Antibody-drug conjugate and preparation method and application thereof

Abstract

The invention relates to an antibody-drug conjugate as well as a preparation method and application thereof, in particular to an antibody-drug conjugate for treating HER3 positive cancer. The antibody-drug conjugate contains a fully human-derived HER3 antibody, has excellent binding activity to HER3 positive cells, and can efficiently deliver a drug to the HER3 positive cells. The antibody-drug conjugate has a better drug-antibody coupling ratio, and has a very good targeted killing effect on colon cancer, gastric cancer, breast cancer and lung cancer (such as non-small cell lung cancer, particularly lung adenocarcinoma). Therefore, the invention further provides a preparation method of the antibody-drug conjugate and an application of the antibody-drug conjugate in treating HER3 positive cancer.

Description

Antibody-drug conjugates and preparation methods and uses thereof

[0001] This application is based on and claims priority to Chinese application No. 202311130165.6, filed on September 1, 2023, and Chinese application No. 202411158958.3, filed on August 22, 2024. The disclosed contents of the Chinese applications are incorporated herein by reference in their entirety. Technical Field

[0002] The present application relates to the field of targeted therapy, and specifically to antibody-drug conjugates and preparation methods and uses thereof. Background Art

[0003] Cancer is one of the leading causes of death today. It is a disease caused by the malignant transformation of healthy cells. Genetic alterations, such as chromosomal translocations and mutations in tumor suppressor genes and growth factor receptors, lead to malignant cell proliferation. Defective apoptosis, or programmed cell death, further promotes the malignant transformation that leads to cancer.

[0004] Human epidermal growth factor receptor 3 (also known as HER3 and ErbB3) is a receptor protein tyrosine kinase that belongs to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases, which also includes HER1 (also known as EGFR), HER2, and HER4. Similar to the EGFR, the transmembrane receptor HER3 is composed of a ligand-binding extracellular domain (ECD), a dimerization domain within the ECD, a transmembrane domain, and a carboxy-terminal phosphorylation domain. In addition to these domains, HER1, HER2, and HER4 also contain an intracellular protein tyrosine kinase domain (TKD), while HER3 lacks this domain and cannot autophosphorylate.

[0005] Ligand regulatory protein (HRG) binds to the extracellular domain of HER3 and activates receptor-mediated signal transduction pathways by promoting dimerization with other human epidermal growth factor receptor (HER) family members and transphosphorylation of their intracellular domains. Dimer formation of HER3 with other HER family members expands the signal transduction potential of HER3 and not only serves as a means of signal diversification, but also as a means of signal amplification. For example, HER2 / HER3 heterodimers induce one of the most important mitogenic signals in HER family members. HER3 is overexpressed in various types of cancers, such as breast cancer, gastrointestinal cancer, and pancreatic cancer. Interestingly, the relationship between the expression of HER2 / HER3 and the progression from the non-invasive stage to the invasive stage has been shown. Therefore, it is desirable to interfere with the signal transduction of HER3 mediation.

[0006] Currently, HER3-targeted indications under clinical investigation cover both hematologic malignancies and solid tumors. Major treatment strategies focus on monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates (ADCs). Two anti-HER3 antibody-drug conjugates are currently under development internationally, with one (Daiichi Sankyo U3-1402) entering clinical trials for the treatment of NSCLC, MBC, and CRC.

[0007] ADC drugs consist of antibodies, bioactive molecules, and linkers. The bioactive molecules are covalently coupled to the antibodies via the linkers. Antibodies (such as monoclonal antibodies) can specifically recognize targets on the surface of tumor cells, guiding the ADC to the cancer cell surface and allowing the ADC to enter the cancer cell through endocytosis. The bioactive molecules are then released inside the cancer cell, killing the cancer cells while minimizing damage to normal tissue cells.

[0008] U3-1402, developed by Daiichi Sankyo, uses a GGFG tetrapeptide as an enzymatic linker and the cytotoxic agent Dxd. A phase I dose expansion trial (5.6 mg / kg, Q3W) in the treatment of EGFR-mutated NSCLC reportedly resulted in an ORR of 39%, a DCR of 72%, and a median progression-free survival of 8.2 months. Similar efficacy was observed in patients with brain metastases. Safety concerns were primarily hematologic toxicity (thrombocytopenia, neutropenia, etc.) and interstitial pneumonitis (5-7%).

[0009] Summary of the Invention

[0010] In one aspect, the present application provides an antibody-drug conjugate having the formula Ab-[MLED] x The structure shown, wherein:

[0011] Ab is an antibody or antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor 3 (HER3, also known as Erbb3);

[0012] M is a linker site connected to the antibody or its antigen-binding fragment;

[0013] L is the structural fragment connecting M and E;

[0014] E is a structural fragment connecting L and D;

[0015] D is the cytotoxic drug fragment;

[0016] x is selected from 1 to 10.

[0017] In some embodiments, Ab is an antibody or antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor 3 (HER3);

[0018] M is a linker site that is connected to the antibody or its antigen-binding fragment and is Wherein, ring A is a 5-6 membered alicyclic heterocyclic ring or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl group substituted; M1 is selected from a single bond and substituted or unsubstituted following groups: C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkynylidene;

[0019] L is a structural fragment connecting M and E, and is selected from the structure consisting of one or more substituted or unsubstituted groups as follows: C 1-6 Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), 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:53), Gly-Gly-Gly-Gly-Gly (SEQ ID NO:54), Where R' represents hydrogen, C 1-6 Alkyl or containing -(CH2CH2O) r -alkyl; r is selected from an integer of 1-10; s is selected from an integer of 1-20;

[0020] E is a structural fragment connecting L and D, and is a single bond or a substituted or unsubstituted structure selected from the following: -NH-CH2-, -NH-CH2-O-CH2-CO-,

[0021] D is a fragment corresponding to a cytotoxic drug obtained by linking a cytotoxic drug to E, wherein the cytotoxic drug is selected from a microtubule inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor, and pharmaceutically acceptable salts, esters, or analogs thereof; preferably, the microtubule inhibitor is an auristatin compound or a maytansine compound; preferably, the DNA intercalator is a pyrrolobenzodiazepine (PBD); preferably, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); preferably, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analog thereof;

[0022] x is selected from 1 to 10.

[0023] In some embodiments, D is a fragment corresponding to a cytotoxic drug obtained by linking a cytotoxic drug to E, wherein the cytotoxic drug is selected from the group consisting of a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0025] (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:

[0026] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0027] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0028] (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 36 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 37 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0029] wherein the variant described in any one of (1a), (1b), and (1c) 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 with the sequence from which it is derived, or the variant has 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 it is derived; preferably, the substitutions are conservative substitutions; provided that the amino acid sequences of the CDRs of the variant have 100% sequence identity with the amino acid sequences of the corresponding CDRs of VH and VL of (1a), (1b), and (1c), and the variant binds to HER3;

[0030] or,

[0031] (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:

[0032] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 9 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0033] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0034] (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 39 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 40 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0035] wherein the variant described in any one of (2a), (2b), and (2c) 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 with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; provided that the amino acid sequences of the CDRs of the variant have 100% sequence identity with the amino acid sequences of the corresponding CDRs of VH and VL of (2a), (2b), and (2c), and the variant binds to HER3;

[0036] or,

[0037] (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:

[0038] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 10 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 11 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 14 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0039] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 27 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 28 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 29 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 30 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0040] (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 41 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 42 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 43 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 44 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0041] wherein the variant described in any one of (3a), (3b), and (3c) 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 with the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions; provided that the amino acid sequences of the CDRs of the variant have 100% sequence identity with the amino acid sequences of the corresponding CDRs of VH and VL of (3a), (3b), and (3c), and the variant binds to HER3.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0043] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 having an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0044] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0045] (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 36 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 37 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0046] wherein the variant described in any one of (1a), (1b), and (1c) 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 it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; provided that the amino acid sequences of the CDRs of the variant have 100% sequence identity with the amino acid sequences of the corresponding CDRs of VH and VL of (1a), (1b), and (1c), and the variant binds to HER3

[0047] or,

[0048] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 9 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0049] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0050] (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 39 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 40 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0051] wherein the variant described in any one of (2a), (2b), and (2c) 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 with the sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; provided that the amino acid sequences of the CDRs of the variant have 100% sequence identity with the amino acid sequences of the corresponding CDRs of VH and VL of (2a), (2b), and (2c), and the variant binds to HER3;

[0052] or,

[0053] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 10 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 11 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 14 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or,

[0054] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 27 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 28 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 29 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 30 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or,

[0055] (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 41 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 42 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 43 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 44 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof;

[0056] wherein the variant described in any one of (3a), (3b), and (3c) 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 with the sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions; provided that the amino acid sequences of the CDRs of the variant have 100% sequence identity with the amino acid sequences of the corresponding CDRs of VH and VL of (3a), (3b), and (3c), and the variant binds to HER3.

[0057] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0058] (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 1, CDR-H2 with an amino acid sequence of SEQ ID NO: 2, and CDR-H3 with an amino acid sequence of SEQ ID NO: 3; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4, CDR-L2 with an amino acid sequence of SEQ ID NO: 5, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6; or,

[0059] (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19, CDR-H2 with an amino acid sequence of SEQ ID NO: 20, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24; or,

[0060] (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 36, CDR-H2 with an amino acid sequence of SEQ ID NO: 37, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52;

[0061] (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 7, CDR-H2 with an amino acid sequence of SEQ ID NO: 8, and CDR-H3 with an amino acid sequence of SEQ ID NO: 9; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4, CDR-L2 with an amino acid sequence of SEQ ID NO: 5, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6; or,

[0062] (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25, CDR-H2 with an amino acid sequence of SEQ ID NO: 26, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24; or,

[0063] (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 39, CDR-H2 with an amino acid sequence of SEQ ID NO: 40, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52;

[0064] or,

[0065] (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 10, CDR-H2 with an amino acid sequence of SEQ ID NO: 11, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13, CDR-L2 with an amino acid sequence of SEQ ID NO: 14, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6; or

[0066] (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 27, CDR-H2 with an amino acid sequence of SEQ ID NO: 28, and CDR-H3 with an amino acid sequence of SEQ ID NO: 29; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 30, CDR-L2 with an amino acid sequence of SEQ ID NO: 31, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24; or

[0067] (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:41, CDR-H2 with an amino acid sequence of SEQ ID NO:42, and CDR-H3 with an amino acid sequence of SEQ ID NO:43; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:44, CDR-L2 with an amino acid sequence of SEQ ID NO:31, and CDR-L3 with an amino acid sequence of SEQ ID NO:52.

[0068] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (1a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 1, CDR-H2 with an amino acid sequence of SEQ ID NO: 2, and CDR-H3 with an amino acid sequence of SEQ ID NO: 3; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4, CDR-L2 with an amino acid sequence of SEQ ID NO: 5, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6.

[0069] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19, CDR-H2 with an amino acid sequence of SEQ ID NO: 20, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24.

[0070] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 36, CDR-H2 with an amino acid sequence of SEQ ID NO: 37, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52.

[0071] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 7, CDR-H2 with an amino acid sequence of SEQ ID NO: 8, and CDR-H3 with an amino acid sequence of SEQ ID NO: 9; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4, CDR-L2 with an amino acid sequence of SEQ ID NO: 5, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6.

[0072] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25, CDR-H2 with an amino acid sequence of SEQ ID NO: 26, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24.

[0073] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 39, CDR-H2 with an amino acid sequence of SEQ ID NO: 40, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45, CDR-L2 with an amino acid sequence of SEQ ID NO: 23, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52.

[0074] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (3a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 10, CDR-H2 with an amino acid sequence of SEQ ID NO: 11, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13, CDR-L2 with an amino acid sequence of SEQ ID NO: 14, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6.

[0075] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 27, CDR-H2 with an amino acid sequence of SEQ ID NO: 28, and CDR-H3 with an amino acid sequence of SEQ ID NO: 29; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 30, CDR-L2 with an amino acid sequence of SEQ ID NO: 31, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24.

[0076] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:41, CDR-H2 with an amino acid sequence of SEQ ID NO:42, and CDR-H3 with an amino acid sequence of SEQ ID NO:43; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:44, CDR-L2 with an amino acid sequence of SEQ ID NO:31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO:52.

[0077] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0078] (a) comprising the VH shown in SEQ ID NO: 15 or a variant thereof, and / or comprising the VL shown in SEQ ID NO: 16 or a variant thereof;

[0079] (b) comprises the VH shown in SEQ ID NO: 32 or a variant thereof, and / or comprises the VL shown in SEQ ID NO: 33 or a variant thereof; or

[0080] (c) comprising the VH shown in SEQ ID NO: 46 or a variant thereof, and / or comprising the VL shown in SEQ ID NO: 47 or a variant thereof;

[0081] 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 it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions, provided that the amino acid sequences of the CDRs of the variant have 100% sequence identity with the amino acid sequences of the corresponding CDRs of VH and VL of (a), (b) and (c), and the variant binds to HER3.

[0082] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0083] (a) comprising the VH shown in SEQ ID NO: 15 or a variant thereof, and comprising the VL shown in SEQ ID NO: 16 or a variant thereof;

[0084] (b) comprising the VH shown in SEQ ID NO: 32 or a variant thereof, and comprising the VL shown in SEQ ID NO: 33 or a variant thereof; or

[0085] (c) comprising the VH shown in SEQ ID NO: 46 or a variant thereof, and comprising the VL shown in SEQ ID NO: 47 or a variant thereof;

[0086] 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 it is derived, or the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

[0087] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0088] (a) comprising the VH set forth in SEQ ID NO: 15, and, comprising the VL set forth in SEQ ID NO: 16;

[0089] (b) comprising the VH set forth in SEQ ID NO: 32 and, comprising the VL set forth in SEQ ID NO: 33; or

[0090] (c) comprising the VH represented by SEQ ID NO: 46, and, comprising the VL represented by SEQ ID NO: 47.

[0091] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0092] (a) comprises the VH shown in SEQ ID NO: 15, and, comprises the VL shown in SEQ ID NO: 16.

[0093] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0094] (b) comprises the VH shown in SEQ ID NO: 32, and, comprises the VL shown in SEQ ID NO: 33.

[0095] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0096] (c) comprising the VH represented by SEQ ID NO: 46, and, comprising the VL represented by SEQ ID NO: 47.

[0097] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3, wherein the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 described in VH, respectively including the amino acid sequence of SEQ ID NO: 15; and a light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 described in VL, respectively including the amino acid sequence of SEQ ID NO: 16.

[0098] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3, wherein the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 described in VH, respectively, including the amino acid sequence of SEQ ID NO: 32; and a light chain variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 described in VL, respectively, including the amino acid sequence of SEQ ID NO: 33.

[0099] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 described in VH comprising the amino acid sequence of SEQ ID NO: 46, respectively; and a light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3, wherein the CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 described in VL comprising the amino acid sequence of SEQ ID NO: 47, respectively. In some embodiments, the antibody or antigen-binding fragment thereof further comprises:

[0100] (a) a heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; and

[0101] (b) a light chain constant region (CL) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived.

[0102] 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 antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 50, or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared to SEQ ID NO: 50.

[0103] In some embodiments, the light chain constant region is a kappa light chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as set forth in SEQ ID NO: 51 or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions (e.g., up to 15, up to 10, or up to 5 conservative amino acid substitutions; e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared to SEQ ID NO: 51.

[0104] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO:50 and a light chain constant region (CL) as shown in SEQ ID NO:51.

[0105] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0106] (1) a heavy chain comprising the VH sequence of SEQ ID NO: 15 and the heavy chain constant region (CH) of SEQ ID NO: 50, and a light chain comprising the VL sequence of SEQ ID NO: 16 and the light chain constant region (CL) of SEQ ID NO: 51;

[0107] (2) a heavy chain comprising the VH sequence of SEQ ID NO: 32 and the heavy chain constant region (CH) of SEQ ID NO: 50, and a light chain comprising the VL sequence of SEQ ID NO: 33 and the light chain constant region (CL) of SEQ ID NO: 51; or

[0108] (3) A heavy chain comprising the VH sequence of SEQ ID NO: 46 and the heavy chain constant region (CH) of SEQ ID NO: 50, and a light chain comprising the VL sequence of SEQ ID NO: 47 and the light chain constant region (CL) of SEQ ID NO: 51.

[0109] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising a VH having an amino acid sequence as shown in SEQ ID NO: 15 and a heavy chain constant region (CH) as shown in SEQ ID NO: 50, and a light chain comprising a VL having an amino acid sequence as shown in SEQ ID NO: 16 and a light chain constant region (CL) as shown in SEQ ID NO: 51.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (2) a heavy chain comprising a VH having an amino acid sequence as set forth in SEQ ID NO: 32 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 50, and a light chain comprising a VL having an amino acid sequence as set forth in SEQ ID NO: 33 and a light chain constant region (CL) as set forth in SEQ ID NO: 51; or

[0111] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (3) a heavy chain comprising a VH having an amino acid sequence as shown in SEQ ID NO: 46 and a heavy chain constant region (CH) as shown in SEQ ID NO: 50, and a light chain comprising a VL having an amino acid sequence as shown in SEQ ID NO: 47 and a light chain constant region (CL) as shown in SEQ ID NO: 51.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 17, and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 18.

[0113] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 34, and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 35.

[0114] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 48, and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 49.

[0115] In certain embodiments of the antibodies or antigen-binding fragments 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 some embodiments of the antibodies or antigen-binding fragments thereof, the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid.

[0116] In the antibody-drug conjugate, the cytotoxic drug can be linked to the antibody or antigen-binding fragment thereof via a linker (such as the "MLE" fragment shown in this application).

[0117] In some embodiments, M is Wherein ring A is a 5-6 membered alicyclic heterocyclic ring, or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl group substituted; M1 is selected from a single bond and substituted or unsubstituted following groups: C 1- 20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkynylidene.

[0118] In some embodiments, M is wherein Ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more 6-membered heteroaromatic rings to a benzene ring via a single bond, wherein the alicyclic heterocycle is optionally substituted with one or more selected from oxy (=O), halogen and C 1-4 Alkyl group substituted; M1 is selected from a single bond and substituted or unsubstituted following groups: C 3-10 Alkylene, C 3-10 Alkenylene or C 3-10 Alkynylidene.

[0119] In some embodiments, M is wherein ring A is selected from M1 is selected from a single bond and C 5-8 Alkylene, C 5-8 Alkenylene or C 5-8 Alkynylidene.

[0120] In some embodiments, M is selected from the following structures:

[0121] In some embodiments, M is selected from the following structures:

[0122] In some embodiments, M is

[0123] Wherein, ring A is a 5-20 membered aromatic ring system, and the aromatic ring system is optionally substituted by one or more selected from halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl group substitution; M1 is substituted or unsubstituted C 2-20 Alkynylidene.

[0124] In some embodiments, M is

[0125] Wherein, ring A is a 6-membered heteroaromatic ring, and the aromatic ring system is optionally substituted by one or more selected from halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl group substitution; M1 is C 3-10 Alkynylidene.

[0126] In some embodiments, M is

[0127] In some embodiments, L is selected from the structure consisting of one or more substituted or unsubstituted groups: C 1-6 Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), 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-G ly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, Where R' represents hydrogen, C 1-6 Alkyl or alkyl containing -(CH2CH2O)r-; r is selected from an integer of 1-10; s is selected from an integer of 1-20.

[0128] In some embodiments, L is selected from the structure consisting of one or more substituted or unsubstituted groups: C 1-6 Alkylene, -NH-, Phe, Lys, Ala-Ala-Ala, Lys(COCH2CH2(OCH2CH2) sOCH3), Gly, Gly-Gly-Phe-Gly, wherein s is selected from an integer of 1-20.

[0129] In some embodiments, L is selected from the structure consisting of one or more substituted or unsubstituted groups: C 1-6 Alkylene, -NH-, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), Gly, Gly-Gly-Phe-Gly, wherein s is selected from an integer of 1-20.

[0130] In some embodiments, L is selected from the following substituted or unsubstituted structures:

[0131] In some embodiments, L is selected from the following structures:

[0132] wherein s is selected from an integer of 1-20.

[0133] Preferably, L is selected from the following structures:

[0134] In some embodiments, L is selected from the following structures:

[0135] In some embodiments, L is selected from the following structures:

[0136] In some embodiments, L is selected from the following structures:

[0137] In some embodiments, L is selected from the group consisting of one or more of the following: Val, Cit, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), 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 and Gly-Gly-Gly-Gly-Gly; wherein s is selected from an integer of 1-20.

[0138] In some embodiments, L consists of one or more substituted or unsubstituted Ala.

[0139] In some embodiments, L consists of one or more Ala.

[0140] In some embodiments, L is substituted or unsubstituted Ala-Ala-Ala.

[0141] In some embodiments, L is

[0142] In some embodiments, E is a single bond or a substituted or unsubstituted structure selected from the group consisting of: -NH-CH2-, -NH-CH2-O-CH2-CO-,

[0143] In some embodiments, E is a single bond or a substituted or unsubstituted structure selected from the group consisting of: -NH-CH2-, -NH-CH2-O-CH2-CO-, In some embodiments, E is substituted or unsubstituted -NH-CH2-O-CH2-CO-,

[0144] In some embodiments, E is -NH-CH2-O-CH2-CO- or In some embodiments, E is -NH-CH2-O-CH2-CO or -NH-CH2-.

[0145] In some embodiments, M is selected from the following substituted or unsubstituted structures:

[0146] L is selected from the following substituted or unsubstituted structures:

[0147] E is -NH-CH2-O-CH2-CO-,

[0148] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0149] In some embodiments, Selected from the following structures:

[0150] In some embodiments, Selected from the following structures:

[0151] In some embodiments, the cytotoxic drug is selected from microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors and RNA polymerase inhibitors. In some embodiments, the microtubule inhibitor is an auristatin compound or a maytansine compound. In some embodiments, the DNA intercalator is a pyrrolobenzodiazepine (PBD). In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, multicarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester or analog thereof.

[0152] The cytotoxic drugs disclosed in this application generally contain a variety of functional groups, such as hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH), primary amino (-NH2), secondary amine (-NR A H) or tertiary amine (-NR B R C ), where R A 、R B 、R C These represent only non-hydrogen substituents on N, through which the cytotoxic drug can be attached to the linker in the conjugate.

[0153] In some embodiments, the cytotoxic drug is linked to E in the antibody-drug conjugate through a -OH, -SH, primary amino group, secondary amine group, or tertiary amine group on the cytotoxic drug.

[0154] In some embodiments, the cytotoxic drug is selected from the following Formula I and Formula II:

[0155] Wherein, R1, R2 are each independently selected from C 1-6 Alkyl and halogen;

[0156] R3 is selected from H and -CO-CH2OH;

[0157] R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring;

[0158] R6 is selected from hydrogen and -C 1-4 Alkylene-NR a R b ;

[0159] R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ;

[0160] where R a 、R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6 alkyl.

[0161] In some embodiments, the cytotoxic drug is selected from the following compounds:

[0162] In some embodiments, the cytotoxic drug is selected from the following compounds:

[0163] The fragment of the cytotoxic drug obtained by connecting the cytotoxic drug to the linker is the formula Ab-[MLED] x In some embodiments, D is a monovalent structure obtained by losing one H from -OH, -NH2 or a secondary amine group on the cytotoxic drug.

[0164] In some embodiments, D is selected from the following structures:

[0165] In some embodiments, the cytotoxic drug is:

[0166] In some embodiments, the cytotoxic drug is:

[0167] In some embodiments, D is:

[0168] In some embodiments, D is:

[0169] In some embodiments, the antibody-drug conjugate is selected from ADC A-01 to ADC A-25, ADC B-01 to ADC B-05 shown below:

[0170] Wherein, HA in each antibody-drug conjugate represents an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO: 15 and VL as shown in SEQ ID NO: 16, for example, an antibody or antigen-binding fragment thereof comprising VH as shown in SEQ ID NO: 15 and CH as shown in SEQ ID NO: 50, and VL as shown in SEQ ID NO: 16 and CL as shown in SEQ ID NO: 51;

[0171] in, Indicates the specific connection method between the thiol group in the antibody or its antigen-binding fragment and the linker.

[0172] In some embodiments, x is an integer from 1-10.

[0173] In some embodiments, the antibody-drug conjugate is selected from:

[0174] Wherein, the HA in each antibody-drug conjugate is selected from:

[0175] (1) an antibody or antigen-binding fragment thereof comprising the VH of SEQ ID NO: 15 and the VL of SEQ ID NO: 16, for example, an antibody or antigen-binding fragment thereof comprising the VH of SEQ ID NO: 15 and the CH of SEQ ID NO: 50, and the VL of SEQ ID NO: 16 and the CL of SEQ ID NO: 51;

[0176] (2) an antibody or antigen-binding fragment thereof comprising the VH set forth in SEQ ID NO:32 and the VL set forth in SEQ ID NO:33, for example, an antibody or antigen-binding fragment thereof comprising the VH set forth in SEQ ID NO:32 and the CH set forth in SEQ ID NO:50, and the VL set forth in SEQ ID NO:33 and the CL set forth in SEQ ID NO:51; and

[0177] (3) an antibody or antigen-binding fragment thereof comprising the VH of SEQ ID NO: 46 and the VL of SEQ ID NO: 47, for example, an antibody or antigen-binding fragment thereof comprising the VH of SEQ ID NO: 46 and the CH of SEQ ID NO: 50, and the VL of SEQ ID NO: 47 and the CL of SEQ ID NO: 51;

[0178] x is 3-8, for example, 3-4 or 7-8.

[0179] In some embodiments, x is an integer from 3-8.

[0180] In some embodiments, the antibody-drug conjugate is:

[0181] Wherein, the HA represents an antibody or antigen-binding fragment thereof comprising the HC shown in SEQ ID NO: 17 and the LC shown in SEQ ID NO: 18;

[0182] x is 6 to 8.

[0183] In some embodiments, the antibody-drug conjugate is:

[0184] HA includes:

[0185] (1) An antibody or antigen-binding fragment thereof comprising the VH of SEQ ID NO: 15 and the VL of SEQ ID NO: 16, for example, an antibody or antigen-binding fragment thereof comprising the VH of SEQ ID NO: 15 and the CH of SEQ ID NO: 50, and the VL of SEQ ID NO: 16 and the CL of SEQ ID NO: 51; x is an integer from 3 to 8, for example, from 3 to 4 or from 7 to 8, for example, from 3 to 8.

[0186] In some embodiments, the antibody-drug conjugate is:

[0187] HA includes:

[0188] (2) An antibody or antigen-binding fragment thereof comprising the VH shown in SEQ ID NO: 32 and the VL shown in SEQ ID NO: 33, for example, an antibody or antigen-binding fragment thereof comprising the VH shown in SEQ ID NO: 32 and the CH shown in SEQ ID NO: 50, and the VL shown in SEQ ID NO: 33 and the CL shown in SEQ ID NO: 51; x is an integer from 3 to 8, for example, from 3 to 4 or from 7 to 8, for example, from 3 to 8.

[0189] In some embodiments, the antibody-drug conjugate is:

[0190] HA includes:

[0191] (3) An antibody or antigen-binding fragment thereof comprising the VH shown in SEQ ID NO:46 and the VL shown in SEQ ID NO:47, for example, an antibody or antigen-binding fragment thereof comprising the VH shown in SEQ ID NO:46 and the CH shown in SEQ ID NO:50, and the VL shown in SEQ ID NO:47 and the CL shown in SEQ ID NO:51; x is an integer from 3 to 8, for example, from 3 to 4 or from 7 to 8, for example, from 3 to 8.

[0192] In some embodiments, the antibody-drug conjugate is:

[0193] Wherein, the HA represents an antibody or antigen-binding fragment thereof comprising the HC shown in SEQ ID NO: 17 and the LC shown in SEQ ID NO: 18;

[0194] x is 6 to 8.

[0195] In some embodiments, x is an integer from 6-8.

[0196] In some embodiments, the antibody-drug conjugate is:

[0197] wherein the HA represents an antibody or antigen-binding fragment thereof comprising the HC shown in SEQ ID NO: 34 and the LC shown in SEQ ID NO: 35;

[0198] x is 6 to 8, for example, an integer of 6-8.

[0199] In some embodiments, the antibody-drug conjugate is:

[0200] wherein said HA represents an antibody or antigen-binding fragment thereof comprising HC shown in SEQ ID NO: 48 and LC shown in SEQ ID NO: 49;

[0201] x is 6 to 8, for example, an integer of 6-8.

[0202] In some embodiments, the antibody-drug conjugate:

[0203] (i) The heavy chain lacks a lysine residue at the C-terminus;

[0204] (ii) the N-terminus of the heavy chain is glutamine, glutamic acid or pyroglutamic acid; or,

[0205] (iii) The heavy chain C-terminus lacks a lysine residue and the heavy chain N-terminus is glutamine, glutamic acid, or pyroglutamic acid.

[0206] In some embodiments, in the formula Ab-[MLED] x In the antibody-drug conjugate shown, Ab is conjugated to the remaining moiety in the formula via a cysteine ​​residue.

[0207] Also provided herein are pharmaceutical compositions comprising the antibody-drug conjugates described herein and one or more pharmaceutically acceptable excipients.

[0208] Also provided herein are methods for treating a HER3-overexpressing cancer in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate described herein or a pharmaceutical composition thereof. In specific embodiments, the cancer comprises a solid tumor or a hematological malignancy. In further embodiments, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma. In further embodiments, the lung cancer is non-small cell lung cancer. In further embodiments, the lung cancer is lung adenocarcinoma.

[0209] Also provided herein is the use of an antibody-drug conjugate or pharmaceutical composition thereof described herein in the preparation of a medicament for treating a cancer that overexpresses HER3. In specific embodiments, the cancer comprises a solid tumor or a hematological malignancy. In further embodiments, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma. In further embodiments, the lung cancer is non-small cell lung cancer. In further embodiments, the lung cancer is lung adenocarcinoma.

[0210] Also provided herein is use of the antibody-drug conjugates described herein or pharmaceutical compositions thereof in treating cancers that overexpress HER3.

[0211] In specific embodiments, the cancer comprises a solid tumor or a hematological malignancy. In further embodiments, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma. In further embodiments, the lung cancer is non-small cell lung cancer. In further embodiments, the lung cancer is lung adenocarcinoma.

[0212] Also provided herein are antibody-drug conjugates or pharmaceutical compositions described herein for use in treating cancers that overexpress HER3. In specific embodiments, the cancer comprises a solid tumor or a hematological malignancy. In further embodiments, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma. In further embodiments, the lung cancer is non-small cell lung cancer. In further embodiments, the lung cancer is lung adenocarcinoma.

[0213] In one embodiment, the HER3-highly expressed cancer includes a solid tumor or a hematological malignancy, such as colon cancer, gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, particularly lung adenocarcinoma) or lymphoma.

[0214] The present invention also provides a pharmaceutically acceptable salt or solvate of any of the above-mentioned antibody-drug conjugates.

[0215] The present invention further provides a composition comprising any one of the aforementioned antibody-drug conjugates and a pharmaceutically acceptable carrier or excipient.

[0216] Antibody preparation

[0217] The antibodies described herein can be prepared by various methods known in the art, for example, by genetic engineering and recombinant techniques. For example, DNA molecules encoding the heavy and light chains of the antibodies described herein are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions and express the antibodies of the present invention.

[0218] Conjugate

[0219] In another aspect, the present application provides a method of conjugating the drug-linker described herein to the antibody described herein to prepare the antibody-drug conjugate (ADC) described herein.

[0220] In certain embodiments, an antibody described herein is conjugated to a drug-linker described herein via coupling to a lysine in the antibody.

[0221] In certain embodiments, the antibodies described herein are conjugated to the drug-linkers described herein via cysteine ​​residues in the antibodies. In certain embodiments, the cysteine ​​residues are derived from reduced intrachain disulfide bonds in the antibodies. In certain embodiments, the cysteine ​​residues are derived from reduced interchain disulfide bonds in the antibodies.

[0222] In some embodiments, the antibody is conjugated to the drug-linker via reduced interchain disulfide bonds in the antibody. For example, an IgG1 antibody is composed of four polypeptide chains, two heavy chains comprising VH, CH1, and Fc (e.g., hinge, CH2, and CH3) domains, and two light chains comprising VL and CL domains, connected by interchain cysteine ​​disulfide bonds (-SS-) (e.g., two heavy chain-light chain interchain disulfide bonds and two hinge heavy chain-heavy chain interchain disulfide bonds). In certain embodiments, when these disulfide bonds are broken under reducing conditions, eight (8) reactive cysteine ​​thiol moieties are generated. In certain embodiments, each of the eight reactive cysteine ​​thiol moieties is a site of attachment for a drug-linker, such that a maximum of eight (x=8) drug-linkers can be attached to the reduced antibody. In certain embodiments, any one of the four disulfide bonds is broken under reducing conditions, resulting in two (2) reactive cysteine ​​thiol moieties. In further embodiments, each of the two reactive cysteine ​​thiol moieties is a site of attachment for a drug-linker, such that two (x=2) drug-linkers can be attached to the reduced antibody. In certain embodiments, any two of the four disulfide bonds are cleaved under reducing conditions, resulting in four (4) reactive cysteine ​​thiol moieties. In further embodiments, each of the four reactive cysteine ​​thiol moieties is a site of attachment for a drug-linker, such that four (x=4) drug-linkers can be attached to the reduced antibody. In certain embodiments, any three of the four disulfide bonds are cleaved under reducing conditions, resulting in six (6) reactive cysteine ​​thiol moieties. In further embodiments, each of the six reactive cysteine ​​thiol moieties is a site of attachment for a drug-linker, such that six (x=6) drug-linkers can be attached to the reduced antibody.

[0223] In some embodiments, the interchain disulfide bond is located between two cysteine ​​residues, which break under reducing conditions to produce two reactive cysteine ​​sulfhydryl moieties. In a further embodiment, the interchain disulfide bond in the antibody is located between the heavy chain and the light chain, for example, between C220 of the heavy chain according to EU numbering and C214 of the kappa light chain, or between C220 of the heavy chain according to EU numbering and C214 of the lambda light chain according to Kabat numbering. In addition, optionally, the interchain disulfide bond in the antibody is located between the two heavy chains, for example, between C226 and / or C229 of the first heavy chain according to EU numbering and C226 and / or C229 of the second heavy chain. In some embodiments, the cysteine ​​residue is located in the hinge region of the antibody. In some embodiments, according to EU numbering, the cysteine ​​residue is located at any one or more of positions 220, 226, or 229 in the heavy chain (also referred to herein as C220, C226, or C229, respectively). In some embodiments, the cysteine ​​residue is located at position 214 of the light chain (also referred to herein as C214, e.g., at position 214 of a kappa light chain according to EU and / or Kabat numbering, or at position 214 of a lambda light chain according to Kabat numbering) according to EU and / or Kabat numbering. In one embodiment, the cysteine ​​residue is located at position 220, 226, and 229 of the heavy chain according to EU and / or Kabat numbering, or at position 214 of a light chain according to EU or Kabat numbering. In one embodiment, the cysteine ​​residue is located at position 220, 226, and 229 of the heavy chain according to EU and / or Kabat numbering, or at position 214 of a kappa light chain according to EU and Kabat numbering. In one embodiment, the cysteine ​​residue is located at position 220, 226, and 229 of the heavy chain according to EU and Kabat numbering, or at position 214 of a lambda light chain according to Kabat numbering. In one embodiment, the cysteine ​​residue is located at any one or more of the following positions:

[0224] (i) any one, any two, any three, or all four of positions 220, 226, and 229 in the first heavy chain, according to EU numbering;

[0225] (ii) any one, any two, any three, or all four of positions 220, 226, and 229 in the second heavy chain, according to EU numbering;

[0226] (iii) position 214 in the first light chain according to Kabat numbering; and / or

[0227] (iv) Position 214 in the second light chain according to Kabat numbering.

[0228] As used herein, C220, C226, and C229 refer to amino acid residues of immunoglobulins identified according to EU numbering (cysteine, Cys, C). As will be appreciated by those skilled in the art, such numbering correspondingly represents amino acid residues of polypeptides that align with the defined amino acid residues of immunoglobulins, as shown in www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0229] In certain embodiments, the antibodies described herein comprise four interchain disulfide bonds in the hinge region that can be reduced, thereby breaking and revealing reactive sulfhydryl moieties that can be conjugated to a maleimide moiety on a drug-linker, e.g., a maleimide moiety on a drug-linker described herein.

[0230] In one embodiment, the present invention provides a method for preparing the ADC described herein, comprising the following steps:

[0231] a) providing a solution comprising an antibody;

[0232] b) contacting the solution of a) with a reducing agent;

[0233] c) contacting the solution of b) with a solution comprising a drug-linker or a salt thereof as described herein to prepare the ADC.

[0234] In one embodiment, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0235] Composition

[0236] In another aspect, the present application provides a composition of an antibody-drug conjugate (ADC) as described herein. Such a composition may comprise a plurality of ADCs as described herein, wherein each ADC comprises a drug-linker as described herein, wherein x is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In other words, each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 drug-linkers. Thus, the composition is characterized in that the "drug-antibody" ratio (DAR) is in the range of about 1 to about 10. Methods for determining DAR are well known to those skilled in the art, including methods using reverse phase chromatography or HPLC-MS.

[0237] For example, in any embodiment, the ADC compositions described herein have a DAR of about 1 to about 10, or any subrange therebetween, e.g., about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10, or about 9 to 10.

[0238] In certain embodiments, the DAR of the ADC compositions described herein is about 3 to 9, e.g., about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 8. .0, about 4.5 to 5.0, about 4.5 to 5.5, about 4.5 to 6.0, about 4.5 to 6.5, about 4.5 to 7.0, about 4.5 to 7.5 about 5.0 to 8.0, about 5.5 to 6.0, about 5.5 to 6.5, about 5.5 to 7.0, about 5.5 to 7.5, about 5.5 to 8.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.5, about 7.0 to 7.5.

[0239] Pharmaceutical composition

[0240] In another aspect, the present application provides a pharmaceutical composition comprising one or more antibody-drug conjugates as described above, and one or more pharmaceutical excipients.

[0241] Pharmaceutical excipients include, for example, pharmaceutically acceptable carriers and / or excipients.

[0242] In some embodiments, the average DAR value (drug-antibody coupling ratio) of the pharmaceutical composition is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4- 10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-9, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3.5 ~6.0,3.5~6.5,3.5~7.0,3.5~7.5,3.5~8.0,4.0~4.5,4.0~5.0,4.0~5.5,4.0~6.0,4.0~6.5,4.0~7.0,4.0~7.5,4.0~8.0,4.5~5.0,4.5~5.5,4.5~6.0,4.5~6.5,4.5~7.0,4.5~7.5,4.5~8.0,5.0~5 .5, 5.0~6.0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0, 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.5, 6.5~7.0, 6.5~7.5, 6.5~8.5, 7.0~7.5, 7.0~9.0 or 7.5~9.0.

[0243] The antibody-drug conjugates described herein are typically formulated in a unit injectable form together with a pharmaceutically acceptable parenteral vehicle for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc. Optionally, the antibody-drug conjugate having the desired purity is mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer in the form of a lyophilized agent or solution (Remington's Pharmaceutical Sciences (1980) 16 th The antibody-drug conjugates described herein, or pharmaceutical compositions containing the same, can be administered by any route appropriate to the individual to be treated.

[0244] The ADCs and pharmaceutical compositions described herein can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended route of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under production and storage conditions. The preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: the necessary dose of the antibody of the present invention is incorporated into a suitable solvent, and other required ingredients (including but not limited to pH regulators, surfactants, adjuvants, ionic strength enhancers, etc., penetrants, preservatives, diluents, or any combination thereof) are optionally added, followed by filtration sterilization. In addition, for ease of storage and use, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying). Such a sterile lyophilized powder can be dispersed in a suitable carrier, such as sterile, pyrogen-free water, before use.

[0245] In addition, the ADC described herein can be present in a pharmaceutical composition in a unit dosage form for administration by any suitable method known in the art, including but not limited to oral, oral, sublingual, ophthalmic, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powder, ointment or drops) or intranasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). The skilled artisan will appreciate that the route and / or mode of administration will vary depending on the intended purpose. In some preferred embodiments, the ADC and pharmaceutical compositions described herein are administered by intravenous infusion or injection.

[0246] In certain embodiments, the pharmaceutical composition may further comprise other pharmaceutically active agents. In certain embodiments, the other pharmaceutically active agents are drugs with anti-tumor activity. In certain embodiments, the other pharmaceutically active agents are selected from EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic agents, or any combination thereof. In certain embodiments, the ADC described herein and the other pharmaceutically active agents are provided as separate components or as mixed components. Therefore, the ADC described herein and the other pharmaceutically active agents can be administered simultaneously, separately, or sequentially.

[0247] How to use

[0248] The antibody-drug conjugates described herein, the drug-linkers described herein, or pharmaceutical compositions thereof can be used to treat various diseases or conditions, such as cancers with high HER3 expression, including solid tumors or hematological malignancies, such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma), or lymphoma.

[0249] Therefore, the present application provides use of an antibody-drug conjugate (ADC), a drug-linker, or a pharmaceutical composition comprising the same as described in any of the aforementioned embodiments in the preparation of a drug for treating HER3-overexpressing cancers.

[0250] At the same time, the present application also provides a method for treating HER3-overexpressing cancer, which comprises the step of administering to a subject in need thereof a therapeutically effective amount of an antibody-drug conjugate (ADC), drug-linker, or pharmaceutical composition comprising the same as described in any one of the aforementioned embodiments.

[0251] In certain embodiments, the antibody-drug conjugate (ADC), drug-linker, or pharmaceutical composition is sufficient (e.g., in a subject):

[0252] (1) Inhibit the proliferation of cells (such as tumor cells);

[0253] (2) inhibit tumor growth;

[0254] (3) induce and / or increase antibody-dependent cellular cytotoxicity activity;

[0255] (4) inhibiting HER3-mediated signal transduction;

[0256] (5) preventing and / or treating HER3-mediated diseases / disorders; or

[0257] (6) Any combination of (1) to (5) above.

[0258] In certain embodiments, the HER3-mediated disease / disorder is a tumor, e.g., a tumor expressing HER3. In certain embodiments, the tumor is selected from breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer), colorectal cancer, pancreatic cancer, head and neck squamous cell carcinoma, melanoma, ovarian cancer, prostate cancer, liver cancer, kidney cancer, bladder cancer, or any combination thereof.

[0259] application

[0260] The antibody-drug conjugates or pharmaceutical compositions thereof described herein can be used to treat a variety of diseases or conditions, such as HER3-overexpressing cancers, including solid tumors or hematological malignancies, such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma), or lymphoma.

[0261] Therefore, the present application provides use of any of the above-mentioned antibody-drug conjugates, drug-linkers, or pharmaceutical compositions containing the same in the preparation of drugs for treating HER3-overexpressing cancers.

[0262] At the same time, the present application also provides a method for treating HER3-overexpressing cancer, which comprises the step of administering a therapeutically effective amount of any of the above-described antibody-drug conjugates, drug-linkers, or pharmaceutical compositions containing the same to a subject in need thereof.

[0263] definition

[0264] Unless otherwise defined below, the meanings of all technical and scientific terms used herein are intended to be the same as those generally understood by those skilled in the art. Reference to the techniques used herein is intended to refer to techniques generally understood in the art, including variations of those techniques that are obvious to those skilled in the art or replacements with equivalent techniques. Furthermore, laboratory procedures such as genomics, nucleic acid chemistry, and molecular biology used herein are conventional procedures widely used in the corresponding fields. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0265] The term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen binding site, respectively. The allocation of amino acids in each region or domain can follow various numbering systems known in the art.

[0266] In some embodiments, the term "antibody" further includes a heavy chain constant region comprising a C-terminal lysine, or a C-terminal lysine or C-terminal glycine-lysine dipeptide. The term also includes a variable region wherein the N-terminal amino acid is cyclized to form pyroglutamic acid. Thus, in the antibody-containing compositions disclosed herein, the various antibodies contained therein may independently comprise a C-terminal lysine, a C-terminal lysine deletion, a C-terminal glycine-lysine deletion, and / or comprise an N-terminal glutamine or glutamic acid, or the N-terminal amino acid is cyclized to pyroglutamic acid.

[0267] The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, as defined in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0268] In the present invention, the CDRs contained in an antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art, such as those defined by the Kabat, Chothia, IMGT, or AbM numbering systems. In certain embodiments, the CDRs contained in an antibody or antigen-binding fragment thereof are defined by the Chothia numbering system.

[0269] Andrew CR Martin's group at www.bioinf.org.uk has published the following general rules for defining CDRs in antibody sequences, which include amino acids that specifically interact with amino acids in the epitope comprising the antigen to which the antibody binds. In rare cases, these normally constant features do not occur. However, Cys residues are the most conserved feature.

[0270] The entire amino acid sequence of VH is generally numbered according to Kabat, while the three CDRs of the variable region can be defined according to any of the aforementioned numbering schemes. In specific embodiments, the numbering of amino acid positions in VH can be starting from amino acid position 1 and continuing sequentially to the end of the sequence or according to Kabat numbering. Unless otherwise indicated, amino acid positions in VH and VL herein are defined according to sequential numbering.

[0271] The numbering of amino acid positions in the heavy chain constant region can be started at amino acid position 1 and continued sequentially to the end of the sequence or according to Eu numbering. The IgG1 heavy chain constant region amino acid sequence has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence starts at position 118 and ends at position 447 according to Eu numbering. Unless otherwise indicated, amino acid positions in the heavy and light chains herein are defined according to sequential numbering.

[0272] The term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0273] The term "antigen-binding fragment" of an antibody refers to polypeptides that are fragments of an antibody, such as polypeptides that are fragments of a full-length antibody, which retain the ability to specifically bind to the same antigen bound by the full-length antibody and / or compete with the full-length antibody for specific binding to the antigen, and are also referred to as "antigen-binding portions." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, F(ab')3 fragments, Fd, Fv, scFv, di-scFv, (scFv)2, disulfide-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAbs, nanobodies), and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0274] The term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0275] The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0276] The term "Fc" refers to an antibody fragment formed by disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but is not involved in antigen binding.

[0277] The term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO: 55) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 56) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, a VH-VH-COOH domain comprising NH2-VH-VH-COOH, NH 2- VL-VL-COOH scFv.

[0278] The term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also called nanobodies.

[0279] 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.

[0280] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0281] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0282] The terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably. They refer to an antibody or an antibody fragment from a population of highly homologous antibody molecules, that is, a population of identical antibody molecules except for possible spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies, and these different antibodies typically recognize different epitopes on an antigen. In addition, the modifier "monoclonal" only indicates that the antibody is characterized as being obtained from a highly homologous antibody population and is not to be understood as requiring the antibody to be prepared by any specific method.

[0283] The term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it retains binding activity to the target antigen. For example, the term "chimeric antibody" may include antibodies in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), while the heavy and light chain variable regions of the antibody are derived from a second antibody (e.g., a human antibody). For example, an antibody produced by immunizing a fully human transgenic mouse may be referred to as a chimeric antibody, which consists of a fully human variable region and a murine constant region.

[0284] The term "murine antibody" refers to antibodies obtained by fusing B cells from immunized mice with myeloma cells, screening for murine hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, followed by screening, antibody preparation, and antibody purification; or refers to antibodies secreted by plasma cells formed by the differentiation and proliferation of B cells in mice after antigen invasion.

[0285] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0286] The term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 total positions match). Typically, two sequences are compared when aligned for maximum identity. Such alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0287] The term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0288] The twenty conventional amino acids referred to herein are denoted according to conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0289] The term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), etc.

[0290] As used herein, the term "pharmaceutically acceptable salts" includes acid addition salts and basic salts.

[0291] Exemplary acid addition salts include acetate, ammonium, ascorbate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, fumarate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, methanesulfonate (also known as mesylate), naphthylsulfonate, nitrate, oxalate, phosphate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate (also known as tosylate), and the like. In addition, P. Stahl et al., Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. 2nd Revised Ed. (2011) Zurich: Wiley-VCH; S. Berge et al., Journal of Pharmaceutical Sciences (1977) 66 (1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33 201-217; Anderson et al., The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, DC, their website). These disclosures are incorporated herein by reference. In one embodiment, the acid salt is an ammonium salt or a diammonium salt.

[0292] Exemplary basic salts include ammonium salts, alkali metal salts such as sodium salts, lithium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, salts formed with organic bases (e.g., organic amines) such as dicyclohexylamine, tert-butylamine, choline, and salts formed with amino acids such as arginine and lysine. Basic nitrogen-containing groups can be quaternized with, for example, lower alkyl halides (e.g., methyl, ethyl or butyl chloride, bromide or iodide), dialkyl sulfates (e.g., dimethyl sulfate, diethyl sulfate and dibutyl sulfate), long-chain halides (e.g., chloride, bromide or iodide of decyl, lauryl or stearyl), aralkyl halides (e.g., benzyl or phenethyl bromide), and the like.

[0293] All such acid salts and base salts are intended as pharmaceutically acceptable salts within the scope of the disclosure and are considered equivalent to the free forms of the corresponding compounds for purposes of the disclosure.

[0294] The terms "comprises," "comprising," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0295] The term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently. The therapeutically effective amount of an ADC may vary depending on the severity of the disease to be treated, the general state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently.

[0296] The term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, alleviating symptoms, alleviating the extent of the disease, stabilizing (i.e., not worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. Additionally, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0297] The term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (such as a human) suffers from a tumor, or has a risk of suffering from the above-mentioned disease.

[0298] The terms "cancer" and "tumor" are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade neighboring tissues and may travel to distant parts of the body (metastasis) via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes blood cancers, particularly hematologic malignancies.

[0299] The term "hematologic malignancies" includes lymphomas, leukemias, myelomas or lymphoid malignancies, as well as splenic cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphomas and T-cell lymphomas. B-cell lymphomas include, for example, Hodgkin's lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphoma. Hematologic malignancies also include leukemias, such as secondary leukemias or acute lymphoblastic leukemia. Hematologic malignancies also include myeloma (e.g., multiple myeloma) and other blood and / or B-cell or T-cell related cancers.

[0300] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, for example, "C 1-20 Alkyl", "C 1- 10 Alkyl", "C 1-6 Alkyl", "C 1-4 Alkyl", "C 1-3 alkyl”, etc., specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0301] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight or branched hydrocarbon group, for example, "C 1-20 Alkylene", "C 1-10 Alkylene", "C 3-10 Alkylene", "C 5-8 Alkylene", "C 1-6 Alkylene", "C 1-4 Alkylene", "C 1-3 Specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene.

[0302] The term "alkenylene" refers to a divalent group derived from a straight or branched hydrocarbon group containing at least one carbon-carbon double bond losing two hydrogen atoms, including, for example, "C 2-20 Alkenylene", "C 3-10 Alkenylene", "C 5-8Examples include, but are not limited to, vinylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 1,3-butadienylene, 1-pentenylene, 2-pentenylene, 3-pentenylene, 1,3-pentadienylene, 1,4-pentadienylene, 1-hexenylene, 2-hexenylene, 3-hexenylene, 1,4-hexadienylene, and the like.

[0303] The term "alkynylene" refers to a divalent group derived from a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond that loses two hydrogen atoms. 2-20 Alkynylidene", "C 3-10 Alkynylidene", "C 5-8 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, and the like.

[0304] The term "aliphatic heterocycle" 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 heterocycles, 5-6 membered nitrogen-containing aliphatic heterocycles, 5-6 membered oxygen-containing aliphatic heterocycles, and the like, such as tetrahydrofuran, pyrrolidine, piperidine, tetrahydropyran, and the like.

[0305] 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.

[0306] The term "aromatic ring system" refers to a monocyclic or polycyclic ring system comprising at least one aromatic ring (e.g., a benzene ring, etc.) or heteroaromatic ring (e.g., a pyrimidine ring, etc.), two or more aromatic rings and / or heteroaromatic rings may form a fused ring or be connected by a single bond (e.g., dipyrimidinylphenyl, etc.), 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.

[0307] The term "linker" refers to a structural fragment that connects a biologically active molecule (eg, a cytotoxic drug fragment) and a targeting moiety (eg, an antibody or antigen-binding fragment thereof).

[0308] The term "substituted" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogen atoms on a designated compound or structural fragment with a substituent, provided that the normal valence of the designated atom in the present context is not exceeded and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form stable compounds. For example, the substituents are each independently composed of one or more of the following structures: -O-, -S-, -NR'-, halogen, -CN, -OH, -NH2, -NO2, -CN, =O, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkylene, 3-8 membered heterocyclyl, C6-C 10 (E)aryl and 5-10 membered (E)heteroaryl, etc. wherein R' is hydrogen or C1-C6 alkyl.

[0309] If a functional group or structural fragment is described as "substituted or unsubstituted", the functional group or structural fragment can be (1) unsubstituted or (2) substituted. When the functional group is substituted, the substituents replacing the functional group are each independently composed of one or more of the following structures: -O-, -S-, -NR'-, halogen, -CN, -OH, -NH2, -NO2, -CN, =O, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkylene, 3-8 membered heterocyclyl, C6-C 10 (E)aryl and 5-10 membered (E)heteroaryl, etc. wherein R' is hydrogen or C1-C6 alkyl.

[0310] As used herein, the terms "about" or "approximately" when used in conjunction with a numerical variable generally mean that the value of the variable is within the range of experimental error (e.g., within a 95% confidence interval about the mean) or within ±10% or wider.

[0311] It should be noted that if there is a discrepancy between a described structure and the name of that structure, the described structure will be given greater weight.

[0312] The present application relates to antibody-drug conjugates for treating HER3-positive cancers, and exemplarily discloses an antibody-drug conjugate having a structure as shown in the general formula Ab-[MLED]x and using the fully human antibody 22B6D2-hIgG1 as a targeting moiety. The results show that the conjugate has a good drug-antibody ratio, has good binding activity to HER3-positive cells, and has a good targeted killing effect on HER3-positive cancers, such as colon cancer, gastric cancer, breast cancer, and lung cancer (such as non-small cell lung cancer, especially lung adenocarcinoma). Therefore, the present application provides an antibody-drug conjugate for treating cancers that overexpress HER3, a pharmaceutical composition comprising the antibody-drug conjugate, and its use in treating cancers that overexpress HER3. BRIEF DESCRIPTION OF THE DRAWINGS

[0313] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0314] FIG1 shows the results of affinity testing of anti-human HER3 antibody-drug conjugates to MDA-MB-453 cells.

[0315] FIG2 shows the results of affinity testing of anti-human HER3 antibody-drug conjugates to NCI-N87 cells.

[0316] Figure 3 shows the results of the endocytic activity test of anti-human HER3 antibody-drug conjugates in MDA-MB-453 cells

[0317] FIG4 shows the results of the endocytosis activity test of the anti-human HER3 antibody-drug conjugate in HCC1569 cells.

[0318] FIG5 shows the results of in vitro cytotoxicity test of anti-human HER3 antibody-drug conjugates against MDA-MB-453 cells.

[0319] FIG6 shows the results of in vitro cytotoxicity assays of anti-human HER3 antibody-drug conjugates against 293T-HER3 cells.

[0320] FIG7A shows the in vivo efficacy test results of the anti-human HER3 antibody-drug conjugate.

[0321] FIG7B shows the changes in mouse body weight during the in vivo efficacy test of the anti-human HER3 antibody-drug conjugate. DETAILED DESCRIPTION

[0322] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and in no way constitutes any limitation on the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0323] The information of the sequences involved in the present invention is described in the following table:

[0324] The abbreviations used in this document have the following meanings:

[0325] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0326] Nuclear magnetic resonance (NMR) 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).

[0327] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0328] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0329] Mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0330] 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)

[0331] Compound A-01-1 (0.40 g, 640.59 μmol, its synthesis is described in patent application CN 111936169A) and isotecan mesylate (0.37 g, 704.65 μmol) were dissolved in DMF (8 mL). HATU (0.32 g, 832.77 μmol) and DIPEA (0.25 g, 1.92 mmol) were added and reacted at 25°C for 4 hours. DIPEA was removed under reduced pressure, and the mixture was freeze-dried with water to remove most of the DMF to obtain a crude product. The crude product was purified by preparative HPLC (conditions as follows) to obtain 273 mg of the title compound.

[0332] Column: Waters XBridge Prep C18 OBD 45 mm × 450 mm × 8.0 μm

[0333] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0334] The structural characterization data are as follows:

[0335] ESI-MS (m / z): 1034.4 [M+H] + .

[0336] Example 2 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)

[0337] Step 1: Synthesis of 1-chloro-3-bromo-2-methyl-5-nitrobenzene (1-8-2)

[0338] Compound 1-8-1 (5.00 g, 29.14 mmol) was dissolved in n-heptane (25 mL) at 25°C, concentrated sulfuric acid (25 mL) was added, and the mixture was heated to 50°C. NBS (6.22 g, 34.97 mmol) was added in batches at 50°C, and the reaction was maintained at 50°C for 2 hours. The reaction was monitored by thin-layer chromatography (ethyl acetate: petroleum ether = 1:10). The reaction solution was cooled to room temperature and then added dropwise to ice water. The mixture was extracted with toluene, and the organic phases were combined and washed with sodium sulfite solution, water, and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography (conditions as follows). The preparative solution was freeze-dried to obtain 4.88 g of the title compound.

[0339] Chromatographic column: C18 ODS 45mm×450mm×8.0μm

[0340] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0341] Step 2: Synthesis of 3-chloro-5-bromo-4-methylaniline (1-8-3)

[0342] Compound 1-8-2 (4.88 g, 19.48 mmol) was dissolved in ethyl acetate (100 mL) at 25°C, and platinum on carbon (2.00 g, 19.48 mmol, 5% content) was added. After hydrogen replacement, the mixture was reacted at 60°C under hydrogen protection for 4 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was filtered and concentrated to obtain 3.68 g of the crude title compound, which was used directly in the next reaction without further purification.

[0343] Step 3: Synthesis of N-(3-chloro-5-bromo-4-methylphenyl)acetamide (1-8-4)

[0344] Compound 1-8-3 (3.63 g, 14.82 mmol) was dissolved in ethyl acetate (70 mL) at 20°C, and triethylamine (4.50 g, 44.45 mmol) and acetic anhydride (2.27 g, 22.23 mmol) were added. The reaction was maintained at 20°C for 20 h, and the reaction was monitored by high-performance liquid chromatography-mass spectrometry. Water was added to the reaction solution, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was slurried in a mixed solvent of ethyl acetate: petroleum ether = 1:5 to obtain 2.86 g of the title compound.

[0345] Step 4: Synthesis of (Z)-4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid (1-8-5)

[0346] At 20°C, compound 1-8-4 (1.80 g, 6.86 mmol) was dissolved in THF (20 mL) and water (5 mL). Vinyl acetic 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 purged with nitrogen and heated to 70°C for 5 h. The reaction was monitored by HPLC-MS / MS. 1N sodium hydroxide solution was added to the reaction solution to adjust the pH to 8, and ethyl acetate was added for extraction. The aqueous phase was adjusted to pH 3 with 1N hydrochloric acid and extracted with ethyl acetate. 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 used directly in the next step.

[0347] Step 5: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)butyric acid (1-8-6)

[0348] Compound 1-8-5 (2.60 g, 9.71 mmol) was dissolved in THF (50 mL) at 20°C, and Pd / C (0.52 g, 10%) was added. The system was replaced with hydrogen and reacted under a hydrogen balloon at 40°C for 2 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was filtered and the filtrate was concentrated to obtain 2.43 g of the title compound, which was used directly in the next reaction without further purification.

[0349] Step 6: Synthesis of N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-7)

[0350] Compound 1-8-6 (2.43 g, 9.01 mmol) was dissolved in trifluoroacetic acid (10 mL), cooled to 5°C, and trifluoroacetic anhydride (3.78 g, 18.02 mmol, 2.50 mL) was added dropwise. The reaction temperature was maintained at 5°C for 4 h, and the reaction was monitored by HPLC-MS / MS. The reaction solution was added to water, the pH was adjusted to 9 with 10N sodium hydroxide solution, and ethyl acetate was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified on a silica gel column (ethyl acetate:petroleum ether = 0-20%) to obtain 1.53 g of the title compound.

[0351] 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)

[0352] At 5°C, potassium tert-butoxide (1.50 g, 13.37 mmol) was dissolved in THF (16 mL) and tert-butanol (4 mL). A THF solution (16 mL) of compound 1-8-7 (1.53 g, 6.08 mmol) was added dropwise. After 10 minutes, amyl nitrite (1.14 g, 9.73 mmol) was added dropwise. The reaction was maintained at 5°C for 1 hour and monitored by HPLC-MS / MS. The reaction solution was adjusted to pH 5 with 1N hydrochloric acid and extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was slurried with methyl tert-butyl ether to obtain 1.20 g of the title compound.

[0353] Step 8: Synthesis of N-(7-amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-9)

[0354] Compound 1-8-8 (0.50 g, 1.78 mmol) was dissolved in methanol (8 mL) and 2N hydrochloric acid (8 mL) at 20°C. Pd / C (0.15 g, 10%) was added. The system was replaced with hydrogen and maintained at 5°C under a hydrogen balloon for 2 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was filtered and concentrated to obtain 0.52 g of the hydrochloride salt of the title compound, which was used directly in the next reaction without further purification.

[0355] Step 9: Synthesis of N,N'-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (1-8-10)

[0356] Compound 1-8-9 (0.52 g, 1.70 mmol) was dissolved in pyridine (5 mL) at 20°C, and acetic anhydride (2 mL) was added. The reaction was maintained at 20°C for 2 h, and the reaction was monitored by HPLC-MS / MS. The reaction solution was added to water and extracted with ethyl acetate. The organic phases were washed with water, combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified on a silica gel column (ethyl acetate:petroleum ether = 0-30%) to obtain 0.22 g of the title compound.

[0357] Step 10: Synthesis of N-(8-amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-8-11)

[0358] Compound 1-8-10 (450.97 mg, 1.46 mmol) was dissolved in methanol (16 mL) at 20°C, 2N hydrochloric acid (16 mL) was added, and the mixture was heated to 60°C for 2 h. The reaction was monitored by HPLC-MS / MS. Saturated sodium bicarbonate solution was added to the cooled reaction solution to adjust the pH to 8, and the mixture was extracted with ethyl acetate. 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 used directly in the next step without further purification.

[0359] 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)

[0360] Compound 1-8-11 (230.00 mg, 0.78 mmol) was dissolved in toluene (10 mL), and (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. The mixture was heated to 140°C for 5 h and monitored by HPLC-MS / MS. The reaction solution was concentrated and the crude product was purified on a silica gel column (methanol:dichloromethane = 0-10%) to obtain 150.00 mg of the title compound.

[0361] 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)

[0362] 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. The reaction was monitored by HPLC-MS / MS. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (conditions as follows). The preparative solution was freeze-dried to obtain the hydrochloride salt of the title compound 1-2. The hydrochloride salt of 1-2 was separated under the following purification conditions to yield two isomers, designated 1-2-A (trifluoroacetate salt 5.00 mg, retention time 9.85 min) and 1-2-B (trifluoroacetate salt 7.00 mg, retention time 10.62 min) based on their retention times.

[0363] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0364] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0365] The structural characterization data are as follows:

[0366] 1-2-A:

[0367] 1 H NMR(400MHz,DMSO-d6)δ8.42(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=7.2Hz,3H).

[0368] ESI-MS (m / z): 452.1 [M+H] + .

[0369] 1-2-B:

[0370] 1 H NMR(400MHz,DMSO-d6)δ8.42(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=7.2Hz,3H).

[0371] ESI-MS (m / z): 452.0 [M+H] + .

[0372] Step 13: 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 Synthesis of (1-8-13-A and 1-8-13-B)-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

[0373] At 25 ° C, the hydrochloride of compound 1-2 (40.00 mg, 81.91 μmol) was dissolved in N, N-dimethylformamide (1 mL), and 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 in sequence. The reaction was maintained at 25 ° C for 0.5 hour and the reaction was monitored by high performance liquid chromatography-mass spectrometry. After the reaction is completed, water is added to the reaction solution, and the mixture is extracted with dichloromethane / methanol (v / v=10 / 1). The organic phases are combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product is purified by preparative thin layer chromatography (dichloromethane:methanol=20:1) to separate and obtain two isomers. The two isomers are named 1-8-13-A (15.00 mg, Rf value 0.3) and 1-8-13-B (12.00 mg, Rf value 0.35) based on the Rf value.

[0374] 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)

[0375] At 25°C, 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. A mixture of tetrabutylammonium fluoride (1 M solution in tetrahydrofuran) and glacial acetic acid (v / v = 13 / 1) (50 μL) was added dropwise. The reaction was maintained at 25°C for 0.5 hours and monitored by HPLC-MS / MS. After completion of the reaction, the reaction solutions were purified by preparative HPLC, and the preparative solutions were lyophilized to give the title compounds 1-8-A (6.94 mg) and 1-8-B (4.00 mg), respectively.

[0376] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0377] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0378] The structural characterization data of 1-8-A are as follows:

[0379] 1H NMR (400MHz, DMSO-d6) δ8.43(d,J=8.8Hz,1H),8.16(s,1H),7.31(s,1H),6.55(s,1H),5.65-5.36(m,4H),5.21(q,J=19.0H z,2H),3.95(d,J=5.7Hz,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=7.3Hz,3H).

[0380] ESI-MS (m / z): 510.1 [M+H] + .

[0381] The structural characterization data of 1-8-B are as follows:

[0382] 1H NMR (400MHz, DMSO-d6) δ8.45(d,J=8.9Hz,1H),8.15(s,1H),7.31(s,1H),6.54(s,1H),5.64-5.35(m,4H),5.19(q,J=19.0H z,2H),3.97(d,J=5.2Hz,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=7.3Hz,3H).

[0383] ESI-MS (m / z): 510.1 [M+H] +.

[0384] 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-ynamide and N-((1 0S)-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)hex-5-ynamide (A-07-A and A-07-B)

[0385] Step 1: Synthesis of (S)-10-benzyl-23-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosane-22-ynecarboxylic acid (A-07-3)

[0386] Compound A-07-2 (30.00 mg, 0.07 mmol) was dissolved in DMF (0.2 mL) at 25°C, and 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (A-07-1, 28.00 mg, 0.08 mmol) was added. The mixture was reacted at 30°C for 1 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly purified by preparative HPLC (conditions as follows) and the preparative solution was freeze-dried to obtain 20.00 mg of the title compound.

[0387] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

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

[0389] The structural characterization data are as follows:

[0390] ESI-MS (m / z): 691.0 [M+H2O] +.

[0391] Step 2: 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-ynamide and N-((10 Synthesis of S)-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)hex-5-ynamide (A-07-A and A-07-B)

[0392] At 25°C, the hydrochloride salt of 1-2 (30.00 mg, 61.43 μmol) was dissolved in N,N-dimethylformamide (1 mL). 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 sequentially. The mixture was maintained at 25°C for 0.5 hours and monitored by HPLC-MS / MS. After completion, the reaction solution was purified by preparative HPLC (conditions listed below) and freeze-dried to obtain the title compound A-07. A-07 was separated under the following purification conditions to yield two isomers, designated A-07-A (11.04 mg, retention time 7.5 min) and A-07-B (19.42 mg, retention time 8.0 min) based on their retention times.

[0393] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0394] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0395] The structural characterization data are as follows:

[0396] A-07-A:

[0397] ESI-MS (m / z): 1107.3 [M+H] + .

[0398] A-07-B:

[0399] ESI-MS (m / z): 1107.3 [M+H] + .

[0400] Example 4 (S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)

[0401] Step 1: Synthesis of 3-(isopropylamino)-1-(6-nitrobenzo[d][1,3]dioxin-5-yl)propan-1-one (2-2-2)

[0402] 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 for 1 hour. 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.

[0403] The structural characterization data are as follows:

[0404] ESI-MS (m / z): 281.1 [M+H] + .

[0405] Step 2: Synthesis of 1-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-(isopropylamino)propan-1-one (2-2-3)

[0406] The formate salt of compound 2-2-2 (1.25 g, 4.46 mmol) was added to tetrahydrofuran (20 mL), followed by 10% palladium on carbon (125.00 mg). The mixture was replaced with hydrogen three times and allowed to react at 25°C for 16 hours. The reaction mixture was filtered through celite, and the filtrate was concentrated to dryness under reduced pressure to obtain 895.00 mg of the crude title compound, which was used directly in the next reaction without purification.

[0407] The structural characterization data are as follows:

[0408] ESI-MS (m / z): 251.1 [M+H] + .

[0409] Step 3: Synthesis of ((S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)

[0410] 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 mixture was reacted at 140°C for 12 hours. The solvent was evaporated under reduced pressure to obtain a crude product of the title compound, which was purified by HPLC (mobile phase A: acetonitrile, mobile phase B: 0.05% formic acid aqueous solution). 3 drops of 3M 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.

[0411] The structural characterization data are as follows:

[0412] ESI-MS (m / z): 478.2 [M+H] + .

[0413] 1 H-NMR (400MHz, DMSO-d6): δ9.38(brs,2H),7.85(s,1H),7.52(s,1H),7.25(s,1H),6.30(d,J=2.0Hz,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=6.4Hz,6H),0.87(t,J=7.6Hz,3H).

[0414] Example 5 Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropyl-N-methylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (B-01)

[0415] Step 1: Synthesis of (S)-4-ethyl-11-(2-(N-isopropyl-N-methylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxy-3,9-diazapentatriacontamido)benzyl)carbonate (B-01-2)

[0416] At room temperature, compound B-01-1 (413.40 mg, 0.251 mmol, its synthesis reference patent CN111295389B) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), and cuprous bromide (72.95 mg, 0.503 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (95.10 mg, 0.302 mmol) were added. The reaction was stirred for 1 h and then filtered. The filtrate was purified by preparative high performance liquid chromatography (conditions as follows) to obtain 30.00 mg of the title compound.

[0417] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0418] Mobile phase A: acetonitrile; mobile phase B: water

[0419] The structural characterization data are as follows:

[0420] ESI-MS(m / z):815.9[(M-273) / 2+H] + .

[0421] Step 2: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropyl-N-methylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (B-01)

[0422] Compound B-01-2 (30.00 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL). Trifluoroacetic acid (0.2 mL) was added to the reaction mixture and allowed to react at room temperature for 30 min. The reaction mixture was concentrated under reduced pressure and purified by preparative HPLC (conditions as follows) to obtain 20.00 mg of the trifluoroacetate salt of the title compound.

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

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

[0425] The structural characterization data are as follows: ESI-MS (m / z): 816.0 [M / 2+H] + .

[0426] 1H NMR (400MHz, DMSO-d6) δ10.18(s,1H),9.10(s,2H),8.38(t,J=5.56Hz,1H),8.32(d,J=8.40Hz,1H),8.22- 8.20(m,2H),8.09(t,J=5.68Hz,1H),7.91-7.87(m,2H),7.82-7.78(m,1H),7.69(brs,3H),7.61(d,J=8.5 6Hz,2H),7.32(d,J=8.56Hz,2H),7.06(s,1H),5.56(d,J=16.96Hz,1H),5.51(d,J=16.96Hz,1H),5.47(d, J=19.28Hz,1H),5.42(d,J=19.28Hz,1H),5.14(d,J=12.20Hz,1H),5.07(d,J=12.16Hz,1H),4.48(t,J=5. 24Hz,2H),4.46-4.43(m,1H),4.29(d,J=5.60Hz,2H),4.08-3.95(m,5H),3.79(t,J=5.28Hz,2H),3.51-3. 43(m,32H),3.40(s,3H),3.39-3.35(m,2H),3.30-3.26(m,2H),3.00(s,3H),2.82-2.74(m,2H),2.56(t,J =7.08Hz,2H),2.29(t,J=7.36Hz,2H),2.23-2.13(m,2H),1.82(p,J=7.24Hz,2H),1.78-1.63(m,2H),1.61 -1.49(m,2H),1.42-1.27(m,2H),1.15(d,J=6.80Hz,3H),1.13(d,J=6.76Hz,3H),0.90(t,J=7.32Hz,3H).

[0427] Example 6 (2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(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)

[0428] 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)

[0429] 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 (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 with stirring. After nitrogen substitution, the mixture was allowed to react at room temperature in the dark for 12 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:3) to obtain 12.80 g of the title compound.

[0430] The structural characterization data are as follows:

[0431] ESI-MS (m / z): 503 [M+18] + .

[0432] 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)

[0433] Compound B-03-3 (2.20 g, 4.53 mmol) was dissolved in ethyl acetate and tetrahydrofuran (50 mL each), and PtO2 (0.20 g) was added. The reaction system was then purged with a hydrogen balloon three times and allowed to react under a hydrogen atmosphere for 2 hours. The reaction was monitored by HPLC-MS / MS. The reaction mixture was filtered, the filter cake was rinsed with ethyl acetate, and the filtrate was evaporated to dryness under reduced pressure to obtain 2.02 g of the crude title compound, which was used directly in the next reaction.

[0434] The structural characterization data are as follows:

[0435] ESI-MS (m / z): 456.1 [M+1] + .

[0436] Step 3: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(2-(2-(2-(9H-fluoren-9-ylmethoxycarbonyl)amino)ethoxy)ethoxy)acetamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-5)

[0437] 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, 2.00 mmol) was added with stirring. The mixture was stirred for 2 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (methanol:dichloromethane = 1:20) to obtain 507.00 mg of the title compound.

[0438] The structural characterization data are as follows:

[0439] ESI-MS (m / z): 823.3 [M+1] + .

[0440] Step 4: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(2-(2-(2-(9H-fluoren-9-ylmethoxycarbonyl)amino)ethoxy)ethoxy)acetamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-6)

[0441] Compound B-03-5 (507.00 mg, 616.18 μmol) and diisopropylethylamine (238.91 mg, 1.85 mmol) were dissolved in dichloromethane (20 mL). p-Nitrophenyl chloroformate (372.60 mg, 1.85 mmol) was then dissolved in dichloromethane (1 mL) and slowly added dropwise to the reaction mixture. The mixture was allowed to react at room temperature for 15 h. The reaction was monitored by HPLC-MS / MS. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (methanol:dichloromethane = 1:20) to obtain 496.00 mg of the title compound.

[0442] The structural characterization data are as follows:

[0443] ESI-MS (m / z): 988.5 [M+1] + .

[0444] Step 5: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(2-(2-(2-(9H-fluoren-9-ylmethoxycarbonyl)amino)ethoxy)ethoxy)acetamido)-4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxy-8,10,11,13-tetrahydro-7H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-7)

[0445] 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), and diisopropylethylamine (32.48 mg, 0.25 mmol) was added dropwise. The mixture was stirred for 12 h and monitored by HPLC-MS / MS. Water and ethyl acetate were added and stirred, and the mixture was allowed to stand for separation. The organic phase was washed with saturated brine and dried, and concentrated under reduced pressure to obtain 100.00 mg of the crude title compound, which was directly used for the next step.

[0446] The structural characterization data are as follows:

[0447] ESI-MS (m / z): 1326.2 [M+1] + .

[0448] Step 6: Synthesis of (2S,3S,4S,5R,6S)-6-(2-(2-(2-(aminoethoxy)ethoxy)acetamido)-4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxy-8,10,11,13-tetrahydro-7H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (B-03-8)

[0449] Compound B-03-7 (100.00 mg, 0.08 mmol) was dissolved in MeOH (5 mL), and 1 drop of dichloromethane was added dropwise. An aqueous solution of lithium hydroxide monohydrate (15.82 mg, 0.377 mmol) (1 mL) was then added dropwise. The mixture was stirred and reacted for 2 hours. The reaction was monitored by HPLC-MS / MS. 3N aqueous hydrochloric acid was added dropwise to adjust the pH of the reaction solution to 4. The solution was concentrated under reduced pressure and purified by preparative HPLC (conditions as follows). The preparative solution was freeze-dried to obtain 27.00 mg of the title compound.

[0450] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

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

[0452] The structural characterization data are as follows:

[0453] ESI-MS (m / z): 964.2 [M+1] + .

[0454] Step 7: Synthesis of (2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(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)

[0455] Compound B-03-8 (27.00 mg, 0.03 mmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoate (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 with stirring. The mixture was allowed to react at room temperature for 4 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was purified by preparative HPLC (conditions as follows) and the preparative solution was freeze-dried to obtain 11.70 mg of the title compound.

[0456] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0457] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0458] The structural characterization data are as follows:

[0459] ESI-MS (m / z): 1214.4 [M+1] + .

[0460] Example 7 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)

[0461] Step 1: Synthesis of 3-bromo-4-chloro-5-fluoroaniline (1-5-02)

[0462] Compound 1-5-01 (2.00 g, 10.53 mmol) was dissolved in N,N-dimethylformamide (30 mL), and N-chlorosuccinimide (1.69 g, 12.63 mmol) was slowly added. The reaction was allowed to react at room temperature for 16 hours, and the reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was then purified on a flash silica gel column (ethyl acetate:petroleum ether = 0-25%) to obtain 0.95 g of the title compound.

[0463] The structural characterization data are as follows:

[0464] 1 H NMR (400MHz, DMSO-d6) δ6.77 (dd, J=2.5, 1.4Hz, 1H), 6.51 (dd, J=11.7, 2.5Hz, 1H), 5.84 (s, 2H).

[0465] Step 2: Synthesis of N-(3-bromo-4-chloro-5-fluorophenyl)acetamide (1-5-03)

[0466] Compound 1-5-02 (0.95 g, 4.23 mmol) was dissolved in ethyl acetate (20 mL). Acetic anhydride (648.13 mg, 6.35 mmol) was added under nitrogen. After addition, the temperature was raised to 50°C and the reaction mixture was reacted for 15 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was quenched with methanol (5 mL) and evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified on a flash silica gel column (ethyl acetate:petroleum ether = 0-40%) to obtain 1.01 g of the title compound.

[0467] The structural characterization data are as follows:

[0468] ESI-MS (m / z): 265.9 [M+H] + .

[0469] Step 3: Synthesis of (E)-4-(5-acetamido-2-chloro-3-fluorophenyl)-3-butenoic acid (1-5-04)

[0470] Compound 1-5-03 and 3-butenoic acid (387.65 mg, 4.50 mmol) were dissolved in a mixed solvent of 1,4-dioxane (24 mL) and water (8 mL). N,N-diisopropylethylamine (1.45 g, 11.26 mmol), tris(o-methylphenyl)phosphine (114.21 mg, 375.24 μmol), and palladium acetate (42.12 mg, 187.62 μmol) were then added. After the addition, the reaction system was purged with nitrogen three times and heated to 100°C under a nitrogen atmosphere for 16 hours. The reaction was monitored by high-performance liquid chromatography-mass spectrometry. After the reaction solution was cooled to room temperature, 1N aqueous sodium hydroxide solution (60 mL) and ethyl acetate (50 mL) were added and the layers were shaken. After separating the lower aqueous phase, the pH was adjusted to about 3 with a 4 mol / L hydrochloric acid aqueous solution, then extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 1.00 g of a crude product of the title compound.

[0471] The structural characterization data are as follows:

[0472] ESI-MS (m / z): 272.0 [M+H] + .

[0473] Step 4: Synthesis of 4-(5-acetamido-2-chloro-3-fluorophenyl)butyric acid (1-5-05)

[0474] The crude product of compound 1-5-04 (1.00 g, 3.68 mmol) was dissolved in tetrahydrofuran (15 mL), and 10% palladium on carbon (0.10 g) was added. After the addition was complete, the reaction system was purged with a hydrogen balloon three times and allowed to react under a hydrogen atmosphere for 4 hours. The reaction was monitored by HPLC-MS / MS. 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.

[0475] The structural characterization data are as follows:

[0476] ESI-MS (m / z): 274.0 [M+H] + .

[0477] Step 5: Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-06)

[0478] 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 complete, the reaction was maintained at 5°C for 2 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was slowly poured into water, then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by flash silica gel column to obtain 0.43 g of the title compound.

[0479] The structural characterization data are as follows:

[0480] ESI-MS (m / z): 256.1 [M+H] + .

[0481] Step 6: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-07)

[0482] Tetrahydrofuran (16 mL) and tert-butanol (4 mL) were added to a reaction flask. 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 slowly added dropwise to the reaction solution. After 10 minutes, isoamyl nitrite (315.24 mg, 2.69 mmol) was added. After the addition was complete, the reaction was maintained at 5°C for 1 hour. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was quenched with 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. The filtrate was concentrated under reduced pressure to obtain 455.00 mg of a crude product of the title compound.

[0483] The structural characterization data are as follows:

[0484] ESI-MS (m / z): 285.0 [M+H] + .

[0485] Step 7: Synthesis of N-(7-amino-4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-08)

[0486] The crude product of compound 1-5-07 (0.40 g, 1.41 mmol) was dissolved in methanol (10 mL), and then 3 mol / L aqueous hydrochloric acid solution (1 mL) and 10% palladium on carbon (40.00 mg) were added. After the addition, the reaction system was replaced with hydrogen three times and reacted at room temperature under a hydrogen atmosphere for 1 hour. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 0.43 g of the crude hydrochloride salt of the title compound.

[0487] The structural characterization data are as follows:

[0488] ESI-MS (m / z): 271.0 [M+H] + .

[0489] Step 8: Synthesis of (9H-fluoren-9-ylmethyl)(8-acetamido-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (1-5-09)

[0490] The crude hydrochloride of compound 1-5-08 (0.43 g, 1.19 mmol) was dissolved in 1,4-dioxane (15 mL), followed by the addition of sodium bicarbonate (400.35 mg, 4.77 mmol), water (5 mL), and 9-fluorenylmethyl-N-succinimidyl carbonate (481.81 mg, 1.43 mmol). The mixture was stirred at room temperature for 2 hours and monitored by HPLC-MS / MS. The reaction mixture was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified on a C18 reverse-phase column (acetonitrile: 0.05% formic acid in water = 20%-100%) to obtain 301.00 mg of the title compound.

[0491] The structural characterization data are as follows:

[0492] ESI-MS (m / z): 493.2 [M+H] + .

[0493] Step 9: Synthesis of (9H-fluoren-9-ylmethyl)(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (1-5-10)

[0494] Compound 1-5-09 (300.00 mg, 608.61 μmol) was dissolved in dioxane (5 mL) and 12 mol / L concentrated hydrochloric acid (1 mL) was added. After addition, the temperature was raised to 60°C and the reaction mixture was reacted for 2 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified on a flash silica gel column (ethyl acetate:petroleum ether = 0-50%) to obtain 198.00 mg of the title compound.

[0495] The structural characterization data are as follows:

[0496] ESI-MS (m / z): 451.1 [M+H] + .

[0497] Step 10: Synthesis of (9H-fluoren-9-ylmethyl)((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)carbamate (1-5-11)

[0498] (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), and then p-toluenesulfonic acid (75.53 mg, 439.13 μmol) was added. After the addition was completed, the temperature was raised to 140 ° C. and the reaction was reacted for 4 hours. The reaction solution was directly evaporated to dryness under reduced pressure at 140 ° C. to obtain a crude product, which was purified by flash silica gel column (methanol: dichloromethane = 0-5%) to obtain 256.00 mg of the title compound.

[0499] The structural characterization data are as follows:

[0500] ESI-MS (m / z): 678.1 [M+H] + .

[0501] 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)

[0502] Compound 1-5-11 (201.18 mg, 296.67 μmol) was dissolved in N,N-dimethylformamide (4 mL), and diethylamine (108.49 mg, 1.48 mmol) was added. After the addition was complete, the mixture was reacted at room temperature for 0.5 hours, and the reaction was monitored by HPLC-MS / MS. The ethylenediamine was evaporated under reduced pressure, and the pH of the reaction solution was adjusted to 2-3 with 1 mol / L aqueous hydrochloric acid. The reaction solution was then directly purified by preparative HPLC to obtain the title compounds 1-5-A (44.00 mg) and 1-5-B (43.00 mg).

[0503] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0504] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0505] 1-5-A (LCMS peak at the front at 6 min, retention time: 1.276 min)

[0506] The structural characterization data are as follows:

[0507] 1 H NMR (400MHz, DMSO-d6) δ8.00(d,J=10.3Hz,1H),7.33(s,1H),6.54(s,1H),5.62(d,J=19.3Hz,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=7.3Hz,3H).

[0508] ESI-MS (m / z): 456.1 [M+H] + .

[0509] The structural characterization data of 1-5-B (6 min LCMS peak later, retention time: 1.300 min) are as follows:

[0510] 1 H NMR (400MHz, DMSO-d6) δ7.98(d,J=10.3Hz,1H),7.32(s,1H),5.61(d,J=19.4Hz,1H),5.44(s,2H),5.32(d,J=19 .4Hz,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=7.3Hz,3H).

[0511] ESI-MS (m / z): 456.1 [M+H] + .

[0512] 6 min LCMS conditions:

[0513] Column: Waters SunFire C18 OBD 4.6 mm × 50 mm × 5.0 μm

[0514] Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid)

[0515] Step 12: N-((S)-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-ynamide and N-((S)- Synthesis of 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)

[0516] The single-configuration compound 1-5-A (36.00 mg, 79.70 μmol) and compound A-07-3 (64.43 mg, 95.64 μmol) were dissolved in N,N-dimethylformamide (2 mL). 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were then added. The mixture was reacted at room temperature for 1 hour. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly purified by HPLC to obtain the single-configuration title compound 1-5-12-A (51.00 mg).

[0517] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0518] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0519] The structural characterization data are as follows:

[0520] ESI-MS (m / z): 1111.0 [M+H] + .

[0521] The single-configuration compound 1-5-B (36.00 mg, 79.70 μmol) and compound A-07-3 (64.43 mg, 95.64 μmol) were dissolved in N,N-dimethylformamide (2 mL). 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were then added. The mixture was reacted at room temperature for 1 hour. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly purified by HPLC to obtain the single-configuration title compound 1-5-12-B (52.00 mg).

[0522] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0523] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0524] The structural characterization data are as follows:

[0525] ESI-MS (m / z): 1111.0 [M+H] + .

[0526] 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)

[0527] 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). A 4 mol / L hydrochloric acid solution in ethyl acetate (1 mL) was then added. The mixture was allowed to react at room temperature for 0.5 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly concentrated to dryness 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.

[0528] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

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

[0530] The structural characterization data are as follows:

[0531] 1 H NMR (400MHz, DMSO-d6) δ8.50(d,J=8.9Hz,1H),8.05(d,J=10.3Hz,1H),7.33(s,1H),6.55(s,1H),5.67-5.60(m,1H),5.49(t,J=5.8Hz, 1H),5.43(s,2H),5.21(s,2H),3.96(d,J=5.8Hz,2H),3.32-3.22(m,2H),2.28-2.15(m,2H),1.93-1.80(m,2H),0.87(t,J=7.3Hz,3H).

[0532] ESI-MS (m / z): 514.0 [M+H] + .

[0533] 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). 4 mol / L hydrochloric acid and ethyl acetate (1 mL) were then added. The mixture was allowed to react at room temperature for 0.5 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly concentrated under reduced pressure to dryness to obtain a crude product, which was purified by HPLC to obtain the title compound 1-11-B (8.24 mg) in a single configuration.

[0534] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0535] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0536] The structural characterization data are as follows:

[0537] 1 H NMR (400MHz, DMSO-d6) δ8.52(d,J=9.0Hz,1H),8.05(d,J=10.3Hz,1H),7.34(s,1H),6.55(s,1H),5.68-5.58(m,1H),5.53(t,J=5.8Hz,1H),5.43 (d,J=2.9Hz,2H),5.20(d,J=7.3Hz,2H),3.97(d,J=5.7Hz,2H),3.31-3.21(m,2H),2.26-2.15(m,2H),1.92-1.82(m,2H),0.87(t,J=7.3Hz,3H).

[0538] ESI-MS (m / z): 514.0 [M+H] + .

[0539] Example 8 N-((10S,19S)-23-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)-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)-1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazatricosan-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-triacontane-38-carboxamide or N -((10S,19S)-23-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)-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)-1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazatricosan-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxatriacontane-38-carboxamide (A-17-A)

[0540] Step 1: Synthesis of (2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38-octadecanecarboxamide)-6-(N-(9H-fluoren-9-ylmethoxycarbonyl)amino)hexanoic acid (A-17-03)

[0541] The hydrochloride salt of compound A-17-02 (389.68 mg, 962.45 μmol) was dissolved in dichloromethane (8 mL). 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 hours. 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 HPLC to obtain the title compound A-17-03 (450.00 mg).

[0542] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0543] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0544] ESI-MS (m / z): 939.3 [M+H] + .

[0545] Step 2: Synthesis of 2-((2-((2S)-2-(2-(2-((2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-dodeca-triacontane-38-amido)-6-(N-(9H-fluoren-9-ylmethoxycarbonyl)amino)hexanamido)acetamido)acetamido)-3-phenylpropionamido)acetamido)methoxy)acetic acid (A-17-04)

[0546] Compound A-17-03 (50.00 mg, 118.09 μmol) was dissolved in DMF (2.5 mL), and 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. The mixture was reacted at 25°C for 1 hour. The solvent was evaporated under reduced pressure, and the concentrate was purified by preparative HPLC to obtain the title compound A-17-04 (40.00 mg).

[0547] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0548] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0549] ESI-MS (m / z): 1344.4 [M+H] + .

[0550] Step 3: (9H-fluoren-9-ylmethyl)((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- Synthesis of [b]quinolin-1-yl)amino]-1,6,9,12,15-pentaoxy-3-oxa-5,8,11,14-tetraazahexadec-16-yl)carbamoyl)-38-oxo-2,5,8,11,14,17,20,23,26,29,35-dodeca-39-aza-44-yl)carbamate (A-17-05)

[0551] Compound A-17-04 (29.86 mg, 59.50 μmol) was dissolved in DMF (3 mL), and HATU (27.15 mg, 71.40 μmol), compound 1-5-A (80.00 mg, 59.50 μmol), and DIPEA (38.45 mg, 297.51 μmol, 52.96 μL) were added. The mixture was reacted at 25°C for 1 hour. The solvent was evaporated under reduced pressure, and the concentrate was purified by preparative HPLC to obtain the title compound A-17-05 (50.00 mg).

[0552] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0553] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0554] ESI-MS (m / z): 1781.6 [M+H] + .

[0555] 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]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazatricosan-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoctatriacontane-38-amide (A-17-06)

[0556] 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 hours. The solvent was evaporated 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).

[0557] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0558] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0559] ESI-MS (m / z): 1559.7 [M+H] +.

[0560] Step 5: N-((10S,19S)-23-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)-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)-1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazatricosan-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoctatrioxane-38-amide or N-( (10S,19S)-23-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)-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 Synthesis of (1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazatricosan-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoctatriacontane-38-carboxamide (A-17-A)

[0561] 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. The mixture was reacted at 25°C for 2 hours. The solvent was evaporated under reduced pressure, and the concentrate was purified by preparative HPLC to obtain the title compound A-17-A (5.60 mg).

[0562] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0563] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0564] ESI-MS (m / z): 1809.8 [M+H] + .

[0565] Example 9: N-((S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxyl-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-05)

[0566] Under nitrogen protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (A-07-1, 0.66 g, 1.80 mmol) and A-07-2 (0.75 g, 1.77 mmol) were added to DMF (19 mL), heated to 35 ° C for 16 hours, and (1S, 9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2 ,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (1-4 (i.e., 1-2-A), 1.00 g, 1.77 mmol), ice water was cooled to 5-15°C, DMTMM (0.98 g, 3.53 mmol) was added, and then DIPEA (1.14 g, 8.84 mmol) was added dropwise. The reaction mixture was reacted at 25°C for 16 hours. The reaction solution was poured into a mixture of DCM (600 mL), IPA (60 mL), and water (100 mL) and stirred for 10 minutes. The DCM phase was separated, washed with brine (100 ml), and concentrated to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography and freeze-dried to obtain 0.98 g of compound A-05 (i.e., A-07-A in Example 3).

[0567] A-05 separation and purification method is as follows:

[0568] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0569] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0570] A-05 structural characterization data are as follows:

[0571] MS m / z(ESI):1107.3[M+H] + .

[0572] 1 H NMR (400MHz, DMSO) δ9.10 (s, 2H), 8.66-8.63 (m, 1H), 8.51 (d, J = 8.8Hz, 1H), 8.34-8.31 (m, 1H), 8.21-8.19 (m, 1H), 8.17-8.09 (m, 2 H),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),4.6 4(d,J=6.4Hz,2H),4.45-4.44(m,1H),4.06-3.98(m,2H),3.77-3.52(m,6H),3.41(s,3H),3.25-3.12(m,2H),3.03-3.00(m,1H),2 .83-2.72(m,1H),2.58-2.56(m,2H),2.48(s,3H),2.33-2.30(m,2H),2.21-2.13(m,2H),1.91-1.76(m,4H),0.87(t,J=7.2Hz,3H).

[0573] The following HPLC conditions were used to detect compounds A-07-A and A-07-B prepared in Example 3; as well as compound A-07-A prepared in Example 3 and compound A-05 prepared in Example 9:

[0574] Instrument: Agilent 1260 HPLC with VWD detector

[0575] Chromatographic column: Waters Xbridge C18 4.6*100mm*3.5μm

[0576] Mobile phase A: 0.01M ammonium phosphate aqueous solution / acetonitrile = 90 / 10; Mobile phase B: acetonitrile

[0577] The results showed that the retention times of compound A-07-A and compound A-07-B were 6.6 min and 6.8 min, respectively, indicating that the above HPLC conditions had good separation of isomers;

[0578] The results showed that compound A-07-A prepared in Example 3 and compound A-05 prepared in Example 9 showed one peak in HPLC with a retention time of 6.6 min, indicating that compound A-07-A was compound A-05.

[0579] 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]indolizin[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetrahydro-3-oxo-5,8,11-triazatetradec-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amide (A-26)

[0580] Step 1:

[0581] 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 then added and stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 700 mg of compound A-26-2.

[0582] The preparation method is as follows:

[0583] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0584] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0585] Step 2:

[0586] Compound A-26-2 (500 mg, 0.513 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (75.05 mg, 1.03 mmol) was added. The mixture was reacted at room temperature for 1 hour. After the reaction, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 307 mg of compound A-26-3.

[0587] The preparation method is as follows:

[0588] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0589] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0590] Step 3:

[0591] 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 mixture was stirred at room temperature for 16 hours. The reaction mixture was directly purified by preparative HPLC and freeze-dried to obtain 50.56 mg of compound A-26.

[0592] Its structural characterization data are as follows:

[0593] MS m / z(ESI):1002.4[M+H] + .

[0594] The separation and purification method is as follows:

[0595] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0596] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0597] 1 H NMR (400MHz, DMSO) δ9.11(s,2H),8.68(t,J=6.4Hz,1H),8.49(d,J=8.8Hz,1H),8.16(s,1H),8.10(d,J=7.2H z,1H),8.01(d,J=7.2Hz,1H),7.91(d,J=6.8Hz,1H),7.31(s,1H),6.55(s,1H),5.65-5.55(m,1H),5.43(s,2H ),5.21(s,2H),4.67-4.55(m,2H),4.29-4.15(m,3H),3.98(s,2H),3.41(s,3H),3.25-3.15(m,2H),2.57-2.5 6(m,2H),2.35-2.27(m,2H),2.22-2.12(m,2H),1.91-1.75(m,4H),1.23-1.09(m,9H),0.87(t,J=7.2Hz,3H).

[0598] Example 11: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-04)

[0599] Step 1: Preparation of ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetate (B-04-1)

[0600] Dissolve (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2 g, 3.65 mmol) in DMF (50 mL). Add DIPEA (1.18 g, 9.12 mmol, 1.59 mL) dropwise. Add acetoxyacetyl chloride (548.12 mg, 4.01 mmol, 431.59 μL) dropwise with ice-cooling and stirring. Continue stirring for 1 hour. Add the reaction solution to 0.1 M dilute hydrochloric acid to precipitate a solid, which is then filtered. The filter cake was dissolved in dichloromethane and methanol, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (methanol / dichloromethane = 0% to 5%) and concentrated again to obtain the title compound (1.7 g, 3.077 mmol).

[0601] Its structural characterization data are as follows:

[0602] ESI-MS (m / z): 552.2 [M+1] + .

[0603] Step 2: Preparation of ethyl 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidin-1-yl)amino)-2-oxoacetate (B-04-2)

[0604] Ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetate (500 mg, 0.905 mmol) and DMAP (885.33 mg, 7.25 mmol) were dissolved in dry dichloromethane (5 mL), cooled to 0 ° C under nitrogen protection, and a dichloromethane solution (5 mL) of triphosgene (268.81 mg, 0.905 mmol) was added dropwise, and the reaction was stirred for 0.5 hour. A solution of (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatrinitroamino)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (1.44 g, 1.36 mmol) in dichloromethane was slowly added dropwise and allowed to react at room temperature for 4 hours. The reaction was quenched with water and extracted three times with dichloromethane (100 ml x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification on a silica gel column (MeOH / DCM = 0% to 5%) afforded the title compound (498 mg, 0.304 mmol).

[0605] Its structural characterization data are as follows:

[0606] ESI-MS (m / z): 1352.8 [M+1] + .

[0607] Step 3: Preparation of 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-04-3)

[0608] 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,1 Ethyl 2-oxoacetate (200 mg, 0.122 mmol) (0,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidin[1,2-b]quinolin-1-yl)amino) was dissolved in THF (3 mL) and MeOH (3 mL). A 1 mL aqueous solution of sodium carbonate (25.88 mg, 0.224 mmol) was added dropwise with stirring. Stirring was continued for 1 hour after the addition was complete. Dilute hydrochloric acid was added dropwise to the reaction mixture to neutralize the reaction. After concentration under reduced pressure, the mixture was directly transferred to the next step.

[0609] Its structural characterization data are as follows:

[0610] ESI-MS (m / z): 1596.7 [M+1] + .

[0611] Step 4: Preparation of 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-04-4)

[0612] 4-((S)-35-Azido-2-(4-((4-methoxyphenyl)benzhydryl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (190 mg, 119.04 μmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (0.5 mL) was added and the reaction was continued for 1 hour. Saturated sodium bicarbonate aqueous solution was added dropwise to the reaction solution for neutralization, and the organic phase was concentrated to obtain a crude product, which was purified by reverse phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0% to 50%) and freeze-dried to obtain the title compound (95 mg, 69.35 μmol).

[0613] Its structural characterization data are as follows:

[0614] ESI-MS (m / z): 1323.6 [M+1] + .

[0615] Step 5: Preparation of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-04)

[0616] 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[ [3',4':6,7] indolizino[1,2-b]quinolin-9-yl) carbonate (90 mg, 0.066 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (24.07 mg, 0.079 mmol) were dissolved in DMSO (2 mL) and water (0.2 mL). Cuprous bromide (9.42 mg, 0.066 mmol) was added and stirring continued for 2 hours. The reaction mixture was directly filtered and the concentrated crude product was purified by preparative HPLC and freeze-dried to obtain the title compound (42.2 mg, 24.69 μmol).

[0617] Its structural characterization data are as follows:

[0618] ESI-MS (m / z): 1628.7 [M+1] + .

[0619] The preparative high performance liquid chromatography method is as follows:

[0620] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0621] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0622] Example 12: Preparation of Antibodies

[0623] In the early stage, by immunizing H2L2 mice (provided by Hebo Pharmaceuticals, the antibodies produced by these mice are chimeric antibodies consisting of fully human variable regions and mouse constant regions), human-mouse chimeric antibodies 22B6D2 (heavy chain variable region, SEQ ID NO: 15; light chain variable region, SEQ ID NO: 16), 47A3E3 (heavy chain variable region, SEQ ID NO: 32; light chain variable region, SEQ ID NO: 33) and 100H7D3 (heavy chain variable region, SEQ ID NO: 46; light chain variable region, SEQ ID NO: 47) were obtained through hybridoma screening. The above heavy chain variable region sequences were combined with the human IgG1 heavy chain constant region (SEQ ID NO: 50), and the above light chain variable region sequences were combined with the human IgG1 light chain constant region (SEQ ID NO: 51). NO:51) were combined to form three complete fully human antibodies (see Table 1), and after codon optimization, they were constructed into the pTT5 plasmid. The pTT5 plasmids corresponding to each antibody heavy chain and light chain were simultaneously transfected into CHO-S cells, and the expressed antibodies in the supernatant were purified using protein A to obtain the corresponding antibodies.

[0624] The HER3 control antibody was derived from U1-59 in patent application CN200680049887. After codon optimization, the antibody heavy and light chain nucleotide sequences were synthesized and cloned into the pTT5 vector, and expressed and purified according to the above method.

[0625] Table 1: Sequence information of 22B6D2-hIgG1, 47A3E3-hIgG1, and 100H7D3-hIgG1

[0626] Example 13: Conjugation of a Compound Comprising a Cellular Bioactive Molecule and a Linker to an Antibody

[0627] The antibodies 22B6, 47A3, and 100H7 involved in the antibody-drug conjugates prepared in the following examples are the aforementioned 22B6D2-hIgG1, 47A3E3-hIgG1, and 100H7D3-hIgG1 antibodies, respectively.

[0628] The sample was prepared by coupling as follows:

[0629] 0.46 ml of each of 22B6, 47A3, 100H7, U1-59, and hIgG1 antibodies (all adjusted to 11.0 mg / mL) was diluted with a 0.1 M edetate disodium solution (pH 7.7). The pH was then adjusted to 7.7 with a 1 M Na2HPO4 solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added, mixed, and allowed to stand at room temperature for 90 minutes. A 4.0-10-fold amount of the drug-linker compound dissolved in dimethyl sulfoxide was added to the above solution, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 10 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva). Sucrose and Tween 20 were then added and mixed to obtain the antibody-drug conjugate (ADC), as shown in Table 2.

[0630] The ADC drug / antibody ratio (DAR value) is determined as follows:

[0631] LC-MS determination of ADC sample molecular weight and calculation of drug / antibody ratio DAR value

[0632] ADC samples were subjected to LC-MS molecular weight analysis.

[0633] Chromatographic determination conditions:

[0634] Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

[0635] Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN;

[0636] Flow rate: 0.25 ml / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 2 μl;

[0637] Mass spectrometry conditions:

[0638] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0639] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 250; CE 10; Accumulation time 0.5s;

[0640] m / z 600-4000; Time bins to sum 40.

[0641] Table 2 ADC number and DAR

[0642] 1. Determine the molecular weight of ADC 22B6-A-07-A-1 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 22B6-A-07-A-1 is shown in Table 3.

[0643] Table 3: Measured molecular weight of ADC 22B6-A-07-A-1

[0644] Table 4: DAR values ​​for ADC 22B6-A-07-A-1

[0645] The calculated drug / antibody ratio of ADC 22B6-A-07-A-1 was DAR = 7.99 (see Table 4).

[0646] 2. LC-MS determination of the molecular weight of ADC 22B6-A-07-A-2 and calculation of the drug / antibody ratio (DAR) were performed. The LC-MS molecular weight analysis of ADC 22B6-A-07-A-2 is shown in Table 5.

[0647] Table 5: Measured molecular weight of ADC 22B6-A-07-A-1

[0648] Table 6: DAR values ​​for ADC 22B6-A-07-A-2

[0649] The calculated drug / antibody ratio of ADC 22B6-A-07-A-2 was DAR = 3.56 (see Table 6).

[0650] 3. Determine the molecular weight of ADC 47A3-A-07-A by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 47A3-A-07-A is shown in Table 7.

[0651] Table 7: Measured molecular weight of ADC 47A3-A-07-A

[0652] Table 8: DAR values ​​for ADC 47A3-A-07-A

[0653] The calculated drug / antibody ratio of ADC 47A3-A-07-A was DAR = 6.27 (see Table 8).

[0654] 4. Determine the molecular weight of ADC 100H7-A-07-A by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 100H7-A-07-A is shown in Table 9.

[0655] Table 9: Measured molecular weight of ADC 100H7-A-07-A

[0656] Table 10: DAR values ​​for ADC 100H7-A-07-A

[0657] The calculated drug / antibody ratio of ADC 100H7-A-07-A was DAR = 8.01 (see Table 10).

[0658] 5. Determine the molecular weight of ADC U1-59-A-07-A by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC U1-59-A-07-A is shown in Table 11.

[0659] Table 11: Measured molecular weight of ADC U1-59-A-07-A

[0660] Table 12: DAR values ​​for ADC U1-59-A-07-A

[0661] The calculated drug / antibody ratio of ADC U1-59-A-07-A was DAR=8.02 (see Table 12).

[0662] 6. LC-MS determination of the molecular weight of the ADC hIgG1-A-07-A and calculation of the drug / antibody ratio (DAR) The LC-MS molecular weight analysis of the ADC hIgG1-A-07-A is shown in Table 13.

[0663] Table 13: Measured molecular weight of ADC hIgG1-A-07-A

[0664] Table 14: DAR values ​​of ADC hIgG1-A-07-A

[0665] The drug / antibody ratio of ADC hIgG1-A-07-A(8) was calculated to be DAR=8.03 (see Table 14).

[0666] 7. Determine the molecular weight of ADC 22B6-A-01 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 22B6-A-01 is shown in Table 15.

[0667] Table 15: Measured molecular weight of ADC 22B6-A-01

[0668] Table 16: DAR values ​​for ADC 22B6-A-01

[0669] The drug / antibody ratio of ADC 22B6-A-01 was calculated to be DAR = 7.93 (see Table 16).

[0670] 8. Determine the molecular weight of ADC U1-59-A-01 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC U1-59-A-01 is shown in Table 17.

[0671] Table 17: Measured molecular weight of ADC U1-59-A-01

[0672] Table 18: DAR values ​​for ADC U1-59-A-01

[0673] The calculated drug / antibody ratio of ADC U1-59-A-01 was DAR = 7.89 (see Table 18).

[0674] 9. Determine the molecular weight of ADC 22B6-B-03-1 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 22B6-B-03-1 is shown in Table 19.

[0675] Table 19: Measured molecular weight of ADC 22B6-B-03-1

[0676] Table 20: DAR values ​​for ADC 22B6-B-03-1

[0677] The calculated drug / antibody ratio of ADC 22B6-B-03-1 was DAR = 8.0 (see Table 20).

[0678] 10. Determine the molecular weight of ADC 22B6-B-03-2 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 22B6-B-03-2 is shown in Table 21.

[0679] Table 21: Measured molecular weight of ADC 22B6-B-03-2

[0680] Table 22: DAR values ​​for ADC 22B6-B-03-2

[0681] The calculated drug / antibody ratio of ADC 22B6-B-03-2 was DAR = 3.14, see Table 22.

[0682] 11. LC-MS determination of the molecular weight of ADC hIgG1-B-03-1 and calculation of the drug / antibody ratio (DAR) value. LC-MS molecular weight analysis of ADC hIgG1-B-03-1 is shown in Table 23.

[0683] Table 23: Measured Molecular Weight of ADC hIgG1-B-03-1

[0684] Table 24: DAR values ​​of ADC hIgG1-B-03-1

[0685] The calculated drug / antibody ratio of ADC hIgG1-B-03-1 was DAR = 7.90 (see Table 24).

[0686] 12. LC-MS determination of the molecular weight of ADC hIgG1-B-03-2 and calculation of the drug / antibody ratio (DAR) The LC-MS molecular weight analysis of ADC hIgG1-B-03-2 is shown in Table 25.

[0687] Table 25: Measured Molecular Weight of ADC hIgG1-B-03-2

[0688] Table 26: DAR values ​​of ADC hIgG1-B-03-2

[0689] The calculated drug / antibody ratio of ADC hIgG1-B-03-2 was DAR = 4.20 (see Table 26).

[0690] 13. Determine the molecular weight of ADC 22B6-B-01 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 22B6-B-01 is shown in Table 27.

[0691] Table 27: Measured molecular weight of ADC 22B6-B-01

[0692] Table 28: DAR values ​​for ADC 22B6-B-01

[0693] The calculated drug / antibody ratio of ADC 22B6-B-01 was DAR = 8.07 (see Table 28).

[0694] 14. Determine the molecular weight of ADC hIgG1-B-01 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC hIgG1-B-01 is shown in Table 29.

[0695] Table 29: Measured Molecular Weight of ADC hIgG1-B-01

[0696] Table 30: DAR values ​​of ADC hIgG1-B-01

[0697] The calculated drug / antibody ratio of ADC hIgG1-B-01 was DAR = 8.06 (see Table 30).

[0698] 15. Determine the molecular weight of ADC hIgG1-A-01 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC hIgG1-A-01 is shown in the table below.

[0699] Table 31: DAR values ​​of ADC hIgG1-A-01

[0700] Example 14. Preparation of ADC 22B6-A-26

[0701] 2.667 ml of 22B6 antibody (18.75 mg / mL) was diluted with 133.35 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.63 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 184.19 μL, pH 7.60) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-26 dissolved in dimethyl sulfoxide (341.73 μL, 10 mM) was then slowly added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain ADC 22B6-A-26. The DAR value was 7.97 as determined by mass spectrometry.

[0702] Example 15. Preparation of ADC hIgG1-A-26

[0703] 2.555 ml of hIgG1 antibody (19.57 mg / mL) was diluted with 127 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.63 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 190.8 μL, pH 7.60) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of A-26 dissolved in dimethyl sulfoxide (354 μL, 10 mM) was then added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain the antibody-drug conjugate (i.e., ADC hIgG1-A-26). The DAR value was determined by mass spectrometry to be 8.06.

[0704] Example 16a. Preparation Example of ADC 22B6-B-04

[0705] 0.854 ml of 22B6 antibody (23.43 mg / mL) was diluted with 42.68 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.57 with 1 M Na₂HPO₄ solution. A 20 mM TCEP (tris(2-carboxyethyl)phosphine, 73.68 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A 12-fold amount of B-04 (164 μL, 10 mM) dissolved in dimethyl sulfoxide was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain ADC 22B6-B-04-1. The DAR value was determined by mass spectrometry to be 8.62.

[0706] Example 16b. Preparation Example of ADC 22B6-B-04

[0707] 2.667 ml of 22B6 antibody (18.75 mg / mL) was diluted with 133.35 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.63 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 184.19 μL, pH 7.60) solution was added and mixed thoroughly. The mixture was allowed to stand at room temperature for 1.5 hours. A 10-fold amount of B-04 (341.73 μL, 10 mM) dissolved in dimethyl sulfoxide was then slowly added and mixed thoroughly. The mixture was allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain ADC 22B6-B-04-2. The DAR value was 6.87 as determined by mass spectrometry.

[0708] Example 16c. Preparation Example of ADC hIgG1-B-04

[0709] The antibody was replaced with hIgG1 antibody, and the hIgG1-B-04 sample was prepared using the same method as in Section 16b above. The DAR value was determined by mass spectrometry to be 7.02.

[0710] Example 17. Detection of Antibody-Drug Conjugate Activity

[0711] 1. Cell affinity testing of anti-human HER3 antibodies and their drug conjugates

[0712] The affinity of the tested ADC to MDA-MB-453 cells or NCI-N87 cells was detected using a flow cytometer (Beckman, model Cytoflex).

[0713] Adherent MDA-MB-453 and NCI-N87 cells were digested with Tryple solution (Gibco). An appropriate number of cells were counted and harvested, washed twice with PBS, and resuspended in 1% BSA (in PBS). The cells were then transferred to a 96-well conical-bottom plate, with 50 μl per well. The candidate antibody and its drug conjugate were diluted in 1% BSA, starting at 15 μg / mL and serially diluted three-fold. 50 μl of the diluted antibody or antibody-drug conjugate was then added to the conical-bottom plate containing the cells and incubated at 4°C for 40 min. The cells were washed twice with PBS, followed by the addition of 50 μl of the diluted secondary antibody to each well, mixing thoroughly, and incubating at 4°C for 30 min. The cells were washed twice with PBS, then resuspended in 200 μl of PBS and analyzed by flow cytometry. Data processing: Mean fluorescence signal values ​​were exported and imported into GraphPad Prism 6 software to calculate the EC50. The results are shown in Table 1 and Figures 1 and 2. The results demonstrate that the ADCs of the present invention exhibit significant cell affinity.

[0714] Table 1 Anti-human HER3 antibody and ADC cell affinity determination results

[0715] 2. Endocytosis activity test of anti-human HER3 antibody-drug conjugates

[0716] The endocytic activity of the ADC or antibody to be tested in MDA-MB-453 and HCC1569 cells was detected using a flow cytometer (Beckman, model Cytoflex).

[0717] The adherent cells were digested with Trypsin-EDTA (0.25%, Shanghai Yuanpei) solution and counted. The cell density was adjusted to 1×10 5 100 μl of cell suspension was added to each well of a 96-well plate (the number of cells was 1×10 4 / well), and the 96-well plate was placed in a 37°C, CO2 constant temperature incubator for 24 hours. The 96-well plate was removed, the culture medium was aspirated, and 50 μl of fresh complete culture medium was added to each well; the bispecific antibody and the control antibody were serially diluted with complete culture medium to a final concentration of 0.55, 1.64, 4.94, 14.81, 44.44, 133.33, 400, and 1200 ng / ml, for a total of 8 concentration points; the pHrodo reagent (Thermo) was diluted with complete culture medium to 12 μg / ml (the final concentration of pHrodo was 3 μg / ml); the serially diluted test antibody was mixed with the diluted Mix the pHrodo reagent in a 1:1 ratio (30 μl:30 μl) and incubate at room temperature in the dark for 30 minutes. Add 50 μl of the test antibody and pHrodo reagent mixture to a 96-well plate and incubate at 37°C, 5% CO2 for 24 hours. Remove the 96-well plate, discard the culture medium, wash once with sterile PBS, add 100 μl of Trypsin-EDTA (0.25%) to each well to digest the cells, and then add 100 μl of complete culture medium to neutralize them. After pipetting and dispersing the cells in the wells, analyze them on a FACS machine. Data processing: Export the average fluorescence signal value and then import it into GraphPad Prism 6 software to calculate the EC. 50 The results are shown in Table 2 and Figures 3 and 4, which indicate that the ADC of the present invention has strong endocytic activity.

[0718] Table 2 Results of endocytic activity assay of anti-human HER3 antibodies and ADCs

[0719] 3. In vitro cytotoxicity of anti-human HER3 antibody-drug conjugates

[0720] MDA-MB-453 or 293T-HER3 cells were digested with TrypLE solution (Gibco), counted, and an appropriate amount of cells was obtained. After dilution with growth medium, 5000 cells / 100 μl / well were plated onto a 96-well plate and incubated overnight at 37°C, 5% CO2. The next day, the ADC was diluted with growth medium, starting at 150 μg / ml and serially diluted 2.5-fold (or starting at 7.5 μg / ml and serially diluted 5-fold). 100 μl of the diluted ADC was then added to the 96-well plate containing the cells. The plate was placed in an incubator and incubated at 37°C, 5% CO2 for 6 days. 20 μl of CCK8 was added to each well and incubated at 37°C for 2-5 hours. The absorbance at OD450 nm was read using a microplate reader. The raw data was imported into Graph Prism 6 to calculate the IC50 value. As shown in Table 3 and Figures 5 and 6, the ADC drugs of the present invention (e.g., 22B6-A-26) have target-specific killing, wherein the IC50 of 22B6-A-26 and hIgG1-A-26 on tumor cells differ by 9.1 times. On overexpressing cells (HEK293T-h HER3), hIgG1-A-26 showed no significant killing, and the IC50 difference between the 22B6-A-26 ADC drug and hIgG1-A-26 was even greater.

[0721] Table 3 In vitro cytotoxicity results of anti-human HER3 ADC

[0722] 4. In vivo efficacy testing of different antibody-drug conjugates in the NCI-H358 model

[0723] Human non-small cell lung cancer cells NCI-H358 (Nanjing Kebai) were cultured in a monolayer in vitro using RPMI-1640 medium supplemented with 10% fetal bovine serum in an incubator at 37°C with 5% CO2. They were digested and passaged 2-3 times per week using trypsin-EDTA. When the cells reached the exponential growth phase, the culture medium was collected for mycoplasma testing, and the cells were collected and counted. Each mouse was inoculated subcutaneously with 5×10 6 NCI-H358 cells (suspended in 0.05 ml PBS + 0.05 ml Matrigel). 3 At the same time, mice with irregular, too small, or too large tumor volumes were eliminated, and the remaining mice were randomly divided into 4 groups according to tumor volume and animal body weight, with 5 mice in each group. The drugs were injected into the tail vein (iv) once a week (QW) for a total of two doses. Tumors were measured with a vernier caliper twice a week after administration, and the tumor volume was calculated according to the following formula: V = 0.5a × b 2, where a and b represent the major and minor diameters of the tumor respectively, and the antitumor drug efficacy is evaluated by the tumor growth inhibition rate TGI (%). The calculation formula is: TGI (%) (tumor volume) = [1 - (T Vt - T V0 ) / (C Vt - C V0 )] × 100%; when the tumor regresses, TGI (%) (tumor volume) = 100% - (T Vt - T V0 ) / T V0 × 100%. T V0 is the average tumor volume of the test drug group during grouped drug administration; T Vt is the average tumor volume of the test drug group at t days after drug administration; C V0 is the average tumor volume of the vehicle group during grouped drug administration; C Vt is the average tumor volume of the vehicle group at t days after drug administration. If the tumor shrinks compared to the initial volume, i.e., V t < V0, it is defined as partial tumor regression (PR); if the tumor completely disappears, it is defined as complete tumor regression (CR). Observe and record the animal death situation every day, and the specific results are shown in Table 4, Figures 7A - 7B.

[0724] Table 4 Analysis of the efficacy of the antibody - conjugated drug on the NCI - H358 cell - bearing mouse model Note: Compared with the vehicle group, * : P < 0.05, and P < 0.05 indicates a significant difference.

[0725] The results show that 22B6 - A - 26 shows obvious drug efficacy, and the animals in each group have good tolerance, showing good efficacy and safety of the above drugs.

[0726] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings that have been disclosed, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody-drug conjugate having the formula Ab-[MLED] x The structure shown, wherein: Ab is an antibody or an antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor 3 (HER3); M is a linker site that is attached to the antibody or antigen-binding fragment thereof and is Wherein, ring A is a 5-6 membered alicyclic heterocyclic ring or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 The alkyl group is substituted; M1 is selected from a single bond and a substituted or unsubstituted group: C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkynylidene; L is a structural fragment connecting M and E, and is selected from the structure consisting of one or more substituted or unsubstituted groups as follows: C 1-6 Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), 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-G ly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, Where R' represents hydrogen, C 1-6 Alkyl or containing -(CH2CH2O) r -alkyl; r is selected from an integer of 1-10; s is selected from an integer of 1-20; E is a structural fragment connecting L and D, and is a single bond or a substituted or unsubstituted structure selected from the following: -NH-CH2-, -NH-CH2-O-CH2-CO-, D is a fragment corresponding to the cytotoxic drug obtained by connecting the cytotoxic drug to E, wherein the cytotoxic drug is selected from a microtubule inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor and an RNA polymerase inhibitor; preferably, the microtubule inhibitor is an auristatin compound or a maytansine compound; preferably, the DNA intercalator is a pyrrolobenzodiazepine (PBD); preferably, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); preferably, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester or analog thereof; x is selected from 1 to 10.

2. The antibody-drug conjugate of claim 1, wherein M is in, Ring A is a 5-20 membered aromatic ring system, which is optionally substituted by one or more selected from halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl radical substitution; M1 is substituted or unsubstituted C 2-20 Alkynylidene; Preferably, M is wherein ring A is a 6-membered heteroaromatic ring, the heteroaromatic ring is optionally substituted by one or more selected from halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl radical substitution; M1 is substituted or unsubstituted C 3-10 Alkynylidene; Preferably, M is 3. The antibody-drug conjugate of claim 1 or 2, wherein L is selected from one or more of the following structures: Val, Cit, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), 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 and Gly-Gly-Gly-Gly-Gly; wherein s is selected from an integer of 1-20; Preferably, L is composed of one or more substituted or unsubstituted Ala; Preferably, L is substituted or unsubstituted Ala-Ala-Ala; Preferably, L is 4. The antibody-drug conjugate of any one of claims 1 to 3, wherein E is a single bond or a substituted or unsubstituted structure selected from the following: -NH-CH2-, -NH-CH2-O-CH2-CO-, Preferably, E is -NH-CH2-O-CH2-CO or -NH-CH2-.

5. The antibody-drug conjugate according to any one of claims 1 to 4, Select from the following structures:

6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein the cytotoxic drug is selected from the compounds represented by formula I and formula II: in, In Formula I, R1, R2 are each independently selected from C 1-6 Alkyl and halogen; R3 is selected from H and -CO-CH2OH; In Formula II, R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring; R6 is selected from hydrogen and -C 1-4 Alkylene-NR a R b ; R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ; Where R a , R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6 alkyl; Preferably, the cytotoxic drug is selected from the following compounds: Preferably, the cytotoxic drug is 7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein D is a monovalent structure obtained by losing one H from -OH, -NH2 or a secondary amine group on the cytotoxic drug; Preferably, D is selected from the following structures: Preferably, D is 8. The antibody-drug conjugate of any one of claims 1 to 7, 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 with an amino acid sequence of SEQ ID NO: 1 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or, (1b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 19 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 20 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or, (1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 36 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 37 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof; in, The variant described in any one of (1a), (1b), and (1c) 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% amino acid sequence identity compared to the amino acid sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the amino acid sequence from which it is derived; preferably, the substitutions are conservative substitutions; provided that the amino acid sequence of the CDR of the variant has 100% sequence identity with the amino acid sequence of the corresponding CDRs of VH and VL of (1a), (1b), and (1c), and the variant binds to HER3; 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 with an amino acid sequence of SEQ ID NO: 7 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 8 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 9 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 4 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 5 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or, (2b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 25 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 22 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or, (2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 39 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 40 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 38 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 45 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 23 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 52 or a variant thereof; wherein the variant described in any one of (2a), (2b), and (2c) 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% amino acid sequence identity compared to the amino acid sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the amino acid sequence from which it is derived; preferably, the substitutions are conservative substitutions; provided that the amino acid sequence of the CDR of the variant has 100% sequence identity with the amino acid sequence of the corresponding CDRs of VH and VL of (2a), (2b), and (2c), and the variant binds to HER3; 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 with an amino acid sequence of SEQ ID NO: 10 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 11 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 12 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 13 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 14 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 6 or a variant thereof; or, (3b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO: 27 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO: 28 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO: 29 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO: 30 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO: 31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO: 24 or a variant thereof; or, (3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with an amino acid sequence of SEQ ID NO:41 or a variant thereof, CDR-H2 with an amino acid sequence of SEQ ID NO:42 or a variant thereof, and CDR-H3 with an amino acid sequence of SEQ ID NO:43 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with an amino acid sequence of SEQ ID NO:44 or a variant thereof, CDR-L2 with an amino acid sequence of SEQ ID NO:31 or a variant thereof, and CDR-L3 with an amino acid sequence of SEQ ID NO:52 or a variant thereof; wherein the variant described in any one of (3a), (3b), and (3c) 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% amino acid sequence identity compared to the amino acid sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; provided that the amino acid sequence of the CDR of the variant has 100% sequence identity with the amino acid sequence of the corresponding CDRs of VH and VL of (3a), (3b), and (3c), and the variant binds to HER3.

9. The antibody-drug conjugate of any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 15 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 16 or a variant thereof; (b) a VH comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof; or (c) a VH comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof, and / or a VL comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof; in, 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% amino acid sequence identity compared to the amino acid sequence from which it is derived, or the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the amino acid sequence from which it is derived; preferably, the substitutions are conservative substitutions; provided that the amino acid sequences of the CDRs of the variant have 100% sequence identity with the amino acid sequences of the corresponding CDRs of VH and VL of (a), (b) and (c), and the variant binds to HER3.

10. The antibody-drug conjugate of any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof further comprises: (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which 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 it is derived; and (b) a light chain constant region (CL) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; Preferably, 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; preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 50 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 50 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; such as 1, 2, 3, 4 or 5 conservative substitutions of amino acids); Preferably, the light chain constant region is a kappa light chain constant region; preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 51 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 51 (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); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO:50 and a light chain constant region (CL) as shown in SEQ ID NO:

51.

11. The antibody-drug conjugate of any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising a VH having an amino acid sequence as set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) having an amino acid sequence as set forth in SEQ ID NO: 50, and a light chain comprising a VL having an amino acid sequence as set forth in SEQ ID NO: 16 and a light chain constant region (CL) having an amino acid sequence as set forth in SEQ ID NO: 51; (2) a heavy chain comprising a VH having an amino acid sequence as set forth in SEQ ID NO:32 and a heavy chain constant region (CH) having an amino acid sequence as set forth in SEQ ID NO:50, and a light chain comprising a VL having an amino acid sequence as set forth in SEQ ID NO:33 and a light chain constant region (CL) having an amino acid sequence as set forth in SEQ ID NO:51; or (3) a heavy chain comprising the VH of the amino acid sequence shown in SEQ ID NO:46 and the heavy chain constant region (CH) of the amino acid sequence shown in SEQ ID NO:50, and a light chain comprising the VL of the amino acid sequence shown in SEQ ID NO:47 and the light chain constant region (CL) of the amino acid sequence shown in SEQ ID NO:

51.

12. The antibody-drug conjugate of any one of claims 1 to 11, wherein the conjugate is: in, The HA in the antibody-drug conjugate is selected from: (1) an antibody or an antigen-binding fragment thereof comprising a VH as set forth in SEQ ID NO: 15 and a VL as set forth in SEQ ID NO: 16, for example, an antibody or an antigen-binding fragment thereof comprising a VH as set forth in SEQ ID NO: 15 and a CH as set forth in SEQ ID NO: 50, and a VL as set forth in SEQ ID NO: 16 and a CL as set forth in SEQ ID NO: 51; (2) an antibody or an antigen-binding fragment thereof comprising the VH set forth in SEQ ID NO:32 and the VL set forth in SEQ ID NO:33, for example, an antibody or an antigen-binding fragment thereof comprising the VH set forth in SEQ ID NO:32 and the CH set forth in SEQ ID NO:50, and the VL set forth in SEQ ID NO:33 and the CL set forth in SEQ ID NO:51; and (3) an antibody or an antigen-binding fragment thereof comprising the VH set forth in SEQ ID NO:46 and the VL set forth in SEQ ID NO:47, for example, an antibody or an antigen-binding fragment thereof comprising the VH set forth in SEQ ID NO:46 and the CH set forth in SEQ ID NO:50, and the VL set forth in SEQ ID NO:47 and the CL set forth in SEQ ID NO:51; x is 3 to 8.

13. The antibody-drug conjugate of claim 12, wherein: x is an integer from 3 to 8.

14. The antibody-drug conjugate of claim 12 or 13, wherein the antibody-drug conjugate is: in, The HA represents an antibody or an antigen-binding fragment thereof comprising the HC shown in SEQ ID NO: 17 and the LC shown in SEQ ID NO: 18; x is 3 to 8.

15. The antibody-drug conjugate of claim 14, wherein: x is an integer from 3 to 8.

16. The antibody-drug conjugate of any one of claims 1 to 15, wherein: (i) The heavy chain lacks a lysine residue at the C-terminus; (ii) the N-terminus of the heavy chain is glutamine, glutamic acid, 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 glutamine, glutamic acid or pyroglutamic acid.

17. The antibody-drug conjugate of any one of claims 1 to 16, wherein the formula is Ab-[MLED] x In the formula, Ab is conjugated to the remainder of the formula via a cysteine ​​residue.

18. A pharmaceutical composition comprising one or more antibody-drug conjugates according to any one of claims 1 to 17, and one or more pharmaceutically acceptable excipients.

19. The pharmaceutical composition of claim 18, wherein the average DAR value (drug-antibody coupling ratio) is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10, preferably 3 to 9, for example, 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.0 to 5.5, 3.0 to 6.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 3.5 to 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.

20. Use of the antibody-drug conjugate according to any one of claims 1 to 17 or the pharmaceutical composition according to claim 18 or 19 in the preparation of a drug for treating cancers with high HER3 expression; Preferably, the HER3-highly expressing cancer includes solid tumors or hematological malignancies, such as colon cancer, gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

21. The antibody-drug conjugate of any one of claims 1 to 17 or the pharmaceutical composition of claim 18 or 19, for use in treating cancers with high HER3 expression; Preferably, the HER3-highly expressing cancer includes solid tumors or hematological malignancies, such as colon cancer, gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

22. A method for treating HER3-overexpressing cancer, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1 to 17 or the pharmaceutical composition of claim 18 or 19; Preferably, the HER3-highly expressing cancer includes solid tumors or hematological malignancies, such as colon cancer, gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

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