

After the binding reaction, the methylsulfonyl group of A-05 is replaced and the cysteine sulfhydryl group in the reference antibody is directly bonded to the pyrimidine ring as shown in ADC A-05.

The DAR values determined by mass spectrometry are shown in Table 2.

The drug/antibody ratio (DAR) of the bound samples was determined as follows: The bound ADC samples were subjected to LC-MS molecular weight analysis. Analytical conditions Liquid chromatography column: Thermo MAbPac RP 3.0*100 mm Mobile phase A: 0.1% FA/H ₂ O; Mobile phase B: 0.1% FA/CAN Flow rate: 0.25 ml/min; Sample chamber temperature: 8 ℃; Column temperature: 60 ℃; Injection volume: 2 μl; Time(min) 2 20 twenty two 25 26 30 Mobile phase A (volume %) 75 60 5 5 75 75 Mobile phase B (volume %) 25 40 95 95 25 25

Mass spectrometry conditions: Mass spectrometer model: AB Sciex Triple TOF 5600+; GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 200; CE 10; accumulation time 0.5 s; m/z 600-4000; time bin for summation 40. Table 2 ADC quality test results Drug-Conjugate Number ADC Number Purity SEC% DAR A-05 64A10HZ-A-05 95.9 8.00 A-05 101A6HZ-A-05 98.8 8.03 B-01 64A10HZ-B-01 92.7 8.01 B-01 101A6HZ-B-01 99.4 7.97 C-01 64A10HZ-C-01 90.0 3.4 C-01 101A6HZ-C-01 89.1 3.96 C-01 Hu24-C-01 92.5 4.08 A-05 101A6HZ-A-05-4 99.2 3.73 A-05 101A6HZm-A-05 97.6 8.05 A-05 hIgG1-A-05 98.5 8.02 A-05 hIgG1m-A-05 97.9 7.18 C-01 hIgG1m-C-01 98.1 2.73 A-26 101A6HZm-A-26 96.7 8.15 B-04 101A6HZm-B-04 93.8 8.47 Example 14 Detection of the activity of antibody-drug conjugates 1. In vivo efficacy detection of different antibody-drug conjugates in the NCI-H358 model

Human non-small cell lung cancer NCI-H358 cells (NANJING COBIOER BIOSCIENCES CO., LTD) were cultured in vitro as monolayers in RPMI1640 medium supplemented with 10% fetal bovine serum and cultured at 37°C in an incubator containing 5% CO2. 5×106 NCI-H358 cells were subcutaneously inoculated into the right armpit of each mouse and suspended in 0.05 ml PBS + 0.05 ml Matrigel. When the average tumor volume grew to 150-200 mm3, mice with irregular tumor volume, too small or too large tumor volume were excluded, and the remaining mice were randomly divided into 8 groups according to tumor volume and animal weight, with 6 mice in each group. The drug was administered IV at a single dose (3 mg/kg); after administration, the tumor was measured twice a week using a vernier caliper, and the tumor volume was calculated according to the following formula: V = 0.5a×b2, where a and b represent the long and short diameters of the tumor, respectively; the anti-tumor drug efficacy was evaluated by the tumor growth inhibition rate TGI (%), and the calculation formula was: TGI (%) (tumor volume) = [1-(TVt- TV0)/(CVt- CV0)]×100%; when the tumor was relieved, TGI(%) (tumor volume) = 100%-(TVt-TV0)/ TV0×100%. TV0 is the mean tumor volume of the test drug group at the time of group dosing; TVt is the mean tumor volume of the test drug group on day t after dosing; CV0 is the mean tumor volume of the vehicle group (normal saline) at the time of group dosing; and CVt is the mean tumor volume of the vehicle group on day t after dosing. If the tumor shrinks compared with the initial volume, that is, Vt < V0, it is defined as a partial tumor response (PR); and if the tumor disappears completely, it is defined as a complete tumor response (CR). Animal deaths were observed and recorded every day, and the specific results are shown in Table 3 and Figures 1A-1B. Table 3 Analysis of the efficacy of different antibody-drug conjugates against human non-small cell lung cancer cells in the NCI-H358 subcutaneous tumor-bearing mouse model (N=6) Drugs V _P0 (mm ³ , mean ± SEM) _VP21 (mm ³ , mean ± SEM) TGI (%) P-value Physiological saline 191.91±11.32 647.23±67.63 / / hIgG1-A-05 190.46±9.11 421.66±34.42 49.22* 0.01398 64A10HZ-A-05 191.59±9.35 268.77±26.92 83.05*** 0.00040 101A6HZ-A-05 190.85±9.52 281.42±24.19 80.11*** 0.00047 64A10HZ-B-01 190.68±7.73 331.38±36.46 69.10** 0.00211 64A10HZ-C-01 188.74±9.49 443.05±33.63 44.15* 0.02219 101A6HZ-C-01 192.06±9.41 465.40±40.01 39.97* 0.04320 Hu24-C-01 189.77±6.92 449.71±50.28 42.91* 0.04107

Note: T test *P<0.05, **P<0.01, ***P<0.001 means significant difference compared with the saline group.

The results showed that: after single intravenous administration, different drug combinations had significant drug effects on human non-small cell lung cancer cells in the NCI-H358 mouse subcutaneous xenograft tumor animal model. TGI (%): A-05 combination was superior to C-01 combination, and animals in each group were better tolerated. 2. In vivo efficacy detection of different antibody-drug combinations in the A431 model

Human epithelial squamous cell carcinoma A431 cells (NANJING COBIOER BIOSCIENCES CO., LTD) were cultured under the following conditions: 10% fetal bovine serum was added to the DMEM medium, and the culture was performed at 37°C in an incubator containing air with 5% CO2. 5×106 A431 cells were subcutaneously inoculated into the right armpit of each mouse and suspended in 0.1 ml PBS. When the average tumor volume grew to about 100-150 mm3, mice with too small or too large tumor volume were excluded, and the remaining mice were randomly divided into 3 groups of 5 mice each according to the tumor volume and animal weight; drugs were given IV once a week for a total of two times. Tumor volume and body weight were measured twice a week after administration, and the specific results are shown in Table 4 and Figures 2A-2B. Table 4 Analysis of the efficacy of antibody-drug conjugates against human epithelial squamous cell carcinoma cells in the A431 subcutaneous tumor-bearing mouse model (N=5) Drugs Drug dosage (mg/kg) V _P0 (mm ³ , mean ± SEM) _VP22 (mm ³ , mean ± SEM) TGI (%) P-value Physiological saline / 128.36±9.62 1675.96±199.83 / / hIgG1-A-05 10 126.58±9.30 1749.78±145.64 -4.89 0.77291 101A6HZ-A-05 10 127.77±9.58 624.86±96.98 67.88** 0.00148

Note: T test **P<0.01 means significant difference compared with the saline group.

The results showed that 101A6HZ-A-05 had significant efficacy at 10 mg/kg. 3. Detection of binding of anti-human PTK7 antibodies and their conjugates to PTK7-overexpressing cells of different genera and species

The ability of antibodies and their conjugates to bind to CHO-PTK7 cells was monitored by flow cytometry (Beckman, model: Cytoflex). CHO-PTK7 cells (human: hPTK7, monkey: cPKT7, mouse: mPTK7, rat: rPTK7) cultured in suspension were counted; cells were collected at a density of 2×105 cells/well, washed twice with 1×PBS, and resuspended in 1% BSA solution; cells were then transferred to 96-well deep plates, 50 μl/well; antibodies and their drug conjugates were diluted with 1% BSA, starting from 10 μg/ml, and diluted in a three-fold gradient; 50 μl of diluted antibodies or antibody-drug conjugates were then added to the deep plates containing cells and incubated at 4°C for 40 min; cells were washed twice with PBS, and 50 μl of the diluted antibodies or antibody-drug conjugates were then added to the deep plates containing cells and incubated at 4°C for 40 min. 100 μl of diluted secondary antibody was added to each well and mixed evenly, and the cells were incubated at 4°C for 30 min; and the cells were washed twice with PBS, then resuspended in 400 μl PBS and detected by flow cytometry. Data processing: The median fluorescence signal value was input into GraphPad Prism 6 software to calculate EC50, and the results are shown in Table 5. The results show that the antibody 101A6HZ and 101A6HZm conjugates of the present invention can bind to human and monkey PTK7 cells overexpressing, but not to mouse and rat PTK7 cells. Table 5 Affinity determination results of anti-human PTK7_ADC cells Drugs _EC50 (ng/ml) hPTK7 cPTK7 mPTK7 rPTK7 101A6HZm-A-05 418.70 551.20 Unbound Unbound 101A6HZ-C-01 147.50 114.70 Unbound Unbound 4. Detection of cell affinity of anti-human PTK7 antibodies and their conjugates

The affinity of anti-human PTK7 antibody and bound ADC to OVCAR3 (ATCC), NCI-H358, HCC1806 (ATCC), A431, NCI-H520 (Nanjing Cobioer Biosciences Co., LTD.), and DU4475 cells was detected by flow cytometry (Beckman, model: Cytoflex). Adherent cells OVCAR3, NCI-H358, HCC1806, A431, and NCI-H520 were digested with trypsin, and suspended cells DU4475 were mixed by pipetting, and the appropriate amount of cells were counted and collected, and the detection steps of flow cytometry were the same as above. The results are shown in Table 6-1, Table 6-2, and Figures 3A-3F. The results show that after the antibodies 101A6HZ and 101A6HZm of the present invention are incorporated into ADC, they can still bind to tumor cells with high affinity. Table 6-1 Affinity test results of anti-human PTK7 ADC for cells Drugs _EC50 (ng/ml) OVCAR3 NCI-H358 HCC1806 A431 101A6HZm-A-05 127.20 123.60 152.0 154.10 101A6HZ-C-01 102.60 108.10 67.07 97.40 Table 6-2 Results of cell affinity detection of anti-human PTK7 ADC Drugs _EC50 (ng/ml) NCI-H520 DU4475 101A6HZm-A-05 157.0 173.0 101A6HZm-A-26 114.3 89.13 101A6HZm-B-04 97.29 74.81 5. Detection of cellular endocytosis of anti-human PTK7 antibodies and their conjugates 5.1 Endocytosis detection method I

HCC1806 and OVCAR3 cells were trypsinized, resuspended and then counted; cells were collected at a density of approximately 2×105 cells/well; cells were suspended with 1% BSA at a volume of 50 μl/well and spread on a deep plate; antibodies and corresponding ADCs were added and incubated at 4°C for 60 min; cells were washed twice with 1% BSA, and then secondary antibodies (anti-human IgG Alexa Fluor 488) were added and incubated at 4°C for 30 min; endocytosis at different temperatures was detected according to the literature (PLoS ONE 10(4): e0124708); and after incubation, 150 μl 1% BSA was added to resuspend and tested on the machine. Data analysis: The fluorescence signal values after quenching at 37°C were used to make fitting curves at different concentration points, and the EC50 was calculated. The results are shown in Table 7. The candidate antibody conjugates have good endocytosis activity. Table 7 Detection of cellular endocytosis of ADC Drugs _EC50 (ng/ml) OVCAR3 NCI-H358 HCC1806 A431 101A6HZm-A-05 168.70 178.40 185.0 96.62 101A6HZ-C-01 65.29 96.20 106.3 68.02 5.2 Endocytosis Detection Method II

Adherent cells NCI-H358, HCC1806, OVCAR3, NCI-H520 were digested by trypsin, and suspended cells DU4475 were mixed by pipetting, then the cells were counted and placed in a 96-well plate, with each well containing 10,000 cells in 100 ml of medium. The cells were then cultured in an incubator at 37°C. The next day, the antibody or ADC sample was diluted with complete medium, starting from 4.8 μg/ml, diluted 3 times, and graded at 8 concentrations. The antibody or ADC sample was labeled with pHrodo red diluted to 12 mg/ml in complete medium. 30 ml of diluted antibody/ADC sample and 30 ml of diluted pHrodo red sample were mixed in a 96-well plate and incubated at room temperature in the dark for 30 minutes. Then 50 ml of medium/well was removed from the cells, 50 ml of labeled antibody or ADC was added to the cells, and the cells were incubated in an incubator at 37°C 5% CO2 for 24 hours. The next day, the culture supernatant was removed, the cells were washed once with PBS, and digested by adding 40 μl 0.25% trypsin/well, then neutralized with 100 ml of complete medium, and mixed thoroughly by pipetting up and down. Flow cytometry was used to detect the fluorescent signal excited by an excitation wavelength of 561 nm. Data processing: The median values were exported and imported into GraphPad Prism 6 software to calculate EC50. The results are shown in Table 8 and Figures 4A-4E. Table 8 Detection of endocytosis of antibodies and antibody-drug conjugates Drugs _EC50 (ng/ml) NCI-H358 HCC1806 OVCAR3 NCI-H520 DU4475 101A6HZm 47.43 137.8 32.94 45.40 42.16 101A6HZm-A-05 58.64 138.0 32.53 45.78 47.50 101A6HZm-A-26 47.34 134.5 24.98 38.73 46.56 101A6HZm-B-04 47.36 176.8 21.20 44.90 44.91 6. In vitro cytotoxicity assay of anti-human PTK7 antibody-drug conjugates

FADU (NANJING COBIOER BIOSCIENCES CO., LTD), HCC1806, DU4475 (NANJING COBIOER BIOSCIENCES CO., LTD) and H358-hPTK7 cells were digested by trypsin; the optimal cell number was selected and plated according to the proliferation curve of each tumor cell, and then cultured overnight at 37°C. Each antibody and conjugate was diluted with culture medium, added to the cells, and cultured continuously for 5-6 days. CCK8 was added to the cells and cultured continuously for 2-4 h; the value at OD450 nm was read with a microplate reader; the killing activity was calculated; and the results are shown in Table 9 and Figures 5A-5C. The killing activity of the conjugate was significantly stronger than that of the negative antibody conjugate, indicating that the killing effect was mediated by the target. Table 9 In vitro cell killing results of anti-human PTK7 ADC Drugs IC ₅₀ (nM) FADU HCC1806 DU4475 H358-hPTK7 hIgGm-A-05 187.54 / 130.17 / 101A6HZm-A-05 22.51 21.59 1.96 1.03

Note: "/" means no killing activity. 7. Hydrophilicity detection of anti-human PTK7 antibodies and conjugates

Agilent 1260 and analytical column TSKgel Butyl-NPR were used to detect the hydrophilicity of antibodies and conjugates. An appropriate amount of test sample was taken and diluted with a diluent (0.75 mol/L (NH4)2SO4) to prepare a 1.0 mg/ml solution as the test sample solution. Control antibodies (hydrophilic control, Tagitanlimab; hydrophobic control, Sacituzumab. Both were produced by Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd.) were taken out and prepared into a 1 mg/ml solution with a diluent as the system suitability solution. Inject about 40 μg of sample; analyze gradient elution; calculate the hydrophobicity of the test sample based on the control sample after detection, and the calculation formula is: (retention time of test sample - retention time of hydrophilic control) / (retention time of hydrophobic control - retention time of hydrophilic control). The smaller the retention time and hydrophobicity value, the better the hydrophilicity of the antibody. The results are shown in Table 10. Antibody 101A6HZm conjugate has good hydrophilicity, which is equivalent to naked satuzumab antibody and can improve the metabolism of ADC drugs in vivo. Table 10 Hydrophilicity detection of anti-human PTK7 antibodies and conjugates antibody Detention time Hydrophobicity Hydrophilic comparison 9.097 0.00 Hydrophobic control 15.962 1.00 101A6HZm-A-05 15.451 0.9 8. Detection of the binding activity of anti-human PTK7 antibody-drug conjugates to Fc receptors

ForteBio (Pall life sciences) was used to detect the dynamic affinity of antibodies 101A6HZm and 101A6HZm-A-05 to human Fc receptor proteins CD16a, CD32a, CD32b, CD64 and FcRn. The specific method includes: capturing the biotinylated protein to be detected by using SA sensor (Pall life sciences) in PBST solution; diluting the antibody and conjugate to be detected by using PBST to reach an initial concentration of 5,000 nM, and performing double dilution until 7 concentration points are reached; binding, dissociation and open detection results in Data Analysis 11.0 software; and analyzing the results by selecting 1:1 mode and global fitting to obtain the binding rate, dissociation rate and affinity constant; and the results are shown in Table 11. Table 11 Detection of binding activity of antibodies and conjugates to Fc receptors antibody KD(M) CD16a CD32a CD32b CD64 FcRn 101A6HZm Not detected Not detected Not detected Not detected 1.66E-8 101A6HZm-A-05 Not detected Not detected Not detected Not detected 1.42E-8

The results showed that the mutant 101A6HZm and its conjugates did not bind to the Fc receptors CD16a, CD32a, CD32b and CD64 proteins, which may reduce nonspecific killing mediated by Fc receptors and improve drug safety. At the same time, 101A6HZm and its conjugates retained FcRn protein binding activity and did not affect the half-life of the drug. 9. In vivo efficacy detection of anti-human PTK7 antibody-drug conjugates in the HCC1806 model

Human breast cancer HCC1806 cells were cultured in monolayer in vitro, and the culture conditions included: 10% fetal bovine serum was added to RPMI 1640 medium, and the culture was carried out at 37°C in an incubator containing air with 5% CO2. 2×106 HCC1806 cells were subcutaneously inoculated into the right armpit of each mouse and suspended in 0.1 ml PBS. When the average tumor volume grew to about 100-150 mm3, mice with tumor volumes that were too small or too large were excluded, and the remaining mice were randomly divided into 5 groups of 5 mice each according to tumor volume and animal weight; drugs were administered IV once every two weeks for a total of two times (P0 and P18). Tumor volume and body weight were measured twice a week after administration, and the specific results are shown in Table 12 and Figures 6A-6B. Table 12 Analysis of the efficacy of antibody-drug conjugates against human breast cancer cells in the HCC1806 subcutaneous tumor-bearing mouse model (N=5) Drugs Dosage (mg/kg) V _P0 (mm ³ , mean ± SEM) _VP31 (mm ³ , mean ± SEM) TGI (%) P-value Physiological saline / 100.87±9.32 2086.11±202.17 / / hIgG1m-A-05 3 100.47±8.76 1470.03±187.50 31.01 0.0559 hIgG1m-A-05 10 102.18±9.99 979.31±147.87 55.82** 0.0040 101A6HZm-A-05 3 100.52±9.40 801.28±213.70 64.70** 0.0024 101A6HZm-A-05 10 102.08±9.06 489.36±83.95 80.49*** 0.0001

Note: T test *P<0.05, **P<0.01, ***P<0.001 means significant difference compared with the saline group.

The results showed that the 101A6HZm-A-05 group had significant drug efficacy at 3 mg/kg and 10 mg/kg, and the anti-tumor effect was significantly better than the anti-tumor effect of the hIgG1m-A-05 group at the same dose. 10. In vivo efficacy detection of anti-human PTK7 antibody-drug conjugate in the OVCAR3 model

Human ovarian cancer OVCAR3 cells were cultured in monolayer in vitro, and the culture conditions included: 20% fetal bovine serum was added to RPMI1640 medium, and the culture was carried out at 37°C in an incubator containing air with 5% CO2. 5×106 OVCAR3 cells were subcutaneously inoculated into the right armpit of each mouse and suspended in 0.05 ml PBS + 0.05 ml matrigel. When the average tumor volume grew to about 100-150 mm3, mice with tumor volumes that were too small or too large were excluded, and the remaining mice were randomly divided into 5 groups of 5 mice each according to tumor volume and animal weight; and the drug was given IV at a single dose. Tumor volume and body weight were measured twice a week after administration, and the specific results are shown in Table 13 and Figures 7A-7B. Table 13 Analysis of the efficacy of antibody-drug conjugates against human ovarian cancer cells in the OVCAR3 subcutaneous tumor-bearing mouse model (N=5) Drugs Dosage (mg/kg) V _P0 (mm ³ , mean ± SEM) _VP36 (mm ³ , mean ± SEM) TGI (%) P -value Tumor remission Physiological saline / 131.40±9.19 2081.67±316.61 / / without hIgG1m-A-05 3 130.66±11.60 1550.40±281.78 27.20 0.2454 without hIgG1m-A-05 10 132.09±9.09 256.56±21.21 93.62*** 0.0004 without 101A6HZm-A-05 3 129.39±11.41 39.57±10.89 169.41*** 0.0002 1/5CR, 4/5PR 101A6HZm-A-05 10 129.93±8.95 45.53±3.11 164.96*** 0.0002 5/5PR

Note: T test **P<0.01, ***P<0.001 means significant difference compared with the saline group.

The results showed that the 101A6HZm-A-05 group had significant drug efficacy at 3 mg/kg and 10 mg/kg, and the anti-tumor effect was significantly better than the anti-tumor effect of the hIgG1m-A-05 group at the same dose. According to tumor regression, under the condition of 3 mg/kg 101A6HZm-A-05, 1 of 5 mice had a complete response (CR) and 4 had a partial response (PR); and under the condition of 101A6HZm-A-05 at 10 mg/kg, all 5 mice achieved a partial response (PR). 11. In vivo efficacy detection of anti-human PTK7 antibody-drug conjugates in the NCI-H146 model

Human small cell carcinoma NCI-H146 cells were cultured in vitro as monolayers, and the culture conditions included: 10% fetal bovine serum was added to RPMI 1640 medium, and the culture was carried out at 37°C in an incubator containing air with 5% CO2. 5×106 NCI-H146 cells were subcutaneously inoculated into the right armpit of each mouse and suspended in 0.05 ml PBS + 0.05 ml matrigel. When the average tumor volume grew to about 150-200 mm3, mice with tumor volumes that were too small or too large were excluded, and the remaining mice were randomly divided into 7 groups according to tumor volume and animal weight, and the drug was given at a single dose. Tumor volume and body weight were measured twice a week after administration, and the specific results are shown in Table 14 and Figures 8A-B. Table 14 Analysis of the efficacy of antibody-drug conjugates against human small cell carcinoma cells in the NCI-H146 subcutaneous tumor-bearing mouse model (N=5) Drugs Dosage (mg/kg) V _P0 (mm ³ , mean ± SEM) _VP35 (mm ³ , mean ± SEM) TGI (%) P-value Physiological saline / 153.66±5.36 1695.16±219.94 / / hIgG1m-A-05 3 153.83±5.18 821.17±77.32 56.71 0.0056 101A6HZm-A-05 1 154.53±7.22 244.21±17.27 94.18*** 0.0002 101A6HZm-A-05 3 154.52±5.49 10.42±3.59 193.26*** 0.0001 Hu24-C-01 1 154.70±5.60 1440.67±136.78 16.58 0.3885 Hu24-C-01 3 154.85±5.10 1412.09±155.26 18.44 0.3525 101A6HZm-A-26 3 154.04±9.57 17.87±2.13 188.40*** 0.0001

Note: T test **P<0.01, ***P<0.001 means significant difference compared with the saline group.

The results showed that the 101A6HZm-A-05 group had significant drug efficacy at 1 mg/kg and 3 mg/kg, and the anti-tumor effect was significantly better than the anti-tumor effect of the hIgG1m-A-05 group and Hu24-C-01 group at the same dose. 101A6HZm-A-05 and 101A6HZm-A-26 had similar drug efficacy at the same dose. At 3 mg/kg of 101A6HZm-A-05 and 3 mg/kg of 101A6HZm-A-26, all 5 mice administered achieved partial response (PR).

Although specific embodiments of the present invention have been described in detail, those skilled in the art should understand that various modifications and substitutions can be made to the 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 attached patent application scope and any equivalent scope.

Figure 1A: Detection of the potency of different antibody-drug conjugates in the NCI-H358 model. Figure 1B: Detection of the weight of different antibody-drug conjugates in the NCI-H358 model. Figure 2A: Detection of the potency of different antibody-drug conjugates in the A431 model. Figure 2B: Detection of the weight of different antibody-drug conjugates in the A431 model. Figure 3A: Detection of the binding of anti-human PTK7 antibody-drug conjugates to OVCAR3 cells. Figure 3B: Detection of the binding of anti-human PTK7 antibody-drug conjugates to NCI-H358 cells. Figure 3C: Detection of the binding of anti-human PTK7 antibody-drug conjugates to HCC1806 cells. Figure 3D: Detection of binding of anti-human PTK7 antibody-drug conjugate to A431 cells. Figure 3E: Detection of binding of anti-human PTK7 antibody-drug conjugate to NCI-H520 cells. Figure 3F: Detection of binding of anti-human PTK7 antibody-drug conjugate to DU4475 cells. Figure 4A: pHrodo detection of endocytosis of antibody-drug conjugate in NCI-H358. Figure 4B: pHrodo detection of endocytosis of antibody-drug conjugate in HCC1806. Figure 4C: pHrodo detection of endocytosis of antibody-drug conjugate in OVCAR3. Figure 4D: pHrodo detection of endocytosis of antibody-drug conjugate in NCI-H520. Figure 4E: pHrodo detection of endocytosis of antibody-drug conjugates in DU4475. Figure 5A: Detection of anti-human PTK7 conjugates in FADU cell killing. Figure 5B: Detection of anti-human PTK7 conjugates in HCC1806 cell killing. Figure 5C: Detection of anti-human PTK7 conjugates in DU4475 cell killing. Figure 6A: Detection of the potency of antibody-drug conjugates in the HCC1806 model. Figure 6B: Detection of the weight detection of antibody-drug conjugates in the HCC1806 model. Figure 7A: Detection of the potency of antibody-drug conjugates in the OVCAR3 model. Figure 7B: Weight detection of antibody-drug conjugates in the OVCAR3 model. Figure 8A: Detection of the efficacy of antibody-drug conjugates in the NCI-H146 model. Figure 8B: Weight detection of antibody-drug conjugates in the NCI-H146 model.

TW202421204A_112139032_SEQL.xml

Claims (73)

An antibody-drug conjugate having a structure shown as the formula Ab-[MLED] _x , wherein: Ab is an antibody or an antigen-binding fragment thereof that specifically binds to human PTK7; M is a linker site linked to the antibody or an antigen-binding fragment thereof; L is a linker between M and E; E is a structural fragment connecting L and D; D is a cytotoxic drug fragment; and x is selected from 1 to 10. The antibody-drug conjugate of claim 1, 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 Chothia numbering system: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence shown as SEQ ID NO: 11 or a variant thereof, CDR-H2 having a sequence shown as SEQ ID NO: 12 or a variant thereof, and CDR-H3 having a sequence shown as SEQ ID NO: 13 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having a sequence shown as SEQ ID NO: 14 or a variant thereof, CDR-L2 having a sequence shown as SEQ ID NO: 15 or a variant thereof, CDR-L3 having a sequence shown as SEQ ID NO: 16 or a variant thereof. 15 or a variant thereof and a CDR-L3 or a variant thereof having a sequence shown as SEQ ID NO: 16; or (1b) a heavy chain variable region (VH) comprising the following three CDRs: a CDR-H1 or a variant thereof having a sequence shown as SEQ ID NO: 27, a CDR-H2 or a variant thereof having a sequence shown as SEQ ID NO: 28, and a CDR-H3 or a variant thereof having a sequence shown as SEQ ID NO: 29; and/or a light chain variable region (VL) comprising the following three CDRs: a CDR-L1 or a variant thereof having a sequence shown as SEQ ID NO: 30, a CDR-L2 or a variant thereof having a sequence shown as SEQ ID NO: 31, and a CDR-L3 or a variant thereof having a sequence shown as SEQ ID NO: 32 or its variants; wherein the variants of any of (1a) and (1b) have at least 70%, 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 the variants are derived, or have undergone substitution, deletion or addition of one or more amino acids compared to the sequence from which the variants are derived; or (2) 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: (2a) a heavy chain variable region (VH) comprising the following 3 CDRs: having a sequence shown as SEQ ID NO: 17 or its variant, CDR-H2 or its variant having a sequence shown as SEQ ID NO: 18 or 19, and CDR-H3 or its variant having a sequence shown as SEQ ID NO: 13; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or its variant having a sequence shown as SEQ ID NO: 14, CDR-L2 or its variant having a sequence shown as SEQ ID NO: 15, and CDR-L3 or its variant having a sequence shown as SEQ ID NO: 16; or (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or its variant having a sequence shown as SEQ ID NO: 33, CDR-H2 or its variant having a sequence shown as SEQ ID NO: 34 or 35 or its variant and CDR-H2 or its variant having the sequence shown as SEQ ID NO: 29 and CDR-H3 or its variant; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or its variant having the sequence shown as SEQ ID NO: 30, CDR-L2 or its variant having the sequence shown as SEQ ID NO: 31 and CDR-L3 or its variant having the sequence shown as SEQ ID NO: 32; wherein the variants of any of (2a) and (2b) have at least 70%, 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 the variants are derived, or undergo substitution, deletion or addition of one or more amino acids compared to the sequence from which the variants are derived; or (3) 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: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having a sequence shown as SEQ ID NO: 20 or a variant thereof, having a sequence shown as SEQ ID NO: 21 or a variant thereof and a CDR-H3 or a variant thereof having a sequence shown as SEQ ID NO: 22; and/or a light chain variable region (VL) comprising the following three CDRs: a CDR-L1 or a variant thereof having a sequence shown as SEQ ID NO: 23, a CDR-L2 or a variant thereof having a sequence shown as SEQ ID NO: 24, and a CDR-L3 or a variant thereof having a sequence shown as SEQ ID NO: 16; or (3b) a heavy chain variable region (VH) comprising the following three CDRs: a CDR-H1 or a variant thereof having a sequence shown as SEQ ID NO: 36, a CDR-H2 or a variant thereof having a sequence shown as SEQ ID NO: 37, and a CDR-H3 or a variant thereof having a sequence shown as SEQ ID NO: 38 or its variant; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or its variant having a sequence shown as SEQ ID NO: 39, CDR-L2 or its variant having a sequence shown as SEQ ID NO: 40, and CDR-L3 or its variant having a sequence shown as SEQ ID NO: 32; wherein the variants of any of (3a) and (3b) have at least 70%, 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 the variants are derived, or undergo substitution, deletion or addition of one or more amino acids compared to the sequence from which the variants are derived; 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 having a sequence shown as SEQ ID NO: 25 or a variant thereof, CDR-H2 having a sequence shown as SEQ ID NO: 30 or a variant thereof, CDR-H3 having a sequence shown as SEQ ID NO: 40 or a variant thereof, CDR-H4 having a sequence shown as SEQ ID NO: 41 or a variant thereof, CDR-H5 having a sequence shown as SEQ ID NO: 43 or a variant thereof, CDR-H6 having a sequence shown as SEQ ID NO: 47 or a variant thereof, CDR-H7 having a sequence shown as SEQ ID NO: 48 or a variant thereof, CDR-H8 having a sequence shown as SEQ ID NO: 26 or a variant thereof and a CDR-H3 or a variant thereof having a sequence shown as SEQ ID NO: 13; and/or a light chain variable region (VL) comprising the following three CDRs: a CDR-L1 or a variant thereof having a sequence shown as SEQ ID NO: 14, a CDR-L2 or a variant thereof having a sequence shown as SEQ ID NO: 15, and a CDR-L3 or a variant thereof having a sequence shown as SEQ ID NO: 16; or (4b) a heavy chain variable region (VH) comprising the following three CDRs: a CDR-H1 or a variant thereof having a sequence shown as SEQ ID NO: 41, a CDR-H2 or a variant thereof having a sequence shown as SEQ ID NO: 42, and a CDR-H3 or a variant thereof having a sequence shown as SEQ ID NO: 29 or its variants; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or its variants having a sequence shown as SEQ ID NO: 30, CDR-L2 or its variants having a sequence shown as SEQ ID NO: 31, and CDR-L3 or its variants having a sequence shown as SEQ ID NO: 32; The variants of any one of (4a) and (4b) have at least 70%, 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 the variants are derived, or have undergone substitution, deletion or addition of one or more amino acids compared to the sequence from which the variants are derived. The antibody-drug conjugate of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: (a) VH or a variant thereof represented by SEQ ID NO: 1, and/or VL or a variant thereof represented by SEQ ID NO: 2; (b) VH or a variant thereof represented by SEQ ID NO: 3, and/or VL or a variant thereof represented by SEQ ID NO: 4; (c) VH or a variant thereof represented by SEQ ID NO: 5, and/or VL or a variant thereof represented by SEQ ID NO: 6; (d) VH or a variant thereof represented by SEQ ID NO: 7, and/or VL or a variant thereof represented by SEQ ID NO: 8; or (e) VH or a variant thereof represented by SEQ ID NO: 9, and/or VL or a variant thereof represented by SEQ ID NO: 10 VL or its variants; wherein the variants have at least 70%, 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 the variants are derived, or have undergone substitution, deletion or addition of one or more amino acids compared to the sequence from which the variants are derived. The antibody-drug conjugate of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof further comprises: (a) a human immunoglobulin heavy chain constant region (CH) or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which the variant is derived; and (b) a human immunoglobulin light chain constant region (CL) or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which the variant is derived. An antibody-drug conjugate as claimed in any one of claims 1 to 4, wherein the antibody or its antigen-binding fragment comprises: (1) a heavy chain comprising a VH sequence shown as SEQ ID NO: 1 and a heavy chain constant region (CH) shown as SEQ ID NO: 43, and a light chain comprising a VL sequence shown as SEQ ID NO: 2 and a light chain constant region (CL) shown as SEQ ID NO: 44; (2) a heavy chain comprising a VH sequence shown as SEQ ID NO: 3 and a heavy chain constant region (CH) shown as SEQ ID NO: 43 or 45, and a light chain comprising a VL sequence shown as SEQ ID NO: 4 and a light chain constant region (CL) shown as SEQ ID NO: 44; (3) a VH sequence shown as SEQ ID NO: 5 and a light chain constant region (CL) shown as SEQ ID NO: NO: 43, and a light chain comprising a VL sequence shown as SEQ ID NO: 6 and a light chain constant region (CL) shown as SEQ ID NO: 44; (4) a heavy chain comprising a VH sequence shown as SEQ ID NO: 7 and a heavy chain constant region (CH) shown as SEQ ID NO: 43, and a light chain comprising a VL sequence shown as SEQ ID NO: 8 and a light chain constant region (CL) shown as SEQ ID NO: 44; or (5) a heavy chain comprising a VH sequence shown as SEQ ID NO: 9 and a heavy chain constant region (CH) shown as SEQ ID NO: 43, and a VL sequence shown as SEQ ID NO: 10 and a light chain constant region (CL) shown as SEQ ID NO: The light chain of the light chain constant region (CL) of 44. The antibody-drug conjugate of any one of claims 1 to 5, wherein M is Ring A is a 5-6 membered aliphatic heterocyclic ring or a 5-20 membered aromatic ring system, and the aliphatic heterocyclic ring and the aromatic ring system are optionally substituted by one or more members selected from the group consisting of a pendooxy group (=O), a halogen, a cyano group, an amino group, a carboxyl group, a hydroxyl group, and a C _1-6 alkyl group; and M ₁ is selected from a single bond, a C _1-20 alkylene group, a C _2-20 alkenyl group, and a C _2-20 alkynyl group. The antibody-drug conjugate of any one of claims 1 to 5, wherein M is , wherein Ring A is a 5-membered aliphatic heterocyclic ring, a 6-membered aromatic heterocyclic ring, or a polycyclic ring formed by connecting one or more 6-membered aromatic heterocyclic rings and a benzene ring via a single bond, and the aliphatic heterocyclic ring is optionally substituted with one or more members selected from the group consisting of a pendooxy group (=O), a halogen, and a C _1-4 alkyl group; and M ₁ is selected from a single bond, a C _3-10 alkylene group, a C _3-10 alkenylene group, and a C _3-10 alkynylene group. The antibody-drug conjugate of any one of claims 1 to 5, wherein M is , wherein ring A is selected from , , and ; and M ₁ is selected from a single bond, a C _5-8 alkylene group, a C _5-8 alkenyl group and a C _5-8 alkynyl group. The antibody-drug conjugate of any one of claims 1 to 5, wherein M is selected from and . The antibody-drug conjugate of any one of claims 1 to 5, wherein M is . The antibody-drug conjugate of any one of claims 1 to 10, wherein L is selected from a structure comprising one or more of the following: C _1-6 alkylene, -N(R')-, carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH ₂ CH ₂ (OCH ₂ CH ₂ ) _s OCH ₃ ), D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly (SEQ ID NO: 48), Gly-Phe-Leu-Gly (SEQ ID NO: 49), Gly-Gly-Val-Ala (SEQ ID NO: 50), Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 51), , , , , , , , , , , and , wherein R' represents hydrogen, C _1-6 alkyl or alkyl containing -(CH ₂ CH ₂ O)r-; r is an integer selected from 1-10; and s is an integer selected from 1-20. The antibody-drug conjugate of any one of claims 1 to 10, wherein L is selected from a structure comprising one or more of the following: C _1-6 alkylene, -NH-, Val, Cit, Phe, Lys, Lys(COCH ₂ CH ₂ (OCH ₂ CH ₂ ) _s OCH ₃ ), Gly, Val-Cit, Gly-Gly-Phe-Gly (SEQ ID NO: 48), , , , , , , , and ; and s is an integer selected from 1-20. The antibody-drug conjugate of any one of claims 1 to 10, wherein L is selected from the following structures: , , , , , , , , , , , and ; s is an integer selected from 1-20. The antibody-drug conjugate of any one of claims 1 to 10, wherein L is selected from the following structures: , , , , and . The antibody-drug conjugate of any one of claims 1 to 10, wherein L is selected from the following structures: , , , and . The antibody-drug conjugate of any one of claims 1 to 15, wherein E is a single bond, -NH-CH ₂ -, -NH-CH ₂ -O-CH ₂ -CO-, , , , or . The antibody-drug conjugate of any one of claims 1 to 15, wherein E is a single bond, -NH-CH ₂ -, -NH-CH ₂ -O-CH ₂ -CO-, , or . The antibody-drug conjugate of any one of claims 1 to 10, wherein E is -NH-CH ₂ -, or . The antibody-drug conjugate of any one of claims 1 to 10, wherein E is -NH-CH ₂ - or . The antibody-drug conjugate of any one of claims 1 to 19, wherein Select from the following structures: , , , , , , , , , , , , , , , , and . The antibody-drug conjugate of any one of claims 1 to 20, wherein the cytotoxic drug is selected from tubulin inhibitors, DNA intercalators, DNA topoisomerase inhibitors and RNA polymerase inhibitors or pharmaceutically acceptable salts, esters or analogs thereof. The antibody-drug conjugate of claim 21, wherein the tubulin inhibitor is an auristatin compound or a maytansinoid compound. The antibody-drug conjugate of claim 21, wherein the DNA intercalator is pyrrolobenzodiazepine (PBD). The antibody-drug conjugate of claim 21, wherein the DNA topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor. The antibody-drug conjugate of claim 24, wherein the topoisomerase I inhibitor is camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan. The antibody-drug conjugate of claim 24, wherein the topoisomerase II inhibitor is doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide. The antibody-drug conjugate of claim 21, wherein the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester or analog thereof. The antibody-drug conjugate of claim 21, wherein the cytotoxic drug is . The antibody-drug conjugate of claim 21, wherein the cytotoxic drug is selected from compounds shown in Formula I and Formula II: , R ₁ and R ₂ are each independently selected from C _1-6 alkyl and halogen; R ₃ is selected from H and -CO-CH ₂ OH; R ₄ and R ₅ are each independently selected from H, halogen and hydroxyl; or R ₄ and R ₅ are connected to the relevant carbon atoms to form a 5-6 membered oxygen-containing heterocyclic ring; R ₆ is selected from hydrogen and -C _1-4 alkylene-NR _a R _b ; R ₇ is selected from hydrogen, C _1-6 alkyl and -C _1-4 alkylene-NR _a R _b ; and _Ra and R _b are each independently selected from H, C _1-6 alkyl, -SO ₂ -C _1-6 alkyl and -CO-C _1-6 alkyl at each occurrence. The antibody-drug conjugate of claim 21, wherein the cytotoxic drug is selected from the following compounds: and ; wherein the corresponding fragment of the cytotoxic drug obtained after the cytotoxic drug is linked to the linker is D as shown in the general formula. The antibody-drug conjugate of claim 30, wherein D is a monovalent structure obtained by losing one H from a -OH, _-NH2 or diamine group on the cytotoxic drug. The antibody-drug conjugate of any one of claims 1 to 31, which is selected from ADC A-01 to ADC A-26, ADC B-01 to ADC B-06 and ADC C01, as shown below: ADC A-01 、ADC A-02 、ADC A-03 、ADC A-04 、ADC A-05 、ADC A-06 、ADC A-07 、ADC A-08 、ADC A-09 、ADC A-10 、ADC A-11 、ADC A-12 、ADC A-13 、ADC A-14 、ADC A-15 、ADC A-16 、ADC A-17 、ADC A-18 、ADC A-19 、ADC A-20 、ADC A-21 、ADC A-22 、ADC A-23 、ADC A-24 、ADC A-25 、ADC A-26 ADC B-01 、ADC B-02 、ADC B-03 、ADC B-04 、ADC B-05 、ADC B-06 and ADC C-01 , wherein the HA in each antibody-drug conjugate represents an antibody or an antigen-binding fragment thereof comprising a VH shown as SEQ ID NO: 1, 3, 5, 7 or 9 and a VL shown as SEQ ID NO: 2, 4, 6, 8 or 10, and It refers to the bond formed by the sulfhydryl group in the antibody or antigen-binding fragment and the linker. The antibody-drug conjugate of any one of claims 1 to 32, wherein the antibody-drug conjugate comprises the formula: , , , or wherein the HA in each antibody-drug conjugate is selected from: (1) an antibody or an antigen-binding fragment thereof comprising a VH having an amino acid sequence as set forth in SEQ ID NO: 1 and a VL having an amino acid sequence as set forth in SEQ ID NO: 2, (2) an antibody or an antigen-binding fragment thereof comprising a VH having an amino acid sequence as set forth in SEQ ID NO: 3 and a VL having an amino acid sequence as set forth in SEQ ID NO: 4, (3) an antibody or an antigen-binding fragment thereof comprising a VH having an amino acid sequence as set forth in SEQ ID NO: 5 and a VL having an amino acid sequence as set forth in SEQ ID NO: 6, (4) an antibody or an antigen-binding fragment thereof comprising a VH having an amino acid sequence as set forth in SEQ ID NO: 7 and a VL having an amino acid sequence as set forth in SEQ ID NO: 8, and (5) an antibody or an antigen-binding fragment thereof comprising a VH having an amino acid sequence as set forth in SEQ ID NO: 9 and a VL having an amino acid sequence as set forth in SEQ ID NO: 10. 9 and a VH having the amino acid sequence as described in SEQ ID NO: 10 and a VL having the amino acid sequence as described in SEQ ID NO: 11 or an antigen-binding fragment thereof, It refers to the bond formed by the sulfhydryl groups in the antibody or antigen-binding fragment and the linker, wherein HA is linked via the sulfhydryl groups in the antibody or antigen-binding fragment to form the antibody-drug conjugate. The antibody-drug conjugate of any one of claims 1 to 32, wherein the antibody-drug conjugate comprises the formula: , , or The HA in each antibody-drug conjugate is selected from: (1) an antibody or antigen-binding fragment thereof comprising a VH having the amino acid sequence of SEQ ID NO: 1 and a CH having the amino acid sequence of SEQ ID NO: 43 or 45, a VL having the amino acid sequence of SEQ ID NO: 2 and a CL having the amino acid sequence of SEQ ID NO: 44; (2) an antibody or antigen-binding fragment thereof comprising a VH having the amino acid sequence of SEQ ID NO: 3 and a CH having the amino acid sequence of SEQ ID NO: 43 or 45, a VL having the amino acid sequence of SEQ ID NO: 4 and a CL having the amino acid sequence of SEQ ID NO: 44; (3) an antibody or antigen-binding fragment thereof comprising a VH having the amino acid sequence of SEQ ID NO: 5 and a CH having the amino acid sequence of SEQ ID NO: 43 or 45, a CH having the amino acid sequence described in SEQ ID NO: 6, and a VL having the amino acid sequence described in SEQ ID NO: 44, or an antigen-binding fragment thereof; (4) an antibody or an antigen-binding fragment thereof comprising a VH having the amino acid sequence described in SEQ ID NO: 7, a CH having the amino acid sequence described in SEQ ID NO: 43 or 45, a VL having the amino acid sequence described in SEQ ID NO: 8, and a CL having the amino acid sequence described in SEQ ID NO: 44; and (5) an antibody or an antigen-binding fragment thereof comprising a VH having the amino acid sequence described in SEQ ID NO: 9, a CH having the amino acid sequence described in SEQ ID NO: 43 or 45, a VL having the amino acid sequence described in SEQ ID NO: 10, and a CL having the amino acid sequence described in SEQ ID NO: An antibody or an antigen-binding fragment thereof against CL of the amino acid sequence described in 44; represents a bond formed by a sulfhydryl group in the antibody or antigen-binding fragment and the linker; wherein HA is linked via one or more sulfhydryl groups in the antibody or antigen-binding fragment to form the antibody-drug conjugate; and x is 3 to 9. The antibody-drug conjugate of any one of claims 1 to 34, wherein the antibody or its antigen-binding fragment comprises VH shown as SEQ ID NO: 3 and CH shown as SEQ ID NO: 43 or 45, VL shown as SEQ ID NO: 4 and CL shown as SEQ ID NO: 44. The antibody-drug conjugate of any one of claims 1 to 33, wherein the antibody-drug conjugate is: , wherein the HA in the antibody-drug conjugate comprises the following antibody or antigen-binding fragment thereof: a VH having the amino acid sequence as described in SEQ ID NO: 3 and a heavy chain constant region (CH) having the amino acid sequence as described in SEQ ID NO: 43 or 45, and a VL having the amino acid sequence as described in SEQ ID NO: 4 and a light chain constant region (CL) having the amino acid sequence as described in SEQ ID NO: 44; represents a bond formed by sulfhydryl groups in the antibody or antigen-binding fragment and the linker; wherein HA is linked via the sulfhydryl groups in the antibody or antigen-binding fragment to form the antibody-drug conjugate; and x is 7 to 9. The antibody-drug conjugate of any one of claims 1 to 33, wherein the antibody-drug conjugate is: , wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) having an amino acid sequence as set forth in SEQ ID NO: 46 and a light chain (LC) having an amino acid sequence as set forth in SEQ ID NO: 47; represents a bond formed by sulfhydryl groups in the antibody or antigen-binding fragment and the linker; wherein HA is linked via the sulfhydryl groups of cysteine in the antibody or antigen-binding fragment to form the antibody-drug conjugate; and x is 7 to 8. The antibody-drug conjugate of any one of claims 1 to 33, wherein the antibody-drug conjugate is: , wherein HA is an antibody comprising a heavy chain (HC) consisting of the amino acid sequence described in SEQ ID NO: 52, 53 or 54 and a light chain (LC) consisting of the amino acid sequence described in SEQ ID NO: 47; represents a bond formed by a sulfhydryl group in the antibody or antigen-binding fragment and the linker; wherein HA is linked via the sulfhydryl groups of the cysteine amino acids in the antibody or antigen-binding fragment to form the antibody-drug conjugate; and x is 7 to 8. The antibody-drug conjugate of any one of claims 1 to 33, wherein the antibody-drug conjugate is: , wherein HA is an antibody consisting of two heavy chains and two light chains, each heavy chain (HC) consists of the amino acid sequence as described in SEQ ID NO: 52 and each light chain (LC) consists of the amino acid sequence as described in SEQ ID NO: 47; and represents a bond formed by a sulfhydryl group in the antibody or antigen-binding fragment and the linker; wherein HA is linked via the sulfhydryl groups of the cysteine amino acids in the antibody or antigen-binding fragment to form the antibody-drug conjugate; and x is 7 to 8. The antibody-drug conjugate of any one of claims 1 to 33, wherein the antibody-drug conjugate is: , wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) having the amino acid sequence as described in SEQ ID NO: 46 and a light chain (LC) having the amino acid sequence as described in SEQ ID NO: 47; represents a bond formed by a sulfhydryl group in the antibody or antigen-binding fragment and the linker; wherein HA is linked via the sulfhydryl groups of the cysteine amino acids in the antibody or antigen-binding fragment to form the antibody-drug conjugate; and x is 8. The antibody-drug conjugate of any one of claims 1 to 33, wherein the antibody-drug conjugate is: Wherein the HA in the antibody-drug conjugate comprises the following antibody or antigen-binding fragment thereof: VH having the amino acid sequence as described in SEQ ID NO: 3 and a heavy chain constant region (CH) having the amino acid sequence as described in SEQ ID NO: 43 or 45, and VL having the amino acid sequence as described in SEQ ID NO: 4 and a light chain constant region (CL) having the amino acid sequence as described in SEQ ID NO: 44; represents a bond formed by sulfhydryl groups in the antibody or antigen-binding fragment and the linker; wherein HA is linked via the sulfhydryl groups in the antibody or antigen-binding fragment to form the antibody-drug conjugate; and x is 7 to 9. The antibody-drug conjugate of any one of claims 1 to 33, wherein the antibody-drug conjugate is: , wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) having the amino acid sequence as described in SEQ ID NO: 46 and a light chain (LC) having the amino acid sequence as described in SEQ ID NO: 47; represents a bond formed by sulfhydryl groups in the antibody or antigen-binding fragment and the linker; wherein HA is linked via the sulfhydryl groups in the antibody or antigen-binding fragment to form the antibody-drug conjugate; and x is 7 to 8. The antibody-drug conjugate of any one of claims 1 to 33, wherein the antibody-drug conjugate is: wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) having the amino acid sequence as described in SEQ ID NO: 46 and a light chain (LC) having the amino acid sequence as described in SEQ ID NO: 47; represents a bond formed by sulfhydryl groups in the antibody or antigen-binding fragment and the linker; wherein HA is linked via the sulfhydryl groups in the antibody or antigen-binding fragment to form the antibody-drug conjugate; and x is 8. The antibody-drug conjugate of any one of claims 1 to 33, wherein the antibody-drug conjugate is: , wherein the HA in the antibody-drug conjugate comprises the following antibody or antigen-binding fragment thereof: a VH having the amino acid sequence as described in SEQ ID NO: 3 and a heavy chain constant region (CH) having the amino acid sequence as described in SEQ ID NO: 43 or 45, and a VL having the amino acid sequence as described in SEQ ID NO: 4 and a light chain constant region (CL) having the amino acid sequence as described in SEQ ID NO: 44; represents a bond formed by sulfhydryl groups in the antibody or antigen-binding fragment and the linker; wherein HA is linked via the sulfhydryl groups in the antibody or antigen-binding fragment to form the antibody-drug conjugate; and x is 7 to 9. The antibody-drug conjugate of any one of claims 1 to 33, wherein the antibody-drug conjugate is: , wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) having the amino acid sequence as described in SEQ ID NO: 46 and a light chain (LC) having the amino acid sequence as described in SEQ ID NO: 47; represents a bond formed by sulfhydryl groups in the antibody or antigen-binding fragment and the linker; wherein HA is linked via the sulfhydryl groups in the antibody or antigen-binding fragment to form the antibody-drug conjugate; and x is 7 to 8. The antibody-drug conjugate of any one of claims 1 to 33, wherein the antibody-drug conjugate is: , wherein HA is an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) having the amino acid sequence as described in SEQ ID NO: 46 and a light chain (LC) having the amino acid sequence as described in SEQ ID NO: 47; represents a bond formed by sulfhydryl groups in the antibody or antigen-binding fragment and the linker; wherein HA is linked via the sulfhydryl groups in the antibody or antigen-binding fragment to form the antibody-drug conjugate; and x is 8. A composition comprising one or more antibody-drug conjugates of any one of claims 1 to 33, wherein the DAR (drug-antibody binding ratio) of the composition is 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. An antibody-drug conjugate as claimed in any one of claims 1 to 47, wherein (i) the C-terminus of the heavy chain lacks a lysine residue; (ii) the N-terminus of the heavy chain is glutamic acid, glutamine, pyroglutamic acid salt or pyroglutamic acid; or (iii) the C-terminus of the heavy chain lacks a lysine residue and the N-terminus of the heavy chain is glutamic acid, glutamine, pyroglutamic acid salt or pyroglutamic acid. The antibody-drug conjugate of any one of claims 1 to 46 and 48, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising a VH having the amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7 and 9. An antibody-drug conjugate as claimed in any one of claims 1 to 46, 48 and 49, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising a VH having the amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7 and 9, wherein the N-terminal glutamic acid or glutamine of the antibody variable region has been cyclized to pyroglutamic acid salt or pyroglutamic acid. The antibody-drug conjugate of any one of claims 1 to 46 and 48 to 50, wherein the antibody or antigen-binding fragment comprises a light chain comprising a VL having the amino acid sequence as described in SEQ ID NO: 8, wherein the N-terminal glutamine of the variable region of the antibody light chain has been cyclized to pyroglutamic acid salt or pyroglutamic acid. The antibody-drug conjugate of any one of claims 1 to 46 and 48 to 51, wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain consisting of the sequence shown as SEQ ID NO: 52 and a light chain consisting of the sequence shown as SEQ ID NO: 47; (b) a heavy chain consisting of the sequence shown as SEQ ID NO: 53 and a light chain consisting of the sequence shown as SEQ ID NO: 47; or (c) a heavy chain consisting of the sequence shown as SEQ ID NO: 54 and a light chain consisting of the sequence shown as SEQ ID NO: 47. An antibody-drug conjugate as claimed in any one of claims 1 to 46 and 48 to 52, wherein the antibody or antigen-binding fragment can be obtained by expressing a nucleic acid molecule encoding the heavy chain and the light chain of the antibody or antigen-binding fragment thereof in a host cell, wherein the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising a VH sequence shown as SEQ ID NO: 1 and a heavy chain constant region (CH) shown as SEQ ID NO: 43, and a light chain comprising a VL sequence shown as SEQ ID NO: 2 and a light chain constant region (CL) shown as SEQ ID NO: 44; (2) a heavy chain comprising a VH sequence shown as SEQ ID NO: 3 and a heavy chain constant region (CH) shown as SEQ ID NO: 43 or 45, and a light chain comprising a VL sequence shown as SEQ ID NO: 44 or 45; 4 and a light chain having a VL sequence of SEQ ID NO: 44 and a light chain having a constant region (CL) of SEQ ID NO: 44; (3) a heavy chain having a VH sequence of SEQ ID NO: 5 and a heavy chain having a constant region (CH) of SEQ ID NO: 43, and a light chain having a VL sequence of SEQ ID NO: 6 and a light chain having a constant region (CL) of SEQ ID NO: 44; (4) a heavy chain having a VH sequence of SEQ ID NO: 7 and a heavy chain having a constant region (CH) of SEQ ID NO: 43, and a light chain having a VL sequence of SEQ ID NO: 8 and a light chain having a constant region (CL) of SEQ ID NO: 44; (5) a heavy chain having a VH sequence of SEQ ID NO: 7 and a heavy chain having a constant region (CH) of SEQ ID NO: 43, and a light chain having a VL sequence of SEQ ID NO: 8 and a light chain having a constant region (CL) of SEQ ID NO: 44; 9 and a heavy chain having a VH sequence as described in SEQ ID NO: 43 and a heavy chain having a heavy chain constant region (CH) as described in SEQ ID NO: 43, and a light chain having a VL sequence as described in SEQ ID NO: 10 and a light chain having a light chain constant region (CL) as described in SEQ ID NO: 44; (6) a heavy chain (HC) having the amino acid sequence as described in SEQ ID NO: 46 and a light chain (LC) having the amino acid sequence as described in SEQ ID NO: 47; (7) a VH having the amino acid sequence as described in SEQ ID NO: 1 and a VL having the amino acid sequence as described in SEQ ID NO: 2; (8) a VH having the amino acid sequence as described in SEQ ID NO: 3 and a VL having the amino acid sequence as described in SEQ ID NO: 4; (9) a VH having the amino acid sequence as described in SEQ ID NO: 5 and a VH having the amino acid sequence described in SEQ ID NO: 6; (10) a VH having the amino acid sequence described in SEQ ID NO: 7 and a VL having the amino acid sequence described in SEQ ID NO: 8; or (11) a VH having the amino acid sequence described in SEQ ID NO: 9 and a VL having the amino acid sequence described in SEQ ID NO: 10. An antibody-drug conjugate comprising an antibody that binds to PTK7, the antibody being conjugated to a drug linker having a structure shown as formula MLED via one or more cysteine or lysine residues or atypical amino acid substitutions of the amino acids of the antibody, wherein M is , wherein Ring A is a 5-6 membered aliphatic heterocyclic ring or a 5-20 membered aromatic ring system, and the aliphatic heterocyclic ring and the aromatic ring system are optionally substituted by one or more members selected from the group consisting of: oxo (=O), halogen, cyano, amine, carboxyl, alkyl and C _1-6 alkyl; _M1 is selected from a single bond, C _1-20 alkylene, C _2-20 alkenyl and C _2-20 alkynyl; L is a linker between M and E; E is a structural fragment connecting L and D; and D is a cytotoxic drug fragment. The antibody-drug conjugate of claim 54, wherein Selected from , , , , , , , , , , , , and . The antibody-drug conjugate of claim 54 or 55, wherein the cytotoxic drug is selected from the following compounds: and ; wherein the corresponding fragment of the cytotoxic drug obtained after the cytotoxic drug is linked to the linker is D as shown in the general formula. The antibody-drug conjugate of claim 54 or 55, wherein the antibody that binds to PTK7 is selected from the group consisting of: (1) 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: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence as described in SEQ ID NO: 11 or a variant thereof, CDR-H2 having an amino acid sequence as described in SEQ ID NO: 12 or a variant thereof, and CDR-H3 having an amino acid sequence as described in SEQ ID NO: 13 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-H1 having an amino acid sequence as described in SEQ ID NO: 11 or a variant thereof, CDR-H2 having an amino acid sequence as described in SEQ ID NO: 12 or a variant thereof, and CDR-H3 having an amino acid sequence as described in SEQ ID NO: 13 or a variant thereof; 14 or a variant thereof, CDR-L1 having an amino acid sequence as described in SEQ ID NO: 15 or a variant thereof, and CDR-L3 having an amino acid sequence as described in SEQ ID NO: 16 or a variant thereof; or (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence as described in SEQ ID NO: 27 or a variant thereof, CDR-H2 having an amino acid sequence as described in SEQ ID NO: 28 or a variant thereof, and CDR-H3 having an amino acid sequence as described in SEQ ID NO: 29 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-H1 having an amino acid sequence as described in SEQ ID NO: 27 or a variant thereof, CDR-H2 having an amino acid sequence as described in SEQ ID NO: 28 or a variant thereof, and CDR-H3 having an amino acid sequence as described in SEQ ID NO: 29 or a variant thereof. 30, or a CDR-L1 or a variant thereof having an amino acid sequence as described in SEQ ID NO: 30, a CDR-L2 or a variant thereof having an amino acid sequence as described in SEQ ID NO: 31, and a CDR-L3 or a variant thereof having an amino acid sequence as described in SEQ ID NO: 32; wherein the variants of any of (1a) and (1b) have at least 70%, 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 the variants are derived, or have undergone substitution, deletion or addition of one or more amino acids compared to the sequence from which the variants are derived; or (2) 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: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence as described in SEQ ID NO: 17 or a variant thereof, CDR-H2 having an amino acid sequence as described in SEQ ID NO: 18 or 19 or a variant thereof, CDR-H3 having an amino acid sequence as described in SEQ ID NO: 13 or a variant thereof; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having an amino acid sequence as described in SEQ ID NO: 14 or a variant thereof, CDR-L2 having an amino acid sequence as described in SEQ ID NO: 15 or a variant thereof and CDR-L3 or a variant thereof having the amino acid sequence described in SEQ ID NO: 16; or (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the amino acid sequence described in SEQ ID NO: 33, CDR-H2 or a variant thereof having the amino acid sequence described in SEQ ID NO: 34 or 35, and CDR-H3 or a variant thereof having the amino acid sequence described in SEQ ID NO: 29; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the amino acid sequence described in SEQ ID NO: 30, CDR-H2 or a variant thereof having the amino acid sequence described in SEQ ID NO: 31 or a variant thereof and a CDR-L3 or a variant thereof having the amino acid sequence as described in SEQ ID NO: 32; wherein the variants of any one of (2a) and (2b) have at least 70%, 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 the variants are derived, or have undergone substitution, deletion or addition of one or more amino acids compared to the sequence from which the variants are derived; or (3) 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: (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having an amino acid sequence as described in SEQ ID NO: 20, CDR-H2 or a variant thereof having an amino acid sequence as described in SEQ ID NO: 21, and CDR-H3 or a variant thereof having an amino acid sequence as described in SEQ ID NO: 22; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having an amino acid sequence as described in SEQ ID NO: 23, CDR-L2 or a variant thereof having an amino acid sequence as described in SEQ ID NO: 24, and CDR-L3 or a variant thereof having the amino acid sequence as described in SEQ ID NO: 16; or (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having an amino acid sequence as described in SEQ ID NO: 20, CDR-H2 or a variant thereof having an amino acid sequence as described in SEQ ID NO: 21, and CDR-H3 or a variant thereof having an amino acid sequence as described in SEQ ID NO: 22; CDR-H1 or its variant having the amino acid sequence described in SEQ ID NO: 36, CDR-H2 or its variant having the amino acid sequence described in SEQ ID NO: 37, and CDR-H3 or its variant having the amino acid sequence described in SEQ ID NO: 38; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or its variant having the amino acid sequence described in SEQ ID NO: 39, CDR-L2 or its variant having the amino acid sequence described in SEQ ID NO: 40, and CDR-L3 or its variant having the amino acid sequence described in SEQ ID NO: 32; wherein the variants of any of (3a) and (3b) have at least 70%, 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 the variants are derived, or undergo substitution, deletion or addition of one or more amino acids compared to the sequence from which the variants are derived; and (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 having an amino acid sequence as described in SEQ ID NO: 25 or a variant thereof, CDR-H2 having an amino acid sequence as described in SEQ ID NO: 26 or a variant thereof and CDR-H3 or a variant thereof having the amino acid sequence described in SEQ ID NO: 13; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or a variant thereof having the amino acid sequence described in SEQ ID NO: 14, CDR-L2 or a variant thereof having the amino acid sequence described in SEQ ID NO: 15, and CDR-L3 or a variant thereof having the amino acid sequence described in SEQ ID NO: 16; or (4b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 or a variant thereof having the amino acid sequence described in SEQ ID NO: 41, CDR-H2 or a variant thereof having the amino acid sequence described in SEQ ID NO: 42, CDR-H3 or a variant thereof having the amino acid sequence described in SEQ ID NO: 42 or its variants and CDR-H3 or its variants having the amino acid sequence as described in SEQ ID NO: 29; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 or its variants having the amino acid sequence as described in SEQ ID NO: 30, CDR-L2 or its variants having the amino acid sequence as described in SEQ ID NO: 31, and CDR-L3 or its variants having the amino acid sequence as described in SEQ ID NO: 32; The variants of any one of (4a) and (4b) have at least 70%, 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 the variants are derived, or have undergone substitution, deletion or addition of one or more amino acids compared to the sequence from which the variants are derived. The antibody-drug conjugate of claim 57, wherein the antibody that binds to PTK7 is selected from the group consisting of: (a) a VH or a variant thereof having an amino acid sequence as described in SEQ ID NO: 1, and/or a VL or a variant thereof having an amino acid sequence as described in SEQ ID NO: 2; (b) a VH or a variant thereof having an amino acid sequence as described in SEQ ID NO: 3, and/or a VL or a variant thereof having an amino acid sequence as described in SEQ ID NO: 4; (c) a VH or a variant thereof having an amino acid sequence as described in SEQ ID NO: 5, and/or a VL or a variant thereof having an amino acid sequence as described in SEQ ID NO: 6; (d) a VH or a variant thereof having an amino acid sequence as described in SEQ ID NO: 7, and/or a VL or a variant thereof having an amino acid sequence as described in SEQ ID NO: 8 or a variant thereof; and (e) a VH or a variant thereof having an amino acid sequence as described in SEQ ID NO: 9, and/or a VL or a variant thereof having an amino acid sequence as described in SEQ ID NO: 10; wherein the variants have at least 70%, 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 the variants are derived, or have undergone substitution, deletion or addition of one or more amino acids compared to the sequence from which the variants are derived. The antibody-drug conjugate of claim 54 or 55, wherein the antibody that binds to PTK7 is selected from the group consisting of: (1) a heavy chain comprising a VH having the amino acid sequence described in SEQ ID NO: 1 and a heavy chain constant region (CH) shown as SEQ ID NO: 43, and a light chain comprising a VL having the amino acid sequence described in SEQ ID NO: 2 and a light chain constant region (CL) having the amino acid sequence described in SEQ ID NO: 44; (2) a heavy chain comprising a VH having the amino acid sequence described in SEQ ID NO: 3 and a heavy chain constant region (CH) having the amino acid sequence described in SEQ ID NO: 43 or 45, and a VL having the amino acid sequence described in SEQ ID NO: 4 and a light chain constant region (CL) having the amino acid sequence described in SEQ ID NO: 44; (3) a light chain comprising a VH having an amino acid sequence as described in SEQ ID NO: 5 and a heavy chain constant region (CH) having the amino acid sequence as described in SEQ ID NO: 43, and a light chain comprising a VL having an amino acid sequence as described in SEQ ID NO: 6 and a light chain constant region (CL) having the amino acid sequence as described in SEQ ID NO: 44; (4) a heavy chain comprising a VH having an amino acid sequence as described in SEQ ID NO: 7 and a heavy chain constant region (CH) having the amino acid sequence as described in SEQ ID NO: 43, and a heavy chain comprising a VL having an amino acid sequence as described in SEQ ID NO: 8 and a light chain constant region (CL) having the amino acid sequence as described in SEQ ID NO: 44; and (5) a light chain comprising a VH sequence having an amino acid sequence as described in SEQ ID NO: 9 and a heavy chain having a heavy chain constant region (CH) having the amino acid sequence as described in SEQ ID NO: 43, and a light chain comprising a VL sequence having an amino acid sequence as described in SEQ ID NO: 10 and a light chain constant region (CL) having the amino acid sequence as described in SEQ ID NO: 44. The antibody-drug conjugate of claim 54 or 55, wherein the antibody that binds to PTK7 comprises: a heavy chain constant region (CH) having the amino acid sequence as described in SEQ ID NO: 43 or 45, and a light chain constant region (CL) having the amino acid sequence as described in SEQ ID NO: 44. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 60 and one or more pharmaceutically acceptable excipients. A method for treating cancer in an individual with high PTK7 expression, comprising administering to the individual a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1 to 60 or the pharmaceutical composition of claim 61. The method of claim 62, wherein the cancer comprises a solid tumor or a hematological malignancy. The method of claim 63, wherein the cancer is lung cancer, breast cancer, epithelial cancer, ovarian cancer, or esophageal cancer. A use of the antibody-drug conjugate of any one of claims 1 to 60 or the pharmaceutical composition of claim 61 for preparing a medicament for treating cancer with high PTK7 expression. The use of claim 65, wherein the cancer comprises a solid tumor or a hematological malignancy. The use of claim 65, wherein the cancer with high PTK7 expression is lung cancer, breast cancer, epithelial cancer, ovarian cancer and esophageal cancer. A use of the antibody-drug conjugate of any one of claims 1 to 60 or the pharmaceutical composition of claim 61 for treating cancer with high PTK7 expression. The use of claim 68, wherein the cancer comprises a solid tumor or a hematological malignancy. The use of claim 69, wherein the cancer with high PTK7 expression is lung cancer, breast cancer, epithelial cancer, ovarian cancer and esophageal cancer. The antibody-drug conjugate of any one of claims 1 to 60 or the pharmaceutical composition of claim 61, for use in treating cancer with high PTK7 expression. The antibody-drug conjugate of claim 71, wherein the cancer comprises a solid tumor or a hematological malignancy. The antibody-drug conjugate of claim 72, wherein the cancer is lung cancer, breast cancer, epithelial cancer, ovarian cancer, or esophageal cancer.