[go: up one dir, main page]

WO2025045015A1 - Antibody-drug conjugate and preparation method therefor and use thereof - Google Patents

Antibody-drug conjugate and preparation method therefor and use thereof Download PDF

Info

Publication number
WO2025045015A1
WO2025045015A1 PCT/CN2024/114694 CN2024114694W WO2025045015A1 WO 2025045015 A1 WO2025045015 A1 WO 2025045015A1 CN 2024114694 W CN2024114694 W CN 2024114694W WO 2025045015 A1 WO2025045015 A1 WO 2025045015A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
variant
cdr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/114694
Other languages
French (fr)
Chinese (zh)
Inventor
胡江江
田强
袁晓曦
柏云
龙虎
蒲钰芝
马玲
张毅涛
宋宏梅
谭淼
谭向阳
葛均友
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Original Assignee
Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sichuan Kelun Biotech Biopharmaceutical Co Ltd filed Critical Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Publication of WO2025045015A1 publication Critical patent/WO2025045015A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

Definitions

  • the present application relates to the field of targeted therapy, and in particular to antibody-drug conjugates and preparation methods and uses thereof.
  • Cancer is one of the leading causes of death in the modern world. It is a class of diseases caused by the malignant transformation of healthy cells, caused by genetic changes such as chromosomal translocations, mutations in tumor suppressor genes, growth factor receptors, leading to malignant proliferation of cells. Defective apoptosis or programmed cell death further promotes the malignant transformation of cells leading to cancer.
  • 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 consists of an extracellular domain (ECD) that binds to the ligand, a dimerization domain within the ECD, a transmembrane domain, and a carboxyl-terminal phosphorylation domain. In addition to these domains, HER1, HER2, and HER4 also carry an intracellular protein tyrosine kinase domain (TKD), while HER3 lacks this domain and cannot autophosphorylate.
  • ECD extracellular domain
  • TKD intracellular protein tyrosine kinase domain
  • Ligand regulatory protein 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.
  • the 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.
  • 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.
  • 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 medicaments of HER3-mediated signal transduction.
  • HER3 target indications under clinical research cover hematological tumors and solid tumors.
  • the main treatment strategies focus on several directions such as monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates (ADCs).
  • ADCs antibody-drug conjugates
  • there are currently two anti-HER3 antibody-drug conjugates under international research and one has entered the clinical research stage (Daiichi Sankyo U3-1402) for the treatment of NSCLC, MBC, and CRC.
  • ADC drugs are composed of antibodies, bioactive molecules and linkers.
  • the bioactive molecules are covalently coupled to the antibodies through linkers; antibodies (such as monoclonal antibodies) can specifically recognize specific targets on the surface of tumor cells, and then guide ADC to the surface of cancer cells, and allow ADC to enter cancer cells through cellular endocytosis; then the bioactive molecules are released inside the cancer cells, killing cancer cells while minimizing damage to normal tissue cells.
  • U3-1402 was developed by Daiichi Sankyo Co., Ltd., using GGFG tetrapeptide as the enzyme cleavage linker and Dxd as the cytotoxic drug. According to reports, in the first-phase dose expansion (5.6 mg/kg, Q3W) of NSCLC with EGFR mutations, ORR 39%, DCR 72%, mPFS 8.2 months, and similar efficacy for patients with brain metastases; in terms of safety, it is mainly blood toxicity (thrombocytopenia, neutropenia, etc.) and interstitial pneumonia (5-7%).
  • the present application provides an antibody-drug conjugate having a structure shown in the formula Ab-[MLED] x , wherein:
  • Ab is an antibody or antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor 3 (HER3, also known as Erbb3);
  • M is a linker site connected to the antibody or antigen-binding fragment thereof
  • L is the structural fragment connecting M and E;
  • E is a structural fragment connecting L and D;
  • D is the cytotoxic drug fragment
  • x is selected from 1 to 10.
  • Ab is an antibody or antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor 3 (HER3);
  • L is a structural fragment connecting M and E, and is selected from a structure consisting of one or more substituted or unsubstituted groups: C 1-6 alkylene, -N(R')-, carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) s OCH 3 ), D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (SEQ ID NO:53), Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:53), Gly-
  • 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-CH 2 -, -NH-CH 2 -O-CH 2 -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 microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors and RNA polymerase inhibitors 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,
  • x is selected from 1 to 10.
  • 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.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having an amino acid sequence of SEQ ID NO:2 or a variant thereof
  • CDR-H3 having an amino acid sequence of SEQ ID NO:3 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with an amino acid sequence of SEQ ID NO:20 or a variant thereof
  • CDR-H3 with an amino acid sequence of SEQ ID NO:21 or a variant thereof
  • VL light chain variable region
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:36 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:37 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO:45 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO:23 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO:52 or a variant thereof;
  • 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 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;
  • amino acid substitutions, deletions, or additions e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having an amino acid sequence of SEQ ID NO:8 or a variant thereof
  • CDR-H3 having an amino acid sequence of SEQ ID NO:9 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof
  • CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having an amino acid sequence of SEQ ID NO:40 or a variant thereof
  • CDR-H3 having an amino acid sequence of SEQ ID NO:38 or a variant thereof
  • VL light chain variable region
  • 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 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 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;
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with an amino acid sequence of SEQ ID NO: 11 or a variant thereof
  • CDR-H3 with an amino acid sequence of SEQ ID NO: 12 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with an amino acid sequence of SEQ ID NO:28 or a variant thereof
  • CDR-H3 with an amino acid sequence of SEQ ID NO:29 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having an amino acid sequence of SEQ ID NO:42 or a variant thereof
  • CDR-H3 having an amino acid sequence of SEQ ID NO:43 or a variant thereof
  • VL light chain variable region
  • 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 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, 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.
  • amino acid substitutions, deletions, or additions e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions
  • the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • CDR-H2 having an amino acid sequence of SEQ ID NO:2 or a variant thereof
  • CDR-H3 having an amino acid sequence of SEQ ID NO:3 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with an amino acid sequence of SEQ ID NO:20 or a variant thereof
  • CDR-H3 with an amino acid sequence of SEQ ID NO:21 or a variant thereof
  • VL light chain variable region
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:36 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:37 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO:45 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO:23 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO:52 or a variant thereof;
  • 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 sequence of the CDR of the variant has 100% sequence identity with the amino acid sequence of the corresponding CDR of VH and VL of (1a), (1b), and (1c), and the variant binds to HER3
  • amino acid substitutions, deletions, or additions e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions
  • VH heavy chain variable region
  • CDR-H2 having an amino acid sequence of SEQ ID NO:8 or a variant thereof
  • CDR-H3 having an amino acid sequence of SEQ ID NO:9 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with an amino acid sequence of SEQ ID NO: 26 or a variant thereof
  • CDR-H3 with an amino acid sequence of SEQ ID NO: 21 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having an amino acid sequence of SEQ ID NO:40 or a variant thereof
  • CDR-H3 having an amino acid sequence of SEQ ID NO:38 or a variant thereof
  • VL light chain variable region
  • 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 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 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;
  • VH heavy chain variable region
  • CDR-H2 with an amino acid sequence of SEQ ID NO: 11 or a variant thereof
  • CDR-H3 with an amino acid sequence of SEQ ID NO: 12 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with an amino acid sequence of SEQ ID NO:28 or a variant thereof
  • CDR-H3 with an amino acid sequence of SEQ ID NO:29 or a variant thereof
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 having an amino acid sequence of SEQ ID NO:42 or a variant thereof
  • CDR-H3 having an amino acid sequence of SEQ ID NO:43 or a variant thereof
  • VL light chain variable region
  • 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 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, 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.
  • amino acid substitutions, deletions, or additions e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions
  • the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or its antigen-binding fragment 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or its antigen-binding fragment 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or its antigen-binding fragment 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or its antigen-binding fragment 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or its antigen-binding fragment 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or its antigen-binding fragment 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or its antigen-binding fragment 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or its antigen-binding fragment 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or its antigen-binding fragment 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment thereof comprises:
  • 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, 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 (a), (b) and (c), and the variant binds to HER3.
  • amino acid substitutions, deletions or additions e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions
  • the antibody or antigen-binding fragment thereof comprises:
  • 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.
  • the antibody or antigen-binding fragment thereof comprises:
  • (c) includes the VH shown in SEQ ID NO: 46, and, includes the VL shown in SEQ ID NO: 47.
  • the antibody or antigen-binding fragment thereof comprises:
  • (a) includes the VH shown in SEQ ID NO: 15, and, includes the VL shown in SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof comprises:
  • (b) includes the VH shown in SEQ ID NO: 32, and, includes the VL shown in SEQ ID NO: 33.
  • the antibody or antigen-binding fragment thereof comprises:
  • (c) includes the VH shown in SEQ ID NO: 46, and, includes the VL shown in SEQ ID NO: 47.
  • the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH), which comprises CDR-H1, CDR-H2 and CDR-H3, and the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 described in VH, which respectively include the amino acid sequence of SEQ ID NO:15; and a light chain variable region (VL), which comprises CDR-L1, CDR-L2 and CDR-L3, and the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 described in VL, which respectively include the amino acid sequence of SEQ ID NO:16.
  • VH heavy chain variable region
  • CDR-H2 and CDR-H3 comprise the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 described in VH, which respectively include the amino acid sequence of SEQ ID NO:15
  • the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH), which comprises CDR-H1, CDR-H2 and CDR-H3, and the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 described in VH, which respectively include the amino acid sequence of SEQ ID NO:32; and a light chain variable region (VL), which comprises CDR-L1, CDR-L2 and CDR-L3, and the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 described in VL, which respectively include the amino acid sequence of SEQ ID NO:33.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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.
  • the antibody or antigen-binding fragment thereof further comprises:
  • 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.
  • 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
  • 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.
  • 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; e.g., 1, 2, 3, 4 or 5 conservative substitutions of amino acids).
  • the light chain constant region is a kappa light chain constant region.
  • 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).
  • the antibody or its antigen-binding fragment 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.
  • CH heavy chain constant region
  • CL light chain constant region
  • the antibody or antigen-binding fragment thereof comprises:
  • a heavy chain comprising a VH sequence shown in SEQ ID NO: 15 and a heavy chain constant region (CH) shown in SEQ ID NO: 50, and a light chain comprising a VL sequence shown in SEQ ID NO: 16 and a light chain constant region (CL) shown in SEQ ID NO: 51;
  • a heavy chain comprising a VH sequence shown in SEQ ID NO: 46 and a heavy chain constant region (CH) shown in SEQ ID NO: 50, and a light chain comprising a VL sequence shown in SEQ ID NO: 47 and a light chain constant region (CL) shown in SEQ ID NO: 51.
  • the antibody or its antigen-binding fragment comprises: (1) a heavy chain comprising a VH with the amino acid sequence shown in SEQ ID NO: 15 and a heavy chain constant region (CH) shown in SEQ ID NO: 50, and a light chain comprising a VL with the amino acid sequence shown in SEQ ID NO: 16 and a light chain constant region (CL) shown in SEQ ID NO: 51.
  • the antibody or antigen-binding fragment thereof comprises: (2) a heavy chain comprising a VH having an amino acid sequence as shown in SEQ ID NO: 32 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: 33 and a light chain constant region (CL) as shown in SEQ ID NO: 51; or
  • the antibody or its antigen-binding fragment comprises: (3) a heavy chain comprising a VH with the amino acid sequence shown in SEQ ID NO: 46 and a heavy chain constant region (CH) shown in SEQ ID NO: 50, and a light chain comprising a VL with the amino acid sequence shown in SEQ ID NO: 47 and a light chain constant region (CL) shown in SEQ ID NO: 51.
  • the antibody or its antigen-binding fragment comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 17, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 18.
  • HC heavy chain
  • LC light chain
  • the antibody or its antigen-binding fragment comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 34, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 35.
  • HC heavy chain
  • LC light chain
  • the antibody or its antigen-binding fragment comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 48, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 49.
  • HC heavy chain
  • LC light chain
  • the heavy chain constant domain may comprise a C-terminal lysine or lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide.
  • the N-terminal amino acid of the antibodies or antigen-binding fragments thereof may be cyclized to pyroglutamic acid.
  • 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).
  • M is wherein ring A is selected from M 1 is selected from a single bond and a C 5-8 alkylene group, a C 5-8 alkenylene group or a C 5-8 alkynylene group.
  • M is selected from the following structures:
  • M is selected from the following structures:
  • M is N
  • ring A is a 5-20 membered aromatic ring system, which is optionally substituted by one or more groups selected from halogen, cyano, amino, carboxyl, thiol and C 1-6 alkyl; M 1 is a substituted or unsubstituted C 2-20 alkynylene group.
  • M is N
  • ring A is a 6-membered heteroaromatic ring, and the aromatic ring system is optionally substituted by one or more groups selected from halogen, cyano, amino, carboxyl, thiol and C 1-6 alkyl; M 1 is a C 3-10 alkynylene group.
  • M is N
  • L is selected from a structure consisting of one or more substituted or unsubstituted groups selected from the group consisting of C 1-6 alkylene, -N(R')-, carbonyl, -O-, Val, Cit, Phe, Lys, Lys( COCH2CH2 ( OCH2CH2 ) sOCH3 ) , 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, Gly-Gly-Gly-Gly-Gly, wherein R' represents hydrogen, C 1-6 alkylene,
  • L is selected from a structure consisting of one or more substituted or unsubstituted groups selected from the group consisting of C 1-6 alkylene, -NH-, Phe, Lys, Ala-Ala-Ala, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) s OCH 3 ), Gly, Gly-Gly-Phe-Gly, wherein s is selected from an integer of 1-20.
  • L is selected from a structure consisting of one or more substituted or unsubstituted groups selected from the group consisting of C 1-6 alkylene, -NH-, Phe, Lys, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) s OCH 3 ), Gly, Gly-Gly-Phe-Gly, wherein s is selected from an integer of 1-20.
  • L is selected from the following substituted or unsubstituted structures:
  • L is selected from the following structures:
  • s is selected from an integer of 1-20.
  • L is selected from the following structures:
  • L is selected from the following structures:
  • L is selected from the following structures:
  • L is selected from the following structures:
  • L is selected from a structure consisting of one or more of the following: Val, Cit, Phe, Lys, Lys( COCH2CH2 ( OCH2CH2 ) sOCH3 ) , 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.
  • L consists of one or more substituted or unsubstituted Ala.
  • L consists of one or more Ala.
  • L is substituted or unsubstituted Ala-Ala-Ala.
  • L is N
  • E is a single bond or a substituted or unsubstituted structure selected from the following: -NH- CH2- , -NH- CH2 -O-CH2 - CO-,
  • E is a single bond or a substituted or unsubstituted structure selected from the following: -NH- CH2- , -NH- CH2 -O-CH2 - CO-, In some embodiments, E is substituted or unsubstituted -NH-CH 2 -O-CH 2 -CO-,
  • E is -NH- CH2 -O- CH2 -CO- or In some embodiments, E is -NH- CH2 -O- CH2 -CO or -NH- CH2- .
  • M is selected from the following substituted or unsubstituted structures:
  • L is selected from the following substituted or unsubstituted structures:
  • E is -NH-CH 2 -O-CH 2 -CO-
  • the cytotoxic drug is selected from microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors and RNA polymerase inhibitors.
  • the microtubule inhibitor is an auristatin compound or a maytansine compound.
  • the DNA intercalator is a pyrrolobenzodiazepine (PBD).
  • 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).
  • the RNA polymerase inhibitor is ⁇ -amanitin or a pharmaceutically acceptable salt, ester or analog thereof.
  • the cytotoxic drugs disclosed in the present 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 RC ), wherein RA , RB , RC here only represent non-hydrogen substituents on N, and the cytotoxic drugs can be connected to the linker in the conjugate through these functional groups.
  • functional groups such as hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH), primary amino ( -NH2 ), secondary amine (-NR A H) or tertiary amine (-NR B RC ), wherein RA , RB , RC here only represent non-hydrogen substituents on N, and the cytotoxic drugs can be connected to the linker in the conjugate through these functional groups.
  • the cytotoxic drug is linked to E in the antibody-drug conjugate through -OH, -SH, primary amino, secondary amine or tertiary amine groups on the cytotoxic drug.
  • the cytotoxic drug is selected from the following Formula I and Formula II:
  • R 1 and R 2 are each independently selected from C 1-6 alkyl and halogen;
  • R 3 is selected from H and -CO-CH 2 OH
  • 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 -C1-4alkylene - NRaRb ;
  • R7 is selected from C1-6 alkyl and -C1-4 alkylene- NRaRb ;
  • Ra , Rb at each occurrence are independently selected from H, C1-6 alkyl, -SO2- C1-6 alkyl and -CO- C1-6 alkyl.
  • the cytotoxic drug is selected from the following compounds:
  • the cytotoxic drug is selected from the following compounds:
  • D is a monovalent structure obtained by losing one H from -OH, -NH2 or a secondary amine group on the cytotoxic drug.
  • D is selected from the following structures:
  • the cytotoxic drug is:
  • the cytotoxic drug is:
  • D is:
  • D is:
  • HA in each antibody-drug conjugate represents an antibody or an 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 an 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;
  • x is an integer from 1-10.
  • the antibody-drug conjugate is selected from:
  • the HA in each antibody-drug conjugate is selected from:
  • an antibody or an antigen-binding fragment thereof comprising the VH shown in SEQ ID NO: 15 and the VL shown in SEQ ID NO: 16, for example, an antibody or an antigen-binding fragment thereof comprising the VH shown in SEQ ID NO: 15 and the CH shown in SEQ ID NO: 50, and the VL shown in SEQ ID NO: 16 and the CL shown in SEQ ID NO: 51;
  • an antibody or an 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 an 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; and
  • an antibody or an 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 an 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 3-8, for example, 3-4 or 7-8.
  • x is an integer from 3-8.
  • the antibody-drug conjugate is:
  • 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 6 to 8.
  • the antibody-drug conjugate is:
  • HA includes:
  • 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;
  • x is 3 to 8, for example, 3 to 4 or 7 to 8, for example, an integer of 3-8.
  • the antibody-drug conjugate is:
  • HA includes:
  • an antibody or an 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 an 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.
  • the antibody-drug conjugate is:
  • HA includes:
  • an antibody or an antigen-binding fragment thereof comprising VH as shown in SEQ ID NO:46 and VL as shown in SEQ ID NO:47, for example, an antibody or an antigen-binding fragment thereof comprising VH as shown in SEQ ID NO:46 and CH as shown in SEQ ID NO:50, and VL as shown in SEQ ID NO:47 and CL as 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.
  • the antibody-drug conjugate is:
  • 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 6 to 8.
  • x is an integer from 6-8.
  • the antibody-drug conjugate is:
  • the HA represents an antibody or an antigen-binding fragment thereof comprising the HC shown in SEQ ID NO: 34 and the LC shown in SEQ ID NO: 35;
  • x is 6 to 8, for example, an integer of 6-8.
  • the antibody-drug conjugate is:
  • the HA represents an antibody or an antigen-binding fragment thereof comprising the HC shown in SEQ ID NO: 48 and the LC shown in SEQ ID NO: 49;
  • x is 6 to 8, for example, an integer of 6-8.
  • the heavy chain lacks a lysine residue at the C-terminus
  • the N-terminus of the heavy chain is glutamine, glutamic acid or pyroglutamic acid; or,
  • 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.
  • Ab is conjugated to the remaining moiety in the formula via a cysteine residue.
  • compositions comprising the antibody-drug conjugates described herein and one or more pharmaceutically acceptable excipients.
  • the cancer comprises a solid tumor or a hematological malignancy.
  • the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma.
  • the lung cancer is non-small cell lung cancer.
  • the lung cancer is lung adenocarcinoma.
  • the cancer comprises a solid tumor or a hematological malignancy.
  • the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma.
  • the lung cancer is non-small cell lung cancer.
  • the lung cancer is lung adenocarcinoma.
  • Also provided herein is use of the antibody-drug conjugate described herein or a pharmaceutical composition thereof in treating cancers that overexpress HER3.
  • the cancer comprises a solid tumor or a hematological malignancy.
  • the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma.
  • the lung cancer is non-small cell lung cancer.
  • the lung cancer is lung adenocarcinoma.
  • the cancer comprises a solid tumor or a hematological malignancy.
  • the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma.
  • the lung cancer is non-small cell lung cancer.
  • the lung cancer is lung adenocarcinoma.
  • the cancer with high HER3 expression includes solid tumors or hematological malignancies, such as colon cancer, gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, particularly lung adenocarcinoma) or lymphoma.
  • solid tumors or hematological malignancies such as colon cancer, gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, particularly lung adenocarcinoma) or lymphoma.
  • the present invention also provides a pharmaceutically acceptable salt or solvate of any of the above antibody-drug conjugates.
  • the present invention further provides a composition comprising any of the aforementioned antibody-drug conjugates and a pharmaceutically acceptable carrier or excipient.
  • 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 obtained 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.
  • 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.
  • an antibody described herein is conjugated to a drug-linker described herein via coupling to a lysine in the antibody.
  • the antibodies described herein are conjugated to the drug-linkers described herein via cysteine in the antibody.
  • the cysteine is from a reduced intrachain disulfide bond in the antibody.
  • the cysteine is from a reduced interchain disulfide bond in the antibody.
  • the antibody is conjugated to a drug-linker via reduced interchain disulfide bonds in the antibody.
  • an IgG1 antibody consists 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 (-S-S-) (e.g., two heavy chain-light chain interchain disulfide bonds and two hinge heavy chain-heavy chain interchain disulfide bonds).
  • -S-S- interchain cysteine disulfide bonds
  • eight (8) reactive cysteine thiol moieties are generated.
  • any one of the four disulfide bonds is broken under reducing conditions, resulting in two (2) reactive cysteine thiol moieties.
  • any two of the four disulfide bonds are cleaved under reducing conditions, resulting in four (4) reactive cysteine thiol moieties.
  • any three of the four disulfide bonds are cleaved under reducing conditions, resulting in six (6) reactive cysteine thiol moieties.
  • the interchain disulfide bond is located between two cysteine residues, which break under reducing conditions to produce two reactive cysteine sulfhydryl moieties.
  • 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 ⁇ light chain, or between C220 of the heavy chain according to EU numbering and C214 of the ⁇ light chain according to Kabat numbering.
  • the interchain disulfide bond in the antibody is located between 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.
  • the cysteine residue is located in the hinge region of the antibody.
  • the cysteine residue is located in any one or more of positions 220, 226 or 229 in the heavy chain (also referred to herein as C220, C226 or C229, respectively).
  • the cysteine residue is located at position 214 of the light chain (also referred to herein as C214, e.g., according to EU and Kabat numbering, at position 214 of a kappa light chain, or according to Kabat numbering, at position 214 of a lambda light chain).
  • the cysteine residue is located at position 220, 226, and 229 of the heavy chain, and according to EU or Kabat numbering, at position 214 of the light chain.
  • the cysteine residue is located at position 220, 226, and 229 of the heavy chain, and according to EU and Kabat numbering, at position 214 of a kappa light chain. In one embodiment, according to EU numbering, the cysteine residue is located at position 220, 226, and 229 of the heavy chain, and according to Kabat numbering, at position 214 of a lambda light chain. In one embodiment, the cysteine residue is located at any one or more of the following positions:
  • C220, C226 and C229 refer to the 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 corresponds to amino acid residues of polypeptides that are aligned with the amino acid residues defined for immunoglobulins, as shown in www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html.
  • the antibodies described herein comprise four interchain disulfide bonds in the hinge region, which can be reduced, thereby breaking and revealing reactive thiol moieties that can be conjugated to a maleimide moiety on a drug-linker, such as a maleimide moiety on a drug-linker described herein.
  • the present invention provides a method for preparing the ADC described herein, comprising the following steps:
  • the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).
  • the present application provides a composition of an antibody-drug conjugate (ADC) as described herein.
  • ADC antibody-drug conjugate
  • 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.
  • each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 drug-linkers. Therefore, the composition is characterized in that the "drug-antibody" ratio (DAR) is in the range of about 1 to about 10.
  • DAR drug-antibody ratio
  • 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.
  • 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.
  • the present application provides a pharmaceutical composition comprising one or more antibody-drug conjugates as described in any one of the preceding items, and one or more pharmaceutical excipients.
  • compositions include, for example, pharmaceutically acceptable carriers and/or excipients.
  • the average DAR value (drug-antibody coupling ratio) of the drug 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
  • the antibody-drug conjugates described herein are generally formulated in a unit injectable form together with a pharmaceutically acceptable parenteral vehicle for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc.
  • a pharmaceutically acceptable parenteral vehicle for parenteral use
  • 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) 16th edition, Osol, A. Ed.).
  • the antibody-drug conjugates described herein or pharmaceutical compositions containing the antibody-drug conjugates can be administered by any route suitable for the individual to be treated.
  • the ADC 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, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions.
  • the preferred dosage form is an injection. This injection can be a sterile injection solution.
  • a sterile injection 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., permeants, preservatives, diluents or any combination thereof) are optionally added, and then filtered and sterilized.
  • the sterile injection can be prepared into a sterile lyophilized powder (for example, by vacuum drying or freeze drying). This sterile lyophilized powder can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.
  • 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.
  • the preferred route of administration/mode is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration e.g., intravenous, subcutaneous, intraperitoneal, intramuscular.
  • the route and/or mode of administration will vary according to the intended purpose.
  • the ADC and pharmaceutical compositions described herein are administered by intravenous infusion or injection.
  • the pharmaceutical composition may also include other pharmaceutically active agents.
  • the other pharmaceutically active agents are drugs with anti-tumor activity.
  • 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 drugs or any combination thereof.
  • the ADC described herein and other pharmaceutically active agents are provided as separate components or as mixed components. Therefore, the ADC described herein and other pharmaceutically active agents can be administered simultaneously, separately or sequentially.
  • 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.
  • 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.
  • 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 cancers with high HER3 expression.
  • ADC antibody-drug conjugate
  • drug-linker a drug-linker
  • pharmaceutical composition comprising the same as described in any of the aforementioned embodiments in the preparation of a drug for treating cancers with high HER3 expression.
  • 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), a drug-linker, or a pharmaceutical composition comprising the same as described in any of the aforementioned embodiments.
  • ADC antibody-drug conjugate
  • drug-linker a drug-linker
  • a pharmaceutical composition comprising the same as described in any of the aforementioned embodiments.
  • the antibody-drug conjugate (ADC), drug-linker, or pharmaceutical composition is sufficient (e.g., in a subject):
  • the HER3-mediated disease/disorder is a tumor, such as a tumor expressing HER3.
  • 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.
  • 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, specifically lung adenocarcinoma), or lymphoma.
  • 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, specifically lung adenocarcinoma), or lymphoma.
  • 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.
  • 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.
  • antibody refers to an immunoglobulin molecule that is 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) and ⁇ (lambda) light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three domains (CH1, CH2, and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementary determining regions
  • FRs framework regions
  • 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 antigen binding sites, respectively.
  • the allocation of amino acids in each region or domain can follow various numbering systems known in the art.
  • 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 N-terminal amino acid cyclized to form pyroglutamic acid. Therefore, in the composition comprising antibodies disclosed herein, the various antibodies contained 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.
  • CDR complementarity determining region
  • CDR1 complementarity determining region
  • CDR2 complementarity determining region
  • CDR3 complementarity determining region
  • the precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as 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; CDR2, CDR3).
  • the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as by Kabat, Chothia, IMGT or AbM numbering systems. In certain embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined by the Chothia numbering system.
  • Andrew CRMartin's group at www.bioinf.org.uk has published the following general rules that can be used to define CDRs in antibody sequences that include amino acids that specifically interact with amino acids in the epitope of the antigen to which the antibody binds. In rare cases, these normally constant features do not appear. However, Cys residues are the most conserved features.
  • the entire amino acid sequence of VH is generally numbered according to Kabat, while the three CDRs of the variable region may be defined according to any of the aforementioned numbering schemes.
  • the numbering of the amino acid positions in VH may be starting from amino acid position 1 and continuing sequentially to the end of the sequence or according to Kabat numbering. Unless otherwise indicated, the amino acid positions in VH and VL herein are defined according to sequential numbering.
  • the numbering of amino acid positions in the heavy chain constant region can be starting from amino acid position 1 and continuing in sequence 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 number 118 and ends at position number 447 according to Eu numbering. Unless otherwise stated, amino acid positions in the heavy and light chains herein are defined according to sequential numbering.
  • 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.
  • antigen-binding fragment of an antibody refers to a polypeptide of a fragment of an antibody, such as a polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion".
  • 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 (sdAb, nanobodies), and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.
  • Fd means an antibody fragment consisting of VH and CH1 domains
  • dAb fragment means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989))
  • Fab fragment means an antibody fragment consisting of VL, VH, CL and CH1 domains
  • F(ab') 2 fragment means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • Fab'fragment means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd fragment (consisting of VH and CH1 domains) of the heavy chain.
  • Fv means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.
  • the Fv fragment is generally considered to be the smallest antibody fragment 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., a 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 a complete binding site.
  • Fc refers to an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain of an antibody bound via a disulfide bond.
  • the Fc fragment of an antibody has a variety of different functions but does not participate in antigen binding.
  • 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.
  • GGGGS amino acid sequence
  • 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.
  • a disulfide bond may also be present between the VH and VL of the scFv.
  • the VH and VL domains may be positioned relative to each other in any suitable arrangement.
  • a scFv comprising NH 2 -VH-VH-COOH, NH 2 -VL-VL-COOH.
  • single-domain antibody 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.
  • 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.
  • antibody includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • Antibody antigen-binding fragments 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 antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.
  • monoclonal antibody and “mAb” have the same meaning and are used interchangeably, and refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously.
  • Monoclonal antibodies have high specificity for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, and generally contain at least two or more different antibodies, which generally recognize different epitopes on an antigen.
  • the modifier "monoclonal” only indicates that the antibody is characterized as being obtained from a highly homologous antibody group, and it should not be understood that the antibody needs to be prepared by any specific method.
  • 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 still retains binding activity to the target antigen.
  • the term “chimeric antibody” may include antibodies in which the heavy chain and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy chain and light chain variable regions of the antibody are derived from a second antibody (e.g., a human antibody).
  • a chimeric antibody which consists of a fully human variable region and a murine constant region.
  • murine antibody refers to antibodies obtained by the following method: fusing B cells of immunized mice with myeloma cells, screening out mouse hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, and then performing screening, antibody preparation and antibody purification; or refers to antibodies secreted by plasma cells formed by differentiation and proliferation of B cells after antigens invade the mouse body.
  • 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.
  • 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).
  • donor antibody non-human antibody
  • 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).
  • non-human primate e.g., cynomolgus monkey
  • expected properties e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and/or ability to enhance immune response.
  • identity is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • 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)
  • 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 being compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • two sequences are compared when they are aligned to produce maximum identity.
  • Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by 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.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI 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.
  • 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.
  • 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.
  • 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
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • 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, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives.
  • 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, which can stabilize 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 their degradation products (such as lactalbumin hydrolysate), etc.
  • 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
  • degradation products such as lactalbumin hydrolysate
  • salts includes acid addition salts and basic salts.
  • 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.
  • P P.
  • the acid salt is an ammonium salt or a diammonium salt.
  • 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.
  • 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
  • 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., decyl, lauryl or stearyl chloride, bromide or iodide), aralkyl halides (e.g., benzyl or phenethyl bromide), and the like.
  • lower alkyl halides e.g., methyl, ethyl or butyl chloride, bromide or iodide
  • dialkyl sulfates e.g., dimethyl sulfate, diethyl sulfate and dibutyl sulfate
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount for preventing 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 who already has the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art.
  • an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.
  • the therapeutically effective amount of 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 simultaneously, etc.
  • treatment refers to a method implemented to obtain a beneficial or desired clinical outcome.
  • a beneficial or desired clinical outcome includes, but is not limited to, alleviation of symptoms, alleviation of the extent of the disease, stabilization (i.e., no longer worsening) of the state of the disease, delay or slowing of the progression of the disease, improvement or alleviation of the state of the disease, and relief of symptoms (whether partial or complete), whether detectable or undetectable.
  • treatment may also refer to prolonged survival compared to the expected survival without treatment.
  • subject refers to a mammal, such as a primate mammal, such as a human.
  • the subject (such as a human) suffers from a tumor, or has a risk of suffering from the above-mentioned disease.
  • 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 (metastasize) via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes blood tumors, especially hematologic malignancies.
  • lymphomas includes lymphomas, leukemias, myelomas or lymphoid malignancies, as well as spleen cancer and lymph node tumors.
  • exemplary lymphomas include B-cell lymphomas and T-cell lymphomas.
  • B-cell lymphomas include, for example, Hodgkin's lymphoma.
  • T-cell lymphomas include, for example, cutaneous T-cell lymphomas.
  • Hematologic malignancies also include leukemias, such as secondary leukemias or acute lymphocytic leukemias.
  • myeloma e.g., multiple myeloma
  • other blood and/or B-cell or T-cell related cancers include myeloma (e.g., multiple myeloma) and other blood and/or B-cell or T-cell related cancers.
  • alkyl refers to a group obtained by removing one hydrogen atom from a straight or branched hydrocarbon group, for example, “ C1-20 alkyl”, “ C1-10 alkyl”, “ C1-6 alkyl”, “ C1-4 alkyl”, “ C1-3 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.
  • 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.
  • alkenylene refers to a divalent group obtained by losing two hydrogen atoms from a straight or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C 2-20 alkenylene”, “C 3-10 alkenylene”, “C 5-8 alkenylene”, etc.
  • Examples include, but are not limited to, vinylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 1,3-butadienylene, 1-pentenylene, 2-pentenylene, 3-pentenylene, 1,3-pentadienylene, 1,4-pentadienylene, 1-hexenylene, 2-hexenylene, 3-hexenylene, 1,4-hexadienylene, etc.
  • alkynylene refers to a divalent group obtained by losing two hydrogen atoms from a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond. Examples include, for example, “C 2-20 alkynylene”, “C 3-10 alkynylene”, “C 5-8 alkynylene”, etc.
  • Examples include, but are not limited to, ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 1,3-butadiynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 1,3-pentadiynylene, 1,4-pentadiynylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 1,4-hexadiynylene, etc.
  • 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.
  • heteromatic 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,
  • aromatic ring system refers to a monocyclic or polycyclic system comprising at least one aromatic ring (e.g., a benzene ring, etc.) or a 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.
  • aromatic ring system may be divalent or higher valent (e.g., trivalent or tetravalent), for example, a 5-20 membered aromatic ring system.
  • 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 an antigen-binding fragment thereof).
  • a biologically active molecule eg, a cytotoxic drug fragment
  • a targeting moiety eg, an antibody or an antigen-binding fragment thereof.
  • substituted refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogens on the specified compound or structural fragment by a substituent, provided that the normal atomic valence of the specified atom in the current situation is not exceeded and the substitution forms a stable compound. Combinations of substituents and/or variables are permitted only when such combinations form stable compounds.
  • the functional group or structural fragment can be (1) unsubstituted or (2) substituted.
  • 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.
  • the present application relates to an antibody-drug conjugate for treating HER3-positive cancers, and exemplarily discloses an antibody-drug conjugate having a structure as shown in the general formula Ab-[M-L-E-D]x and using the fully human antibody 22B6D2-hIgG1 as a targeting portion.
  • 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 highly express HER3, a pharmaceutical composition comprising the antibody-drug conjugate, and its use in treating cancers that highly express HER3.
  • FIG1 shows the results of the affinity test of the anti-human HER3 antibody-drug conjugate to MDA-MB-453 cells.
  • FIG2 shows the results of the affinity test of the anti-human HER3 antibody-drug conjugate to NCI-N87 cells.
  • Figure 3 shows the results of the endocytic activity test of anti-human HER3 antibody-drug conjugates in MDA-MB-453 cells
  • FIG4 shows the results of the endocytic activity test of the anti-human HER3 antibody-drug conjugate in HCC1569 cells.
  • FIG5 shows the results of the in vitro detection of the killing activity of anti-human HER3 antibody-drug conjugates on MDA-MB-453 cells.
  • FIG6 shows the results of in vitro detection of the killing activity of anti-human HER3 antibody-drug conjugates on 293T-HER3 cells.
  • FIG7A shows the in vivo efficacy test results of the anti-human HER3 antibody-drug conjugate.
  • FIG7B shows the changes in mouse body weight during the in vivo efficacy test of the anti-human HER3 antibody-drug conjugate.
  • NMR nuclear magnetic resonance
  • MS Mass spectrometry
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% trifluoroacetic acid)
  • compound 1-8-1 (5.00 g, 29.14 mmol) was dissolved in n-heptane (25 mL), concentrated sulfuric acid (25 mL) was added, and the mixture was heated to 50°C.
  • the reaction solution was cooled to room temperature, and then added dropwise to ice water, extracted with toluene, and the organic phases were combined, 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), and the preparative solution was freeze-dried to obtain 4.88 g of the title compound.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • compound 1-8-2 (4.88 g, 19.48 mmol) was dissolved in ethyl acetate (100 mL), and platinum carbon (2.00 g, 19.48 mmol, content 5%) was added. After hydrogen replacement, the mixture was reacted at 60°C for 4 h under hydrogen protection, and the reaction was monitored by HPLC-MS/MS. The reaction solution was filtered and concentrated to obtain 3.68 g of crude title compound, which was directly used in the next step without further purification.
  • compound 1-8-3 (3.63 g, 14.82 mmol) was dissolved in ethyl acetate (70 mL), triethylamine (4.50 g, 44.45 mmol) and acetic anhydride (2.27 g, 22.23 mmol) were added, and the reaction was maintained at 20°C for 20 h, and the reaction was monitored by HPLC-MS.
  • Step 4 Synthesis of (Z)-4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid (1-8-5)
  • compound 1-8-4 (1.80 g, 6.86 mmol) was dissolved in THF (20 mL) and water (5 mL), and vinylacetic acid (708.31 mg, 8.23 mmol), DIPEA (1.95 g, 15.08 mmol), tri(o-methylphenyl)phosphine (62.60 mg, 0.20 mmol) were added.
  • the reaction system was replaced with nitrogen and heated to 70°C for 5 h.
  • compound 1-8-5 (2.60 g, 9.71 mmol) was dissolved in THF (50 mL), Pd/C (0.52 g, content 10%) was added, the system was replaced with hydrogen and reacted at 40°C for 2 h under the protection of a hydrogen balloon, and 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 directly used in the next step without further purification.
  • Step 6 Synthesis of N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-7)
  • 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)
  • Step 8 Synthesis of N-(7-amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-9)
  • compound 1-8-8 (0.50 g, 1.78 mmol) was dissolved in methanol (8 mL) and 2N hydrochloric acid (8 mL), Pd/C (0.15 g, content 10%) was added, the system was replaced with hydrogen and kept at 5°C for 2 h under the protection of a hydrogen balloon, and the reaction was monitored by HPLC-MS/MS. The reaction solution was filtered and concentrated to obtain 0.52 g of the hydrochloride of the title compound, which was directly used in the next step without further purification.
  • Step 9 Synthesis of N,N'-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (1-8-10)
  • compound 1-8-9 (0.52 g, 1.70 mmol) was dissolved in pyridine (5 mL), acetic anhydride (2 mL) was added, and the reaction was maintained at 20°C for 2 h, and the reaction was monitored by HPLC-MS/MS.
  • Step 10 Synthesis of N-(8-amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-8-11)
  • compound 1-8-10 (450.97 mg, 1.46 mmol) was dissolved in methanol (16 mL), 2N hydrochloric acid (16 mL) was added, and the mixture was heated to 60°C for 2 h, and the reaction was monitored by HPLC-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.
  • 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)
  • 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)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% trifluoroacetic acid)
  • 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 ((1R,9S)-5-chloro-9
  • the hydrochloride of compound 1-2 (40.00 mg, 81.91 ⁇ mol) was dissolved in N,N-dimethylformamide (1 mL), and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (30.91 mg, 98.29 ⁇ mol), HATU (62.25 mg, 163.81 ⁇ mol) and N,N-diisopropylethylamine (42.34 mg, 327.63 ⁇ mol) were added in sequence. The reaction was maintained at 25°C for 0.5 hour, and the reaction was monitored by HPLC-MS/MS.
  • 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)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • 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)
  • compound A-07-2 (30.00 mg, 0.07 mmol) was dissolved in DMF (0.2 mL), 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, and the mixture was reacted at 30°C for 1 h, and 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.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • 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-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide and 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
  • the hydrochloride of 1-2 (30.00 mg, 61.43 ⁇ mol) was dissolved in N, N-dimethylformamide (1 mL), and A-07-3 (49.66 mg, 73.72 ⁇ mol), HATU (35.01 mg, 92.14 ⁇ mol) and N, N-diisopropylethylamine (23.82 mg, 184.29 ⁇ mol) were added in sequence, and the reaction was maintained at 25°C for 0.5 hours, and the reaction was monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative HPLC (conditions as follows), and the preparative solution was freeze-dried to obtain the title compound A-07. A-07 was separated under the following purification conditions to obtain two isomers, which were named A-07-A (11.04 mg, retention time 7.5 min) and A-07-B (19.42 mg, retention time 8.0 min) according to the retention time.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Synthesis of 3-(isopropylamino)-1-(6-nitrobenzo[d][1,3]dioxin-5-yl)propan-1-one (2-2-2)
  • Step 2 Synthesis of 1-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-(isopropylamino)propan-1-one (2-2-3)
  • Step 3 Synthesis of ((S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)
  • 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-diazapentacontriacontamido)benzyl)carbonate (B-01-2)
  • compound B-01-1 (413.40 mg, 0.251 mmol, its synthesis reference patent CN111295389B) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), and cuprous bromide (72.95 mg, 0.503 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (95.10 mg, 0.302 mmol) were added. The reaction was stirred for 1 hour and then filtered. The filtrate was purified by preparative HPLC (conditions as follows) to obtain 30.00 mg of the title compound.
  • Mobile phase A acetonitrile
  • Mobile phase B water
  • 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)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% trifluoroacetic acid)
  • 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)
  • 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)
  • Step 3 Synthesis of (2S,3R,4S,5S,6S)-2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(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)
  • Step 4 Synthesis of (2S,3R,4S,5S,6S)-2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(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)
  • Step 5 Synthesis of (2S,3R,4S,5S,6S)-2-(2-(2-(2-(2-(2-(2-(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]dioxolane[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)
  • 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]dioxolane[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)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • 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]dioxolane[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-(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)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 5 Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-06)
  • Step 6 Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-07)
  • Tetrahydrofuran (16 mL) and tert-butanol (4 mL) were added to the reaction bottle, cooled to 5 ° C in an ice bath, and potassium tert-butoxide (415.18 mg, 3.70 mmol) was added, and then 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 completed, the reaction was maintained at 5 ° C for 1 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry.
  • reaction solution was quenched with saturated aqueous ammonium chloride solution, it was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, the organic phases were dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to obtain 455.00 mg of the crude product of the title compound.
  • Step 7 Synthesis of N-(7-amino-4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-08)
  • Step 8 Synthesis of (9H-fluoren-9-ylmethyl)(8-acetamide-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (1-5-09)
  • the reaction solution was poured into water, and then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product.
  • 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)
  • 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)
  • 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)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Mobile phase A 0.05% acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • 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-tetraazahexadecane-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-1
  • the single-configuration compound 1-5-A (36.00 mg, 79.70 ⁇ mol) and compound A-07-3 (64.43 mg, 95.64 ⁇ mol) were dissolved in N, N-dimethylformamide (2 mL), and then 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 ⁇ mol) and triethylamine (24.19 mg, 239.10 ⁇ mol) were added. After addition, the mixture was reacted at room temperature for 1 hour, and the reaction was detected by HPLC-MS. The reaction solution was directly purified by HPLC to obtain the single-configuration title compound 1-5-12-A (51.00 mg).
  • 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)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Synthesis of (2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38-octadecane-38-amido)-6-(N-(9H-fluoren-9-ylmethoxycarbonyl)amino)hexanoic acid (A-17-03)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • 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)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • 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-
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • A-05 separation and purification method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • A-05 structural characterization data are as follows:
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • 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 solution was stirred at room temperature for 16 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 50.56 mg of compound A-26.
  • the separation and purification method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • 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)
  • 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)
  • 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)
  • Step 3 Preparation of 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 (B-04-3)
  • 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)
  • 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)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • H2L2 mice provided by Hebo Pharmaceuticals, the antibody produced by this mouse is a chimeric antibody composed of a fully human variable region and a mouse constant region
  • 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 HER3 control antibody was derived from U1-59 in patent application CN200680049887. After codon optimization, the heavy and light chain nucleotide sequences of the antibody were synthesized and cloned into the pTT5 vector, and expressed and purified according to the above method.
  • Table 1 Sequence information of 22B6D2-hIgG1, 47A3E3-hIgG1, 100H7D3-hIgG1
  • Example 13 Conjugation of a compound comprising a cell bioactive molecule and a linker with an antibody
  • 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.
  • the sample coupling preparation is as follows:
  • ADC antibody-drug conjugate
  • the ADC drug/antibody ratio (DAR value) is determined as follows:
  • ADC samples were subjected to LC-MS molecular weight analysis.
  • Liquid chromatography column Thermo MAbPac RP 3.0*100mm;
  • Mobile phase A 0.1% FA/H 2 O
  • Mobile phase B 0.1% FA/ACN
  • the buffer was replaced with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain ADC 22B6-B-04-1, and the DAR value was 8.62 determined by mass spectrometry.
  • the buffer was replaced with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain ADC 22B6-B-04-2, and the DAR value was 6.87 determined by mass spectrometry.
  • 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.
  • the endocytic activity of the ADC or antibody to be tested on MDA-MB-453 and HCC1569 cells was detected using a flow cytometer (Beckman, model Cytoflex).
  • the adherent cells were digested with Trypsin-EDTA (0.25%, Shanghai Yuanpei) solution and counted. The cell density was adjusted to 1 ⁇ 10 5 cells/ml with complete culture medium. 100 ⁇ l of cell suspension was added to each well of a 96-well plate (the number of cells was 1 ⁇ 10 4 cells/well). The 96-well plate was placed in a 37°C, CO 2 constant temperature incubator and incubated for 24 h.
  • the 96-well plate was removed, the culture medium was discarded, and 50 ⁇ l of fresh complete culture medium was added to each well; the bispecific antibody and the control antibody were gradiently diluted with complete culture medium, and the final concentrations were 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 to 12 ⁇ g/ml with complete culture medium (the final concentration of PHrodo was 3 ⁇ g/ml); the gradiently diluted antibody to be tested was mixed evenly with the diluted PHrodo reagent in a ratio of 1:1 (30 ⁇ l:30 ⁇ l), and incubated at room temperature in the dark for 30 min; 50 ⁇ l of the mixture of the antibody to be tested and the PHrodo reagent was added to the 96-well plate, and incubated at 37°C, 5% CO 2Cultivate for 24 hours; remove the 96
  • MDA-MB-453 or 293T-HER3 cells were digested with TrypLE (manufacturer Gibco) solution, and an appropriate amount of cells were counted and taken. After diluting with growth medium, 5000 cells/100 ⁇ l/well were spread on a 96-well plate and cultured at 37°C and 5% CO2 overnight; on the second day, ADC was diluted with growth medium, starting at 150 ⁇ g/ml, and 2.5-fold gradient dilution (or starting at 7.5 ⁇ g/ml, 5-fold gradient dilution), and then 100 ⁇ l of the diluted ADC was added to the 96-well plate containing cells, and the plate was placed in an incubator.
  • the ADC drugs of the present invention have target-specific killing, wherein the IC50 of 22B6-A-26 and hIgG1-A-26 on tumor cells differ by 9.1 times; hIgG1-A-26 has no obvious killing on overexpressing cells (HEK293T-h HER3), and the IC50 difference between the 22B6-A-26 ADC drug and hIgG1-A-26 is even greater.
  • NCI-H358 Human non-small cell lung cancer cells NCI-H358 (Nanjing Kebai) were cultured in vitro in monolayers, with RPMI-1640 medium plus 10% fetal bovine serum, and cultured in an incubator at 37°C and 5% CO2 . Digestion and passage were performed with trypsin-EDTA 2-3 times per week. When the cells were in the exponential growth phase, the culture medium was taken for mycoplasma detection, and the cells were collected and counted. 5 ⁇ 10 6 NCI-H358 cells (suspended in 0.05 ml PBS + 0.05 ml Matrigel) were subcutaneously inoculated at the right scapula of each mouse.
  • 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 weight, with 5 mice in each group.
  • T V0 is the average tumor volume of the test drug group at the time of group administration
  • T Vt is the average tumor volume of the test drug group t days after administration
  • C V0 is the average tumor volume of the vehicle group at the time of group administration
  • C Vt is the average tumor volume of the vehicle group t days after administration.
  • the tumor is smaller than the initial volume, that is, when V t ⁇ V 0 , it is defined as partial tumor regression (PR); if the tumor disappears completely, it is defined as complete tumor regression (CR).
  • PR partial tumor regression
  • CR complete tumor regression

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)
  • Peptides Or Proteins (AREA)

Abstract

An antibody-drug conjugate and a preparation method therefor and a use thereof, in particular, an antibody-drug conjugate for treating HER3-positive cancers. The antibody-drug conjugate contains a fully human HER3 antibody, has excellent binding activity to HER3-positive cells, and can efficiently deliver a drug to HER3-positive cells. The antibody-drug conjugate has a good drug-antibody coupling ratio, and has a good targeted killing effect on colon cancer, gastric cancer, breast cancer, and lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma). Therefore, further provided are a method for preparing the antibody-drug conjugate and a use of the antibody-drug conjugate in treatment of HER3-positive cancers.

Description

抗体-药物缀合物及其制备方法和用途Antibody-drug conjugates and preparation methods and uses thereof

本申请是以中国申请号202311130165.6，申请日2023年9月1日的申请，以及中国申请号202411158958.3，申请日2024年8月22日的申请为基础，并主张其优先权，该中国申请的公开内容通过引用整体并入本文。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, and the disclosures of the Chinese applications are incorporated herein by reference in their entirety.

技术领域Technical Field

本申请涉及靶向治疗领域，具体涉及抗体-药物缀合物及其制备方法和用途。The present application relates to the field of targeted therapy, and in particular to antibody-drug conjugates and preparation methods and uses thereof.

背景技术Background Art

癌症是当代人死亡的主要原因之一。它是由健康细胞的恶性转化引起的一类疾病，由遗传改变引起，如染色体易位，肿瘤抑制基因、生长因子受体中的突变，导致细胞恶性增殖。有缺陷的凋亡或程序性细胞死亡进一步促进了导致癌症的细胞恶性转化。Cancer is one of the leading causes of death in the modern world. It is a class of diseases caused by the malignant transformation of healthy cells, caused by genetic changes such as chromosomal translocations, mutations in tumor suppressor genes, growth factor receptors, leading to malignant proliferation of cells. Defective apoptosis or programmed cell death further promotes the malignant transformation of cells leading to cancer.

人表皮生长因子受体3(也称为HER3和ErbB3)是受体蛋白酪氨酸激酶，属于受体蛋白酪氨酸激酶的表皮生长因子受体(EGFR)亚家族，该家族还包括HER1(也称为EGFR)、HER2和HER4。和表皮生长因子受体类似，跨膜受体HER3由与配体结合的胞外结构域(ECD)、ECD内的二聚化结构域、跨膜结构域和羧基端磷酸化结构域构成。除这些结构域以外，HER1、HER2和HER4还携带胞内蛋白酪氨酸激酶结构域(TKD)，而HER3缺乏该结构域，因而不能自磷酸化。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 consists of an extracellular domain (ECD) that binds to the ligand, a dimerization domain within the ECD, a transmembrane domain, and a carboxyl-terminal phosphorylation domain. In addition to these domains, HER1, HER2, and HER4 also carry an intracellular protein tyrosine kinase domain (TKD), while HER3 lacks this domain and cannot autophosphorylate.

配体调节蛋白(HRG)与HER3的胞外结构域相结合，并通过促进与其它人表皮生长因子受体(HER)家族成员二聚化及其胞内结构域的转磷酸作用激活受体介导的信号传导途径。HER3与其它HER家族成员的二聚体形成扩展HER3的信号传导潜力，而且不仅充当信号多样化的手段，还用作信号放大的手段。例如，HER2/HER3杂二聚体诱导HER家族成员中最重要的促有丝分裂信号之一。HER3在多种类型的癌中过表达，例如乳腺癌、肠胃癌和胰腺癌。有趣的是，已显示出HER2/HER3的表达与从非侵袭阶段进展到侵袭阶段之间的关系。因此，期望干扰HER3介导的信号传导的药剂。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. The 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 medicaments of HER3-mediated signal transduction.

目前临床在研HER3靶点适应症涵盖了血液瘤和实体瘤。主要的治疗策略集中在单抗、双抗、抗体偶联药物(ADC)等几个方向。其中抗-HER3抗体偶联药物，目前国际在研2项，1项进入临床研究阶段(第一三共U3-1402)治疗NSCLC、MBC和CRC等。Currently, the HER3 target indications under clinical research cover hematological tumors and solid tumors. The main treatment strategies focus on several directions such as monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates (ADCs). Among them, there are currently two anti-HER3 antibody-drug conjugates under international research, and one has entered the clinical research stage (Daiichi Sankyo U3-1402) for the treatment of NSCLC, MBC, and CRC.

ADC药物由抗体、生物活性分子及连接体(Linker)组成。生物活性分子通过连接体共价偶联到抗体上；抗体(例如单克隆抗体)能够特异性识别肿瘤细胞表面的特异性靶点，进而能够引导ADC到达癌细胞表面，并使ADC通过细胞内吞效应进入癌细胞；然后生物活性分子在癌细胞内释放，达到杀灭癌细胞而尽量不损伤正常组织细胞的作用。ADC drugs are composed of antibodies, bioactive molecules and linkers. The bioactive molecules are covalently coupled to the antibodies through linkers; antibodies (such as monoclonal antibodies) can specifically recognize specific targets on the surface of tumor cells, and then guide ADC to the surface of cancer cells, and allow ADC to enter cancer cells through cellular endocytosis; then the bioactive molecules are released inside the cancer cells, killing cancer cells while minimizing damage to normal tissue cells.

U3-1402是由第一三共公司开发，采用GGFG四肽作为酶切linker，细胞毒性药物为Dxd。据报道，在治疗EGFR突变的NSCLC一期剂量扩展(5.6mg/kg，Q3W)，ORR 39％，DCR 72％，mPFS 8.2个月，对脑转移患者有类似的疗效；安全性方面，主要以血液毒性(血小板减少，中性粒细胞减少等)和间质性肺炎(5～7％)为主。U3-1402 was developed by Daiichi Sankyo Co., Ltd., using GGFG tetrapeptide as the enzyme cleavage linker and Dxd as the cytotoxic drug. According to reports, in the first-phase dose expansion (5.6 mg/kg, Q3W) of NSCLC with EGFR mutations, ORR 39%, DCR 72%, mPFS 8.2 months, and similar efficacy for patients with brain metastases; in terms of safety, it is mainly blood toxicity (thrombocytopenia, neutropenia, etc.) and interstitial pneumonia (5-7%).

发明内容Summary of the invention

在一个方面，本申请提供一种抗体-药物缀合物，其具有式Ab-[M-L-E-D]x所示结构，其中：In one aspect, the present application provides an antibody-drug conjugate having a structure shown in the formula Ab-[MLED] x , wherein:

Ab是与人表皮生长因子受体3(HER3，也称为Erbb3)特异性结合的抗体或其抗原结合片段；Ab is an antibody or antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor 3 (HER3, also known as Erbb3);

M是与抗体或其抗原结合片段连接的接头部位；M is a linker site connected to the antibody or antigen-binding fragment thereof;

L是连接M和E的结构片段；L is the structural fragment connecting M and E;

E是连接L和D的结构片段；E is a structural fragment connecting L and D;

D是细胞毒性药物片段；D is the cytotoxic drug fragment;

x选自1至10。x is selected from 1 to 10.

在一些实施方案中，Ab是与人表皮生长因子受体3(HER3)特异性结合的抗体或其抗原结合片段；In some embodiments, Ab is an antibody or antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor 3 (HER3);

M是与抗体或其抗原结合片段连接的接头部位，并且为其中，环A为5-6元脂杂环、或5-20元芳香族环系，所述脂杂环和芳香族环系任选地被一个或多个选自氧基(＝O)、卤素、氰基、氨基、羧基、巯基和C1-6烷基的基团取代；M1选自单键和取代或未取代的以下基团：C1-20亚烷基、C2-20亚烯基或C2-20亚炔基；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 groups selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 alkyl; M 1 is selected from a single bond and the following substituted or unsubstituted groups: C 1-20 alkylene, C 2-20 alkenylene or C 2-20 alkynylene;

L是连接M和E之间的结构片段，并且选自由下述的一个或多个取代或未取代的基团组成的结构：C1-6亚烷基、-N(R’)-、羰基、-O-、Val、Cit、Phe、Lys、Lys(COCH2CH2(OCH2CH2)sOCH3)、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)、 其中R’代表氢、C1-6烷基或含-(CH2CH2O)r-的烷基；r选自1-10的整数；s选自1-20的整数；L is a structural fragment connecting M and E, and is selected from a structure consisting of one or more substituted or unsubstituted groups: C 1-6 alkylene, -N(R')-, carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) s OCH 3 ), D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly (SEQ ID NO:53), Gly-Gly-Gly-Gly-Gly (SEQ ID NO:54), wherein R' represents hydrogen, C 1-6 alkyl or alkyl containing -(CH 2 CH 2 O) r -; r is selected from integers of 1-10; s is selected from integers of 1-20;

E是连接L和D的结构片段，并且为单键或选自以下取代或未取代的结构：-NH-CH2-，-NH-CH2-O-CH2-CO-， 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-CH 2 -, -NH-CH 2 -O-CH 2 -CO-,

D是细胞毒性药物与E连接后得到的所述细胞毒性药物相应的片段，其中所述细胞毒性药物选自微管蛋白抑制剂、DNA嵌入剂、DNA拓扑异构酶抑制剂和RNA聚合酶抑制剂以及它们药学上可接受的盐、酯或类似物；优选地，所述微管蛋白抑制剂为奥瑞他汀类化合物或美登素类化合物；优选地，所述DNA嵌入剂为吡咯并苯二氮卓(PBD)；优选地，所述DNA拓扑异构酶抑制剂为拓扑异构酶I抑制剂(例如，喜树碱、羟基喜树碱、9-氨基喜树碱、SN-38、伊立替康、拓扑替康、贝洛替康、或卢比替康)或拓扑异构酶II抑制剂(例如，阿霉素、PNU-159682、多卡米星、柔红霉素、米托蒽醌、鬼臼毒素、或依托泊苷)；优选地，所述RNA聚合酶抑制剂为α-鹅膏草碱(α-amanitin)或其药学上可接受的盐、酯或类似物；D is a fragment corresponding to the cytotoxic drug obtained by connecting the cytotoxic drug to E, wherein the cytotoxic drug is selected from microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors and RNA polymerase inhibitors 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;

x选自1至10。x is selected from 1 to 10.

在一些实施方案中，D是细胞毒性药物与E连接后得到的所述细胞毒性药物相应的片段，其中所述细胞毒性药物选自微管蛋白抑制剂、DNA嵌入剂、DNA拓扑异构酶抑制剂和RNA聚合酶抑制剂。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 a microtubule inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor.

在一些实施方案中，所述抗体或其抗原结合片段包含:In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(1)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Chothia编号系统定义：(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:1或其变体的CDR-H1，氨基酸序列为SEQ ID NO:2或其变体的CDR-H2，氨基酸序列为SEQ ID NO:3或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:4或其变体的CDR-L1，氨基酸序列为SEQ ID NO:5或其变体的CDR-L2，氨基酸序列为SEQ ID NO:6或其变体的CDR-L3；或，(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,

(1b)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:19或其变体的CDR-H1，氨基酸序列为SEQ ID NO:20或其变体的CDR-H2，氨基酸序列为SEQ ID NO:21或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:22或其变体的CDR-L1，氨基酸序列为SEQ ID NO:23或其变体的CDR-L2，氨基酸序列为SEQ ID NO:24或其变体的CDR-L3；或，(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:36或其变体的CDR-H1，氨基酸序列为SEQ ID NO:37或其变体的CDR-H2，氨基酸序列为SEQ ID NO:38或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:45或其变体的CDR-L1，氨基酸序列为SEQ ID NO:23或其变体的CDR-L2，氨基酸序列为SEQ ID NO:52或其变体的CDR-L3；(1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:36 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:37 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO:45 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO:23 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO:52 or a variant thereof;

其中，(1a)、(1b)、(1c)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；条件是，所述变体的CDR的氨基酸序列与(1a)、(1b)和(1c)的VH和VL的相应CDR的氨基酸序列具有100％序列同一性，并且所述变体结合HER3；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 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;

或者，or,

(2)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号系统定义：(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:7或其变体的CDR-H1，氨基酸序列为SEQ ID NO:8或其变体的CDR-H2，氨基酸序列为SEQ ID NO:9或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:4或其变体的CDR-L1，氨基酸序列为SEQ ID NO:5或其变体的CDR-L2，氨基酸序列为SEQ ID NO:6或其变体的CDR-L3；或，(2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:7 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:8 or a variant thereof, and CDR-H3 having 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 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,

(2b)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:25或其变体的CDR-H1，氨基酸序列为SEQ ID NO:26或其变体的CDR-H2，氨基酸序列为SEQ ID NO:21或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:22或其变体的CDR-L1，氨基酸序列为SEQ ID NO:23或其变体的CDR-L2，氨基酸序列为SEQ ID NO:24或其变体的CDR-L3；或，(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:39或其变体的CDR-H1，氨基酸序列为SEQ ID NO:40或其变体的CDR-H2，氨基酸序列为SEQ ID NO:38或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:45或其变体的CDR-L1，氨基酸序列为SEQ ID NO:23或其变体的CDR-L2，氨基酸序列为SEQ ID NO:52或其变体的CDR-L3；(2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:39 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:40 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO:45 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO:23 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO:52 or a variant thereof;

其中，(2a)、(2b)、(2c)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；条件是，所述变体的CDR的氨基酸序列与(2a)、(2b)和(2c)的VH和VL的相应CDR的氨基酸序列具有100％序列同一性，并且所述变体结合HER3；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 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 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;

或者，or,

(3)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:10或其变体的CDR-H1，氨基酸序列为SEQ ID NO:11或其变体的CDR-H2，氨基酸序列为SEQ ID NO:12或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:13或其变体的CDR-L1，氨基酸序列为SEQ ID NO:14或其变体的CDR-L2，氨基酸序列为SEQ ID NO:6或其变体的CDR-L3；或，(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:27或其变体的CDR-H1，氨基酸序列为SEQ ID NO:28或其变体的CDR-H2，氨基酸序列为SEQ ID NO:29或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:30或其变体的CDR-L1，氨基酸序列为SEQ ID NO:31或其变体的CDR-L2，氨基酸序列为SEQ ID NO:24或其变体的CDR-L3；或，(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:41或其变体的CDR-H1，氨基酸序列为SEQ ID NO:42或其变体的CDR-H2，氨基酸序列为SEQ ID NO:43或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:44或其变体的CDR-L1，氨基酸序列为SEQ ID NO:31或其变体的CDR-L2，氨基酸序列为SEQ ID NO:52或其变体的CDR-L3；(3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:41 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:42 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO:44 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO:31 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO:52 or a variant thereof;

其中，(3a)、(3b)、(3c)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；条件是，所述变体的CDR的氨基酸序列与(3a)、(3b)和(3c)的VH和VL的相应CDR的氨基酸序列具有100％序列同一性，并且所述变体结合HER3。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 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, 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.

在一些实施方案中，所述抗体或其抗原结合片段包含:In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(1a)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:1或其变体的CDR-H1，氨基酸序列为SEQ ID NO:2或其变体的CDR-H2，氨基酸序列为SEQ ID NO:3或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:4或其变体的CDR-L1，氨基酸序列为SEQ ID NO:5或其变体的CDR-L2，氨基酸序列为SEQ ID NO:6或其变体的CDR-L3；或，(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,

(1b)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:19或其变体的CDR-H1，氨基酸序列为SEQ ID NO:20或其变体的CDR-H2，氨基酸序列为SEQ ID NO:21或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:22或其变体的CDR-L1，氨基酸序列为SEQ ID NO:23或其变体的CDR-L2，氨基酸序列为SEQ ID NO:24或其变体的CDR-L3；或，(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:36或其变体的CDR-H1，氨基酸序列为SEQ ID NO:37或其变体的CDR-H2，氨基酸序列为SEQ ID NO:38或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:45或其变体的CDR-L1，氨基酸序列为SEQ ID NO:23或其变体的CDR-L2，氨基酸序列为SEQ ID NO:52或其变体的CDR-L3；(1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:36 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:37 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO:45 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO:23 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO:52 or a variant thereof;

其中，(1a)、(1b)、(1c)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；条件是，所述变体的CDR的氨基酸序列与(1a)、(1b)和(1c)的VH和VL的相应CDR的氨基酸序列具有100％序列同一性，并且所述变体结合HER3wherein 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 sequence of the CDR of the variant has 100% sequence identity with the amino acid sequence of the corresponding CDR of VH and VL of (1a), (1b), and (1c), and the variant binds to HER3

或者，or,

(2a)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:7或其变体的CDR-H1，氨基酸序列为SEQ ID NO:8或其变体的CDR-H2，氨基酸序列为SEQ ID NO:9或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:4或其变体的CDR-L1，氨基酸序列为SEQ ID NO:5或其变体的CDR-L2，氨基酸序列为SEQ ID NO:6或其变体的CDR-L3；或，(2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:7 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:8 or a variant thereof, and CDR-H3 having 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 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,

(2b)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:25或其变体的CDR-H1，氨基酸序列为SEQ ID NO:26或其变体的CDR-H2，氨基酸序列为SEQ ID NO:21或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:22或其变体的CDR-L1，氨基酸序列为SEQ ID NO:23或其变体的CDR-L2，氨基酸序列为SEQ ID NO:24或其变体的CDR-L3；或，(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:39或其变体的CDR-H1，氨基酸序列为SEQ ID NO:40或其变体的CDR-H2，氨基酸序列为SEQ ID NO:38或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:45或其变体的CDR-L1，氨基酸序列为SEQ ID NO:23或其变体的CDR-L2，氨基酸序列为SEQ ID NO:52或其变体的CDR-L3；(2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:39 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:40 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO:45 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO:23 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO:52 or a variant thereof;

其中，(2a)、(2b)、(2c)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；条件是，所述变体的CDR的氨基酸序列与(2a)、(2b)和(2c)的VH和VL的相应CDR的氨基酸序列具有100％序列同一性，并且所述变体结合HER3；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 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 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;

或者，or,

(3a)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:10或其变体的CDR-H1，氨基酸序列为SEQ ID NO:11或其变体的CDR-H2，氨基酸序列为SEQ ID NO:12或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:13或其变体的CDR-L1，氨基酸序列为SEQ ID NO:14或其变体的CDR-L2，氨基酸序列为SEQ ID NO:6或其变体的CDR-L3；或，(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:27或其变体的CDR-H1，氨基酸序列为SEQ ID NO:28或其变体的CDR-H2，氨基酸序列为SEQ ID NO:29或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:30或其变体的CDR-L1，氨基酸序列为SEQ ID NO:31或其变体的CDR-L2，氨基酸序列为SEQ ID NO:24或其变体的CDR-L3；或，(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:41或其变体的CDR-H1，氨基酸序列为SEQ ID NO:42或其变体的CDR-H2，氨基酸序列为SEQ ID NO:43或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:44或其变体的CDR-L1，氨基酸序列为SEQ ID NO:31或其变体的CDR-L2，氨基酸序列为SEQ ID NO:52或其变体的CDR-L3；(3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:41 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:42 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO:44 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO:31 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO:52 or a variant thereof;

其中，(3a)、(3b)、(3c)任一项中所述的变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；条件是，所述变体的CDR的氨基酸序列与(3a)、(3b)和(3c)的VH和VL的相应CDR的氨基酸序列具有100％序列同一性，并且所述变体结合HER3。 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 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, 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.

在一些实施方案中，所述抗体或其抗原结合片段包含:In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(1a)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:1的CDR-H1，氨基酸序列为SEQ ID NO:2的CDR-H2，氨基酸序列为SEQ ID NO:3的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:4的CDR-L1，氨基酸序列为SEQ ID NO:5的CDR-L2，氨基酸序列为SEQ ID NO:6的CDR-L3；或，(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,

(1b)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:19的CDR-H1，氨基酸序列为SEQ ID NO:20的CDR-H2，氨基酸序列为SEQ ID NO:21的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:22的CDR-L1，氨基酸序列为SEQ ID NO:23的CDR-L2，氨基酸序列为SEQ ID NO:24的CDR-L3；或，(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,

(1c)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:36的CDR-H1，氨基酸序列为SEQ ID NO:37的CDR-H2，氨基酸序列为SEQ ID NO:38的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:45的CDR-L1，氨基酸序列为SEQ ID NO:23的CDR-L2，氨基酸序列为SEQ ID NO:52的CDR-L3；(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;

(2a)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:7的CDR-H1，氨基酸序列为SEQ ID NO:8的CDR-H2，氨基酸序列为SEQ ID NO:9的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:4的CDR-L1，氨基酸序列为SEQ ID NO:5的CDR-L2，氨基酸序列为SEQ ID NO:6的CDR-L3；或，(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,

(2b)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:25的CDR-H1，氨基酸序列为SEQ ID NO:26的CDR-H2，氨基酸序列为SEQ ID NO:21的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:22的CDR-L1，氨基酸序列为SEQ ID NO:23的CDR-L2，氨基酸序列为SEQ ID NO:24的CDR-L3；或，(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,

(2c)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:39的CDR-H1，氨基酸序列为SEQ ID NO:40的CDR-H2，氨基酸序列为SEQ ID NO:38的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:45的CDR-L1，氨基酸序列为SEQ ID NO:23的CDR-L2，氨基酸序列为SEQ ID NO:52的CDR-L3；(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;

或者，or,

(3a)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:10的CDR-H1，氨基酸序列为SEQ ID NO:11的CDR-H2，氨基酸序列为SEQ ID NO:12的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:13的CDR-L1，氨基酸序列为SEQ ID NO:14的CDR-L2，氨基酸序列为SEQ ID NO:6的CDR-L3；或，(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,

(3b)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:27的CDR-H1，氨基酸序列为SEQ ID NO:28的CDR-H2，氨基酸序列为SEQ ID NO:29的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:30的CDR-L1，氨基酸序列为SEQ ID NO:31的CDR-L2，氨基酸序列为SEQ ID NO:24的CDR-L3；或，(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,

(3c)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:41的CDR-H1，氨基酸序列为SEQ ID NO:42的CDR-H2，氨基酸序列为SEQ ID NO:43的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:44的CDR-L1，氨基酸序列为SEQ ID NO:31的CDR-L2，氨基酸序列为SEQ ID NO:52的CDR-L3。(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.

在一些实施方案中，所述抗体或其抗原结合片段包含:(1a)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:1的CDR-H1，氨基酸序列为SEQ ID NO:2的CDR-H2，氨基酸序列为SEQ ID NO:3的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:4的CDR-L1，氨基酸序列为SEQ ID NO:5的CDR-L2，氨基酸序列为SEQ ID NO:6的CDR-L3。In some embodiments, the antibody or its antigen-binding fragment 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.

在一些实施方案中，所述抗体或其抗原结合片段包含:(1b)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:19的CDR-H1，氨基酸序列为SEQ ID NO:20的CDR-H2，氨基酸序列为SEQ ID NO:21的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:22的CDR-L1，氨基酸序列为SEQ ID NO:23的CDR-L2，氨基酸序列为SEQ ID NO:24的CDR-L3。In some embodiments, the antibody or its antigen-binding fragment 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.

在一些实施方案中，所述抗体或其抗原结合片段包含:(1c)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:36的CDR-H1，氨基酸序列为SEQ ID NO:37的CDR-H2，氨基酸序列为SEQ ID NO:38的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:45的CDR-L1，氨基酸序列为SEQ ID NO:23的CDR-L2，氨基酸序列为SEQ ID NO:52的CDR-L3。In some embodiments, the antibody or its antigen-binding fragment 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.

在一些实施方案中，所述抗体或其抗原结合片段包含:(2a)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:7的CDR-H1，氨基酸序列为SEQ ID NO:8的CDR-H2，氨基酸序列为SEQ ID NO:9的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:4的CDR-L1，氨基酸序列为SEQ ID NO:5的CDR-L2，氨基酸序列为SEQ ID NO:6的CDR-L3。In some embodiments, the antibody or its antigen-binding fragment 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.

在一些实施方案中，所述抗体或其抗原结合片段包含:(2b)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:25的CDR-H1，氨基酸序列为SEQ ID NO:26的CDR-H2，氨基酸序列为SEQ ID NO:21的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:22的CDR-L1，氨基酸序列为SEQ ID NO:23的CDR-L2，氨基酸序列为SEQ ID NO:24的CDR-L3。In some embodiments, the antibody or its antigen-binding fragment 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.

在一些实施方案中，所述抗体或其抗原结合片段包含:(2c)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:39的CDR-H1，氨基酸序列为SEQ ID NO:40的CDR-H2，氨基酸序列为SEQ ID NO:38的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:45的CDR-L1，氨基酸序列为SEQ ID NO:23的CDR-L2，氨基酸序列为SEQ ID NO:52的CDR-L3。In some embodiments, the antibody or its antigen-binding fragment 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.

在一些实施方案中，所述抗体或其抗原结合片段包含:(3a)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:10的CDR-H1，氨基酸序列为SEQ ID NO:11的CDR-H2，氨基酸序列为SEQ ID NO:12的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:13的CDR-L1，氨基酸序列为SEQ ID NO:14的CDR-L2，氨基酸序列为SEQ ID NO:6的CDR-L3。In some embodiments, the antibody or its antigen-binding fragment 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.

在一些实施方案中，所述抗体或其抗原结合片段包含:(3b)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:27的CDR-H1，氨基酸序列为SEQ ID NO:28的CDR-H2，氨基酸序列为SEQ ID NO:29的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:30的CDR-L1，氨基酸序列为SEQ ID NO:31的CDR-L2，氨基酸序列为SEQ ID NO:24的CDR-L3。In some embodiments, the antibody or its antigen-binding fragment 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.

在一些实施方案中，所述抗体或其抗原结合片段包含:(3c)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:41的CDR-H1，氨基酸序列为SEQ ID NO:42的CDR-H2，氨基酸序列为SEQ ID NO:43的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:44的CDR-L1，氨基酸序列为SEQ ID NO:31其变体的CDR-L2，氨基酸序列为SEQ ID NO:52的CDR-L3。In some embodiments, the antibody or its antigen-binding fragment 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.

在一些实施方案中，所述抗体或其抗原结合片段包含：In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)包括SEQ ID NO：15所示的VH或其变体，和/或，包括SEQ ID NO：16所示的VL或其变体；(a) comprising the VH or a variant thereof shown in SEQ ID NO: 15, and/or comprising the VL or a variant thereof shown in SEQ ID NO: 16;

(b)包括SEQ ID NO：32所示的VH或其变体，和/或，包括SEQ ID NO：33所示的VL或其变体；或(b) comprising VH or a variant thereof shown in SEQ ID NO: 32, and/or comprising VL or a variant thereof shown in SEQ ID NO: 33; or

(c)包括SEQ ID NO：46所示的VH或其变体，和/或，包括SEQ ID NO：47所示的VL或其变体； (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;

其中，所述变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换，条件是，所述变体的CDR的氨基酸序列与(a)、(b)和(c)的VH和VL的相应CDR的氨基酸序列具有100％序列同一性，并且所述变体结合HER3。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, 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 (a), (b) and (c), and the variant binds to HER3.

在一些实施方案中，所述抗体或其抗原结合片段包含：In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)包括SEQ ID NO：15所示的VH或其变体，和，包括SEQ ID NO：16所示的VL或其变体；(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;

(b)包括SEQ ID NO：32所示的VH或其变体，和，包括SEQ ID NO：33所示的VL或其变体；或(b) comprising VH shown in SEQ ID NO: 32 or a variant thereof, and, comprising VL shown in SEQ ID NO: 33 or a variant thereof; or

(c)包括SEQ ID NO：46所示的VH或其变体，和，包括SEQ ID NO：47所示的VL或其变体；(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;

其中，所述变体与其所源自的序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。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.

在一些实施方案中，所述抗体或其抗原结合片段包含：In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)包括SEQ ID NO：15所示的VH，和，包括SEQ ID NO：16所示的VL；(a) comprising the VH shown in SEQ ID NO: 15, and, comprising the VL shown in SEQ ID NO: 16;

(b)包括SEQ ID NO：32所示的VH，和，包括SEQ ID NO：33所示的VL；或(b) comprising the VH shown in SEQ ID NO: 32, and, comprising the VL shown in SEQ ID NO: 33; or

(c)包括SEQ ID NO：46所示的VH，和，包括SEQ ID NO：47所示的VL。(c) includes the VH shown in SEQ ID NO: 46, and, includes the VL shown in SEQ ID NO: 47.

在一些实施方案中，所述抗体或其抗原结合片段包含：In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(a)包括SEQ ID NO：15所示的VH，和，包括SEQ ID NO：16所示的VL。(a) includes the VH shown in SEQ ID NO: 15, and, includes the VL shown in SEQ ID NO: 16.

在一些实施方案中，所述抗体或其抗原结合片段包含：In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(b)包括SEQ ID NO：32所示的VH，和，包括SEQ ID NO：33所示的VL。(b) includes the VH shown in SEQ ID NO: 32, and, includes the VL shown in SEQ ID NO: 33.

在一些实施方案中，所述抗体或其抗原结合片段包含：In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(c)包括SEQ ID NO：46所示的VH，和，包括SEQ ID NO：47所示的VL。(c) includes the VH shown in SEQ ID NO: 46, and, includes the VL shown in SEQ ID NO: 47.

在一些实施方案中，抗体或其抗原结合片段包含：重链可变区(VH)，其包含CDR-H1、CDR-H2和CDR-H3，所述CDR-H1，CDR-H2和CDR-H3包含分别包括SEQ ID NO:15的氨基酸序列的VH中所述的CDR-H1，CDR-H2和CDR-H3的氨基酸序列；和轻链可变区(VL)，其包含CDR-L1、CDR-L2和CDR-L3，所述CDR-L1，CDR-L2和CDR-L3包含分别括含SEQ ID NO:16的氨基酸序列的VL中所述的CDR-L1，CDR-L2和CDR-L3的氨基酸序列。In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH), which comprises CDR-H1, CDR-H2 and CDR-H3, and the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 described in VH, which respectively include the amino acid sequence of SEQ ID NO:15; and a light chain variable region (VL), which comprises CDR-L1, CDR-L2 and CDR-L3, and the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 described in VL, which respectively include the amino acid sequence of SEQ ID NO:16.

在一些实施方案中，抗体或其抗原结合片段包含：重链可变区(VH)，其包含CDR-H1、CDR-H2和CDR-H3，所述CDR-H1，CDR-H2和CDR-H3包含分别包括SEQ ID NO:32的氨基酸序列的VH中所述的CDR-H1，CDR-H2，和CDR-H3的氨基酸序列；和轻链可变区(VL)，其包含CDR-L1、CDR-L2和CDR-L3，所述CDR-L1，CDR-L2和CDR-L3包含分别包括SEQ ID NO:33的氨基酸序列的VL中所述的CDR-L1，CDR-L2和CDR-L3的氨基酸序列。In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH), which comprises CDR-H1, CDR-H2 and CDR-H3, and the CDR-H1, CDR-H2 and CDR-H3 comprise the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 described in VH, which respectively include the amino acid sequence of SEQ ID NO:32; and a light chain variable region (VL), which comprises CDR-L1, CDR-L2 and CDR-L3, and the CDR-L1, CDR-L2 and CDR-L3 comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 described in VL, which respectively include the amino acid sequence of SEQ ID NO:33.

在一些实施方案中，抗体或其抗原结合片段包含：重链可变区(VH)，其包含CDR-H1、CDR-H2和CDR-H3，所述CDR-H1，CDR-H2和CDR-H3包含分别包括SEQ ID NO:46的氨基酸序列的VH中所述的CDR-H1，CDR-H2，和CDR-H3的氨基酸序列；和轻链可变区(VL)，其包含CDR-L1、CDR-L2和CDR-L3，所述CDR-L1，CDR-L2和CDR-L3包含分别包括SEQ ID NO:47的氨基酸序列的VL中所述的CDR-L1，CDR-L2和CDR-L3的氨基酸序列。在一些实施方案中，所述抗体或其抗原结合片段进一步包含：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:

(a)人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和(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)人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)。(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.

在一些实施方案中，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区，例如人IgG1重链恒定区或人IgG4重链恒定区。在一些实施方案中，所述抗体或其抗原结合片段包含如SEQ ID NO：50所示的重链恒定区(CH)或其变体，所述变体与SEQ ID NO：50相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)。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 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; e.g., 1, 2, 3, 4 or 5 conservative substitutions of amino acids).

在一些实施方案中，所述轻链恒定区是κ轻链恒定区。在一些实施方案中，所述抗体或其抗原结合片段包含如SEQ ID NO：51所示的轻链恒定区(CL)或其变体，所述变体与SEQ ID NO：51相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)。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 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).

在一些实施方案中，所述抗体或其抗原结合片段包含如SEQ ID NO：50所示的重链恒定区(CH)和如SEQ ID NO：51所示的轻链恒定区(CL)。In some embodiments, the antibody or its antigen-binding fragment 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.

在一些实施方案中，所述抗体或其抗原结合片段包含：In some embodiments, the antibody or antigen-binding fragment thereof comprises:

(1)包括SEQ ID NO：15所示序列的VH和SEQ ID NO：50所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：16所示序列的VL和SEQ ID NO：51所示的轻链恒定区(CL)的轻链；(1) a heavy chain comprising a VH sequence shown in SEQ ID NO: 15 and a heavy chain constant region (CH) shown in SEQ ID NO: 50, and a light chain comprising a VL sequence shown in SEQ ID NO: 16 and a light chain constant region (CL) shown in SEQ ID NO: 51;

(2)包括SEQ ID NO：32所示序列的VH和SEQ ID NO：50所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：33所示序列的VL和SEQ ID NO：51所示的轻链恒定区(CL)的轻链；或(2) a heavy chain comprising a VH sequence shown in SEQ ID NO: 32 and a heavy chain constant region (CH) shown in SEQ ID NO: 50, and a light chain comprising a VL sequence shown in SEQ ID NO: 33 and a light chain constant region (CL) shown in SEQ ID NO: 51; or

(3)包括SEQ ID NO：46所示序列的VH和SEQ ID NO：50所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：47所示序列的VL和SEQ ID NO：51所示的轻链恒定区(CL)的轻链。(3) A heavy chain comprising a VH sequence shown in SEQ ID NO: 46 and a heavy chain constant region (CH) shown in SEQ ID NO: 50, and a light chain comprising a VL sequence shown in SEQ ID NO: 47 and a light chain constant region (CL) shown in SEQ ID NO: 51.

在一些实施方案中，所述抗体或其抗原结合片段包含：(1)包括SEQ ID NO：15所示氨基酸序列的VH和SEQ ID NO：50所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：16所示氨基酸序列的VL和SEQ ID NO：51所示的轻链恒定区(CL)的轻链。In some embodiments, the antibody or its antigen-binding fragment comprises: (1) a heavy chain comprising a VH with the amino acid sequence shown in SEQ ID NO: 15 and a heavy chain constant region (CH) shown in SEQ ID NO: 50, and a light chain comprising a VL with the amino acid sequence shown in SEQ ID NO: 16 and a light chain constant region (CL) shown in SEQ ID NO: 51.

在一些实施方案中，所述抗体或其抗原结合片段包含：(2)包括SEQ ID NO：32所示氨基酸序列的VH和SEQ ID NO：50所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：33所示氨基酸序列的VL和SEQ ID NO：51所示的轻链恒定区(CL)的轻链；或In some embodiments, the antibody or antigen-binding fragment thereof comprises: (2) a heavy chain comprising a VH having an amino acid sequence as shown in SEQ ID NO: 32 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: 33 and a light chain constant region (CL) as shown in SEQ ID NO: 51; or

在一些实施方案中，所述抗体或其抗原结合片段包含：(3)包括SEQ ID NO：46所示氨基酸序列的VH和SEQ ID NO：50所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：47所示氨基酸序列的VL和SEQ ID NO：51所示的轻链恒定区(CL)的轻链。In some embodiments, the antibody or its antigen-binding fragment comprises: (3) a heavy chain comprising a VH with the amino acid sequence shown in SEQ ID NO: 46 and a heavy chain constant region (CH) shown in SEQ ID NO: 50, and a light chain comprising a VL with the amino acid sequence shown in SEQ ID NO: 47 and a light chain constant region (CL) shown in SEQ ID NO: 51.

在一些实施方案中，所述抗体或其抗原结合片段包含：包括SEQ ID NO：17所示氨基酸序列的重链(HC)，和，包括SEQ ID NO：18所示氨基酸序列的轻链(LC)。In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 17, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 18.

在一些实施方案中，所述抗体或其抗原结合片段包含：包括SEQ ID NO：34所示氨基酸序列的重链(HC)，和，包括SEQ ID NO：35所示氨基酸序列的轻链(LC)。In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 34, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 35.

在一些实施方案中，所述抗体或其抗原结合片段包含：包括SEQ ID NO：48所示氨基酸序列的重链(HC)，和，包括SEQ ID NO：49所示氨基酸序列的轻链(LC)。In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 48, and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 49.

在本文公开的抗体或抗原结合片段的特定实施方案中，重链恒定结构域可包含C-末端赖氨酸或缺乏C-末端赖氨酸或C-末端甘氨酸-赖氨酸二肽。在抗体或其抗原结合片段的一些实施方案中，抗体或其抗原结合片段的N端氨基酸可以环化成焦谷氨酸。In specific 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.

在所述抗体-药物缀合物中，所述细胞毒性药物可通过连接体(如本申请中所示“M-L-E”片段)连接至所述抗体或其抗原结合片段。 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).

在一些实施方案中，M为其中环A为5-6元脂杂环、或5-20元芳香族环系，所述脂杂环和芳香族环系任选地被一个或多个选自氧基(＝O)、卤素、氰基、氨基、羧基、巯基和C1-6烷基的基团取代；M1选自单键和取代或未取代的以下基团：C1- 20亚烷基、C2-20亚烯基或C2-20亚炔基。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 groups selected from oxy (＝O), halogen, cyano, amino, carboxyl, thiol and C 1-6 alkyl; M 1 is selected from a single bond and the following substituted or unsubstituted groups: C 1-20 alkylene , C 2-20 alkenylene or C 2-20 alkynylene.

在一些实施方案中，M为其中环A为5元脂杂环、6元杂芳环、或由一个以上的6元芳杂环与苯环通过单键连接形成的多环，所述脂杂环任选地被一个或多个选自氧基(＝O)、卤素和C1-4烷基的基团取代；M1选自单键和取代或未取代的以下基团：C3-10亚烷基、C3-10亚烯基或C3-10亚炔基。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 aromatic heterocycles to a benzene ring via a single bond, wherein the alicyclic heterocycle is optionally substituted by one or more groups selected from oxy (=O), halogen, and C 1-4 alkyl; M 1 is selected from a single bond and the following substituted or unsubstituted groups: C 3-10 alkylene, C 3-10 alkenylene, or C 3-10 alkynylene.

在一些实施方案中，M为其中环A选自 M1选自单键和C5-8亚烷基、C5-8亚烯基或C5-8亚炔基。In some embodiments, M is wherein ring A is selected from M 1 is selected from a single bond and a C 5-8 alkylene group, a C 5-8 alkenylene group or a C 5-8 alkynylene group.

在一些实施方案中，M选自以下结构：
In some embodiments, M is selected from the following structures:

在一些实施方案中，M选自以下结构：
In some embodiments, M is selected from the following structures:

在一些实施方案中，M为 In some embodiments, M is

其中，环A为5-20元芳香族环系，所述芳香族环系任选地被一个或多个选自卤素、氰基、氨基、羧基、巯基和C1-6烷基的基团取代；M1为取代或未取代的C2-20亚炔基。Wherein, ring A is a 5-20 membered aromatic ring system, which is optionally substituted by one or more groups selected from halogen, cyano, amino, carboxyl, thiol and C 1-6 alkyl; M 1 is a substituted or unsubstituted C 2-20 alkynylene group.

在一些实施方案中，M为 In some embodiments, M is

其中，环A为6元杂芳环，所述芳香族环系任选地被一个或多个选自卤素、氰基、氨基、羧基、巯基和C1-6烷基的基团取代；M1为C3-10亚炔基。Wherein, ring A is a 6-membered heteroaromatic ring, and the aromatic ring system is optionally substituted by one or more groups selected from halogen, cyano, amino, carboxyl, thiol and C 1-6 alkyl; M 1 is a C 3-10 alkynylene group.

在一些实施方案中，M为 In some embodiments, M is

在一些实施方案中，L选自由下述的一个或多个取代或未取代的基团组成的结构：C1-6亚烷基、-N(R’)-、羰基、-O-、Val、Cit、Phe、Lys、Lys(COCH2CH2(OCH2CH2)sOCH3)、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、Gly-Gly-Gly-Gly-Gly、 其中R’代表氢、C1-6烷基或含-(CH2CH2O)r-的烷基；r选自1-10的整数；s选自1-20的整数。In some embodiments, L is selected from a structure consisting of one or more substituted or unsubstituted groups selected from the group consisting of C 1-6 alkylene, -N(R')-, carbonyl, -O-, Val, Cit, Phe, Lys, Lys( COCH2CH2 ( OCH2CH2 ) sOCH3 ) , 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, Gly-Gly-Gly-Gly-Gly, wherein R' represents hydrogen, C 1-6 alkyl or alkyl containing -(CH 2 CH 2 O)r-; r is selected from an integer of 1-10; and s is selected from an integer of 1-20.

在一些实施方案中，L选自由下述的一个或多个取代或未取代的基团组成的结构：C1-6亚烷基、-NH-、Phe、Lys、Ala-Ala-Ala、Lys(COCH2CH2(OCH2CH2)sOCH3)、Gly、Gly-Gly-Phe-Gly、 其中s选自1-20的整数。In some embodiments, L is selected from a structure consisting of one or more substituted or unsubstituted groups selected from the group consisting of C 1-6 alkylene, -NH-, Phe, Lys, Ala-Ala-Ala, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) s OCH 3 ), Gly, Gly-Gly-Phe-Gly, wherein s is selected from an integer of 1-20.

在一些实施方案中，L选自由下述的一个或多个取代或未取代的基团组成的结构：C1-6亚烷基、-NH-、Phe、Lys、Lys(COCH2CH2(OCH2CH2)sOCH3)、Gly、Gly-Gly-Phe-Gly、 其中s选自1-20的整数。In some embodiments, L is selected from a structure consisting of one or more substituted or unsubstituted groups selected from the group consisting of C 1-6 alkylene, -NH-, Phe, Lys, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) s OCH 3 ), Gly, Gly-Gly-Phe-Gly, wherein s is selected from an integer of 1-20.

在一些实施方案中，L选自以下取代或未取代的结构：
In some embodiments, L is selected from the following substituted or unsubstituted structures:

在一些实施方案中，L选自以下结构：
In some embodiments, L is selected from the following structures:

其中s选自1-20的整数。wherein s is selected from an integer of 1-20.

优选地，L选自以下结构：
Preferably, L is selected from the following structures:

在一些实施方案中，L选自以下结构：

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

在一些实施方案中，L选自以下结构：
In some embodiments, L is selected from the following structures:

在一些实施方案中，L选自以下结构：
In some embodiments, L is selected from the following structures:

在一些实施方案中，L选自由下述的一个或多个组成的结构：Val、Cit、Phe、Lys、Lys(COCH2CH2(OCH2CH2)sOCH3)、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和Gly-Gly-Gly-Gly-Gly；其中s选自1-20的整数。In some embodiments, L is selected from a structure consisting of one or more of the following: Val, Cit, Phe, Lys, Lys( COCH2CH2 ( OCH2CH2 ) sOCH3 ) , 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.

在一些实施方案中，L由一个或多个取代或未取代的Ala组成。In some embodiments, L consists of one or more substituted or unsubstituted Ala.

在一些实施方案中，L由一个或多个Ala组成。In some embodiments, L consists of one or more Ala.

在一些实施方案中，L为取代或未取代的Ala-Ala-Ala。In some embodiments, L is substituted or unsubstituted Ala-Ala-Ala.

在一些实施方案中，L为 In some embodiments, L is

在一些实施方案中，E为单键或选自以下取代或未取代的结构：-NH-CH2-，-NH-CH2-O-CH2-CO-， In some embodiments, E is a single bond or a substituted or unsubstituted structure selected from the following: -NH- CH2- , -NH- CH2 -O-CH2 - CO-,

在一些实施方案中，E为单键或选自以下取代或未取代的结构：-NH-CH2-、-NH-CH2-O-CH2-CO-、 在一些实施方案中，E为取代或未取代的-NH-CH2-O-CH2-CO-、 In some embodiments, E is a single bond or a substituted or unsubstituted structure selected from the following: -NH- CH2- , -NH- CH2 -O-CH2 - CO-, In some embodiments, E is substituted or unsubstituted -NH-CH 2 -O-CH 2 -CO-,

在一些实施方案中，E为-NH-CH2-O-CH2-CO-或在一些实施方案中，E为-NH-CH2-O-CH2-CO或-NH-CH2-。In some embodiments, E is -NH- CH2 -O- CH2 -CO- or In some embodiments, E is -NH- CH2 -O- CH2 -CO or -NH- CH2- .

在一些实施方案中，M选自以下取代或未取代的结构：
In some embodiments, M is selected from the following substituted or unsubstituted structures:

L选自以下取代或未取代的结构：
L is selected from the following substituted or unsubstituted structures:

E为-NH-CH2-O-CH2-CO-、 E is -NH-CH 2 -O-CH 2 -CO-,

在一些实施方案中，选自以下取代或未取代的结构：


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


在一些实施方案中，选自以下结构：
In some embodiments, Select from the following structures:

在一些实施方案中，选自以下结构：
In some embodiments, Select from the following structures:

在一些实施方案中，所述细胞毒性药物选自微管蛋白抑制剂、DNA嵌入剂、DNA拓扑异构酶抑制剂和RNA聚合酶抑制剂。在一些实施方案中，所述微管蛋白抑制剂为奥瑞他汀类化合物或美登素类化合物。在一些实施方案中，所述DNA嵌入剂为吡咯并苯二氮卓(PBD)。在一些实施方案中，所述DNA拓扑异构酶抑制剂为拓扑异构酶I抑制剂(例如，喜树碱、羟基喜树碱、9-氨基喜树碱、SN-38、伊立替康、拓扑替康、贝洛替康、或卢比替康)或拓扑异构酶II抑制剂(例如，阿霉素、PNU-159682、多卡米星、柔红霉素、米托蒽醌、鬼臼毒素、或依托泊苷)。在一些实施方案中，所述RNA聚合酶抑制剂为α-鹅膏草碱(α-amanitin)或其药学上可接受的盐、酯或类似物。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.

本申请中所公开的细胞毒性药物通常含有多种官能团，例如羟基(-OH)、羧基(-COOH)、巯基(-SH)、一级氨基(-NH2)、二级胺基(-NRAH)或三级胺基(-NRBRC)，其中RA、RB、RC在此仅代表N上的非氢取代基，所述细胞毒性药物可通过这些官能团与缀合物中的连接体连接。The cytotoxic drugs disclosed in the present 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 RC ), wherein RA , RB , RC here only represent non-hydrogen substituents on N, and the cytotoxic drugs can be connected to the linker in the conjugate through these functional groups.

在一些实施方案中，所述细胞毒性药物通过其上的-OH、-SH、一级氨基、二级胺基或三级胺基与所述抗体-药物缀合物中的E连接。In some embodiments, the cytotoxic drug is linked to E in the antibody-drug conjugate through -OH, -SH, primary amino, secondary amine or tertiary amine groups on the cytotoxic drug.

在一些实施方案中，所述细胞毒性药物选自以下式I和式II：
In some embodiments, the cytotoxic drug is selected from the following Formula I and Formula II:

其中，R1，R2各自独立地选自C1-6烷基和卤素；Wherein, R 1 and R 2 are each independently selected from C 1-6 alkyl and halogen;

R3选自H和-CO-CH2OH；R 3 is selected from H and -CO-CH 2 OH;

R4和R5各自独立地选自H、卤素和羟基；或者R4和R5与相连碳原子连接成5-6元含氧杂环； 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选自氢和-C1-4亚烷基-NRaRb； R6 is selected from hydrogen and -C1-4alkylene - NRaRb ;

R7选自C1-6烷基和-C1-4亚烷基-NRaRb； R7 is selected from C1-6 alkyl and -C1-4 alkylene- NRaRb ;

其中Ra、Rb在每次出现时各自独立地选自H、C1-6烷基、-SO2-C1-6烷基和-CO-C1-6烷基。wherein Ra , Rb at each occurrence are independently selected from H, C1-6 alkyl, -SO2- C1-6 alkyl and -CO- C1-6 alkyl.

在一些实施方案中，所述细胞毒性药物选自以下化合物：

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

在一些实施方案中，所述细胞毒性药物选自以下化合物：
In some embodiments, the cytotoxic drug is selected from the following compounds:

所述细胞毒性药物与连接体连接后得到的该细胞毒性药物相应的片段即为本申请式Ab-[M-L-E-D]x中的D。在一些实施方案中，D为所述细胞毒性药物上的-OH、-NH2或二级胺基失掉一个H得到的一价结构。The corresponding fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is D in the formula Ab-[MLED] x of the present application. 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.

在一些实施方案中，D选自以下结构：
In some embodiments, D is selected from the following structures:

在一些实施方案中，所述细胞毒性药物为：
In some embodiments, the cytotoxic drug is:

在一些实施方案中，所述细胞毒性药物为：
In some embodiments, the cytotoxic drug is:

在一些实施方案中，所述D为：
In some embodiments, D is:

在一些实施方案中，所述D为：
In some embodiments, D is:

在一些实施方案中，所述抗体-药物缀合物选自下示的ADC A-01～ADC A-25，ADC B-01～ADC B-05：








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:








其中，各抗体-药物缀合物中的HA代表包含如SEQ ID NO:15所示的VH和如SEQ ID NO:16所示的VL的抗体或其抗原结合片段，例如包括SEQ ID NO：15所示的VH和SEQ ID NO：50所示的CH，以及SEQ ID NO：16所示的VL和SEQ ID NO：51所示的CL的抗体或其抗原结合片段；Wherein, HA in each antibody-drug conjugate represents an antibody or an 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 an 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;

其中，表示抗体或其抗原结合片段中的巯基与连接体的具体连接方式。in, It indicates the specific connection mode between the thiol group in the antibody or its antigen-binding fragment and the linker.

在一些实施方案中，x为1-10的整数。In some embodiments, x is an integer from 1-10.

在一些实施方案中，所述抗体-药物缀合物选自：

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

其中，各抗体-药物缀合物中的HA选自：Wherein, the HA in each antibody-drug conjugate is selected from:

(1)包含如SEQ ID NO:15所示的VH和如SEQ ID NO:16所示的VL的抗体或其抗原结合片段，例如包含SEQ ID NO：15所示的VH和SEQ ID NO：50所示的CH，以及SEQ ID NO：16所示的VL和SEQ ID NO：51所示的CL的抗体或其抗原结合片段；(1) an antibody or an antigen-binding fragment thereof comprising the VH shown in SEQ ID NO: 15 and the VL shown in SEQ ID NO: 16, for example, an antibody or an antigen-binding fragment thereof comprising the VH shown in SEQ ID NO: 15 and the CH shown in SEQ ID NO: 50, and the VL shown in SEQ ID NO: 16 and the CL shown in SEQ ID NO: 51;

(2)包含如SEQ ID NO:32所示的VH和如SEQ ID NO:33所示的VL的抗体或其抗原结合片段，例如包含SEQ ID NO：32所示的VH和SEQ ID NO：50所示的CH，以及SEQ ID NO：33所示的VL和SEQ ID NO：51所示的CL的抗体或其抗原结合片段；和(2) an antibody or an 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 an 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; and

(3)包含如SEQ ID NO:46所示的VH和如SEQ ID NO:47所示的VL的抗体或其抗原结合片段，例如含SEQ ID NO：46所示的VH和SEQ ID NO：50所示的CH，以及SEQ ID NO：47所示的VL和SEQ ID NO：51所示的CL的抗体或其抗原结合片段；(3) an antibody or an 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 an 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为3～8，例如3～4或7～8。 x is 3-8, for example, 3-4 or 7-8.

在一些实施方案中，x为3-8的整数。In some embodiments, x is an integer from 3-8.

在一些实施方案中，所述抗体-药物缀合物为：
In some embodiments, the antibody-drug conjugate is:

其中，所述HA代表包括SEQ ID NO：17所示的HC和SEQ ID NO：18所示的LC的抗体或其抗原结合片段；Wherein, 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为6～8。x is 6 to 8.

在一些实施方案中，所述抗体-药物缀合物为：
In some embodiments, the antibody-drug conjugate is:

其中，HA包含：Among them, HA includes:

(1)包含含有如SEQ ID NO:15所示的VH和如SEQ ID NO:16所示的VL的抗体或其抗原结合片段，例如包含SEQ ID NO：15所示的VH和SEQ ID NO：50所示的CH，以及SEQ ID NO：16所示的VL和SEQ ID NO：51所示的CL的抗体或其抗原结合片段；x为3～8，例如3～4或7～8，例如3-8的整数。(1) 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; x is 3 to 8, for example, 3 to 4 or 7 to 8, for example, an integer of 3-8.

在一些实施方案中，所述抗体-药物缀合物为：
In some embodiments, the antibody-drug conjugate is:

其中，HA包含：Among them, HA includes:

(2)包含如SEQ ID NO:32所示的VH和如SEQ ID NO:33所示的VL的抗体或其抗原结合片段，例如包含SEQ ID NO：32所示的VH和SEQ ID NO：50所示的CH，以及SEQ ID NO：33所示的VL和SEQ ID NO：51所示的CL的抗体或其抗原结合片段；x为3～8，例如3～4或7～8，例如3-8的整数。(2) An antibody or an 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 an 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.

在一些实施方案中，所述抗体-药物缀合物为：
In some embodiments, the antibody-drug conjugate is:

其中，HA包含：Among them, HA includes:

(3)包含如SEQ ID NO:46所示的VH和如SEQ ID NO:47所示的VL的抗体或其抗原结合片段，例如含SEQ ID NO：46所示的VH和SEQ ID NO：50所示的CH，以及SEQ ID NO：47所示的VL和SEQ ID NO：51所示的CL的抗体或其抗原结合片段；x为3～8，例如3～4或7～8，例如3-8的整数。(3) An antibody or an antigen-binding fragment thereof comprising VH as shown in SEQ ID NO:46 and VL as shown in SEQ ID NO:47, for example, an antibody or an antigen-binding fragment thereof comprising VH as shown in SEQ ID NO:46 and CH as shown in SEQ ID NO:50, and VL as shown in SEQ ID NO:47 and CL as 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.

在一些实施方案中，所述抗体-药物缀合物为：
In some embodiments, the antibody-drug conjugate is:

其中，所述HA代表包括SEQ ID NO：17所示的HC和SEQ ID NO：18所示的LC的抗体或其抗原结合片段；Wherein, 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为6～8。x is 6 to 8.

在一些实施方案中，x为6-8的整数。In some embodiments, x is an integer from 6-8.

在一些实施方案中，所述抗体-药物缀合物为：
In some embodiments, the antibody-drug conjugate is:

其中，所述HA代表包括SEQ ID NO：34所示的HC和SEQ ID NO：35所示的LC的抗体或其抗原结合片段；Wherein, the HA represents an antibody or an antigen-binding fragment thereof comprising the HC shown in SEQ ID NO: 34 and the LC shown in SEQ ID NO: 35;

x为6～8，例如6-8的整数。x is 6 to 8, for example, an integer of 6-8.

在一些实施方案中，所述抗体-药物缀合物为：
In some embodiments, the antibody-drug conjugate is:

其中，所述HA代表包括SEQ ID NO：48所示的HC和SEQ ID NO：49所示的LC的抗体或其抗原结合片段；Wherein, the HA represents an antibody or an antigen-binding fragment thereof comprising the HC shown in SEQ ID NO: 48 and the LC shown in SEQ ID NO: 49;

x为6～8，例如6-8的整数。x is 6 to 8, for example, an integer of 6-8.

在一些实施方案中，所述的抗体-药物缀合物中：In some embodiments, in the antibody-drug conjugate:

(i)重链C末端缺少赖氨酸残基；(i) The heavy chain lacks a lysine residue at the C-terminus;

(ii)重链N末端是谷氨酰胺、谷氨酸或焦谷氨酸；或者，(ii) the N-terminus of the heavy chain is glutamine, glutamic acid or pyroglutamic acid; or,

(iii)重链C末端缺少赖氨酸残基并且重链N末端是谷氨酰胺、谷氨酸或焦谷氨酸。(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.

在一些实施方案中，在式Ab-[M-L-E-D]x所示的抗体-药物缀合物，Ab通过半胱氨酸残基与式中剩余部分缀合。In some embodiments, in the antibody-drug conjugate represented by the formula Ab-[MLED] x , Ab is conjugated to the remaining moiety in the formula via a cysteine residue.

本文还提供了包含本文所述的抗体-药物缀合物和一种或多种药用辅料的药物组合物。Also provided herein are pharmaceutical compositions comprising the antibody-drug conjugates described herein and one or more pharmaceutically acceptable excipients.

本文还提供了治疗受试者中HER3高表达癌症的方法，包括向受试者施用治疗有效量的本文所述的抗体-药物缀合物或其药物组合物。在具体实施方案中，癌症包括实体瘤或血液恶性肿瘤。在进一步的实施方案中，所述癌症是结肠癌、胃癌、乳腺癌、肺癌或淋巴瘤。在进一步的实施方案中，所述肺癌是非小细胞肺癌。在进一步的实施方案中，所述肺癌是肺腺癌。Also provided herein is a method 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 a specific embodiment, the cancer comprises a solid tumor or a hematological malignancy. In a further embodiment, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma. In a further embodiment, the lung cancer is non-small cell lung cancer. In a further embodiment, the lung cancer is lung adenocarcinoma.

本文还提供本文所述的抗体-药物缀合物或其药物组合物在制备用于治疗高表达HER3的癌症的药物中的用途。在具体实施方案中，所述癌症包括实体瘤或血液恶性肿瘤。在进一步的实施方案中，所述癌症是结肠癌、胃癌、乳腺癌、肺癌或淋巴瘤。在进一步的实施方案中，所述肺癌是非小细胞肺癌。在进一步的实施方案中，所述肺癌是肺腺癌。Also provided herein is the use of an antibody-drug conjugate or a pharmaceutical composition thereof as described herein in the preparation of a medicament for treating a cancer that highly expresses HER3. In a specific embodiment, the cancer comprises a solid tumor or a hematological malignancy. In a further embodiment, the cancer is colon cancer, gastric cancer, breast cancer, lung cancer, or lymphoma. In a further embodiment, the lung cancer is non-small cell lung cancer. In a further embodiment, the lung cancer is lung adenocarcinoma.

本文还提供本文所述的抗体-药物缀合物或其药物组合物在治疗高表达HER3的癌症中的用途。Also provided herein is use of the antibody-drug conjugate described herein or a pharmaceutical composition thereof in treating cancers that overexpress HER3.

在具体实施方案中，所述癌症包括实体瘤或血液恶性肿瘤。在进一步的实施方案中，所述癌症是结肠癌、胃癌、乳腺癌、肺癌或淋巴瘤。在进一步的实施方案中，所述肺癌是非小细胞肺癌。在进一步的实施方案中，所述肺癌是肺腺癌。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.

本文还提供了本文所述的抗体-药物缀合物或本文所述的药物组合物，用于治疗高表达HER3的癌症。在具体实施方案中，所述癌症包括实体瘤或血液恶性肿瘤。在进一步的实施方案中，所述癌症是结肠癌、胃癌、乳腺癌、肺癌或淋巴瘤。在进一步的实施方案中，所述肺癌是非小细胞肺癌。在进一步的实施方案中，所述肺癌是肺腺癌。Also provided herein are antibody-drug conjugates as described herein or pharmaceutical compositions as described herein for treating cancers that highly express 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.

在一种实施方式中，所述HER3高表达的癌症包括实体瘤或血液恶性肿瘤，例如结肠癌、胃癌、乳腺癌、肺癌(例如，非小细胞肺癌，特别是肺腺癌)或淋巴瘤。In one embodiment, the cancer with high HER3 expression includes solid tumors or hematological malignancies, such as colon cancer, gastric cancer, breast cancer, lung cancer (eg, non-small cell lung cancer, particularly lung adenocarcinoma) or lymphoma.

本发明还提供任何一种上述抗体-药物缀合物的药学上可接受的盐或溶剂化物。The present invention also provides a pharmaceutically acceptable salt or solvate of any of the above antibody-drug conjugates.

本发明进一步提供了包含任何一种前述抗体-药物缀合物和药学上可接受的载体或赋形剂的组合物。The present invention further provides a composition comprising any of the aforementioned antibody-drug conjugates and a pharmaceutically acceptable carrier or excipient.

抗体的制备Antibody preparation

本文描述的抗体可以通过本领域已知的各种方法制备，例如，通过遗传工程和重组技术。例如，通过化学合成或PCR扩增获得编码本发明所述抗体的重链和轻链的DNA分子。将得到的DNA分子插入到表达载体中，然后转染到宿主细胞中。然后，转染的宿主细胞在特定条件下培养，并表达本发明的抗体。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 obtained 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.

偶联物Conjugate

另一方面，本申请提供了一种将本文所述的药物-连接体缀合至本文所述的抗体以制备本文所述的抗体-药物缀合物(ADC)的方法。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.

在某些实施方案中，本文所述的抗体通过与抗体中的赖氨酸偶联而与本文所述的药物-连接体缀合。In certain embodiments, an antibody described herein is conjugated to a drug-linker described herein via coupling to a lysine in the antibody.

在某些实施方案中，本文所述的抗体通过抗体中的半胱氨酸与本文所述的药物-连接体缀合。在某些实施方案中，所述半胱氨酸来自所述抗体中还原的链内二硫键。在某些实施方案中，所述半胱氨酸来自所述抗体中还原的链间二硫键。In certain embodiments, the antibodies described herein are conjugated to the drug-linkers described herein via cysteine in the antibody. In certain embodiments, the cysteine is from a reduced intrachain disulfide bond in the antibody. In certain embodiments, the cysteine is from a reduced interchain disulfide bond in the antibody.

在一些实施方案中，所述抗体通过所述抗体中还原的链间二硫键而与药物-连接体缀合。例如，IgG1抗体由四条多肽链组成，两条重链包含VH、CH1和Fc(例如铰链、CH2和CH3)结构域，两条轻链包含VL和CL结构域，通过链间半胱氨酸二硫键(-S-S-)连接(例如两条重链-轻链链间二硫键和两条铰链重链-重链链间二硫键)。在某些实施方案中，当这些二硫键在还原条件下断裂时，产生八(8)个反应性半胱氨酸巯基部分。在某些实施方案中，所述八个反应性半胱氨酸巯基部分中的每一个都是药物-连接体的连接位点，这样最多八个(x＝8)药物-连接体可以连接到还原的抗体上。在某些实施方案中，四个二硫键中的任何一个在还原条件下断裂，产生两(2)个反应性半胱氨酸巯基部分。在进一步的实施方案中，两个反应性半胱氨酸巯基部分中的每一个都是药物-连接体的连接位点，使得两个(x＝2)药物-连接体可以连接到还原的抗体上。在某些实施方案中，四个二硫键中的任何两个在还原条件下断裂，产生四(4)个反应性半胱氨酸巯基部分。在进一步的实施方案中，四个反应性半胱氨酸巯基部分中的每一个都是药物-连接体的连接位点，使得四个(x＝4)药物-连接体可以连接到还原的抗体上。在某些实施方案中，四个二硫键中的任何三个在还原条件下断裂，产生六(6)个反应性半胱氨酸巯基部分。在进一步的实施方案中，六个反应性胱氨酸巯基部分中的每一个都是药物-连接体的连接位点，使得六个(x＝6)药物-连接体可以连接到还原的抗体上。In some embodiments, the antibody is conjugated to a drug-linker via reduced interchain disulfide bonds in the antibody. For example, an IgG1 antibody consists 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 (-S-S-) (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 a 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.

在一些实施例中，链间二硫键位于两个半胱氨酸残基之间，其在还原条件下断裂，产生两个反应性半胱氨酸巯基部分。在进一步的实施方案中，所述抗体中的链间二硫键位于重链和轻链之间，例如位于根据EU编号重链的C220和κ轻链的C214之间，或者位于根据EU编号重链的C220和根据Kabat编号λ轻链的C214之间。此外，可选地，抗体中的链间二硫键位于两条重链之间，例如根据EU编号第一条重链的C226和/或C229与第二条重链的C226和/或C229之间。在一些实施例中，所述半胱氨酸残基位于抗体的铰链区。在一些实施方案中，根据EU编号，所述半胱氨酸残基位于重链中220、226或229位中的任何一个或多个(在本文中也分别称为C220、C226或C229)。在一些实施方案中，根据EU和/或Kabat编号，所述半胱氨酸残基位于轻链的214位(本文也称为C214，例如根据EU和Kabat编号，位于κ轻链的214位，或根据Kabat编号，位于λ轻链的214位)。在一个实施方案中，根据EU编号，所述半胱氨酸残基位于重链的第220、226和229位，根据EU或Kabat编号，位于轻链的第214位。在一个实施方案中，根据EU编号，所述半胱氨酸残基位于重链的第220、226和229位，根据EU和Kabat编号，位于κ轻链的第214位。在一个实施方案中，根据EU编号，所述半胱氨酸残基位于重链的第220、226和229位，根据Kabat编号，位于λ轻链的第214位。在一个实施方案中，所述半胱氨酸残基位于下列任何一个或多个位置: 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 κ light chain, or between C220 of the heavy chain according to EU numbering and C214 of the λ light chain according to Kabat numbering. In addition, optionally, the interchain disulfide bond in the antibody is located between 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 in 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, according to EU and/or Kabat numbering, the cysteine residue is located at position 214 of the light chain (also referred to herein as C214, e.g., according to EU and Kabat numbering, at position 214 of a kappa light chain, or according to Kabat numbering, at position 214 of a lambda light chain). In one embodiment, according to EU numbering, the cysteine residue is located at position 220, 226, and 229 of the heavy chain, and according to EU or Kabat numbering, at position 214 of the light chain. In one embodiment, according to EU numbering, the cysteine residue is located at position 220, 226, and 229 of the heavy chain, and according to EU and Kabat numbering, at position 214 of a kappa light chain. In one embodiment, according to EU numbering, the cysteine residue is located at position 220, 226, and 229 of the heavy chain, and according to Kabat numbering, at position 214 of a lambda light chain. In one embodiment, the cysteine residue is located at any one or more of the following positions:

(i)根据EU编号，第一重链中220、226和229位中的任一个、或任两个、或任三个、或全部四个；(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;

(ii)根据EU编号，第二重链中220、226和229位中的任一个、或任两个、或任三个、或全部四个；(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;

(iii)根据Kabat编号，第一轻链中的第214位；和/或(iii) position 214 in the first light chain according to Kabat numbering; and/or

(iv)根据Kabat编号，第二轻链中的第214位。(iv) Position 214 in the second light chain according to Kabat numbering.

如本文所用，C220、C226和C229指根据EU编号鉴定的免疫球蛋白的氨基酸残基(半胱氨酸、Cys、C)。如本领域技术人员所理解的，这样的编号相应地代表多肽的氨基酸残基，其与免疫球蛋白所确定的氨基酸残基对齐，如www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html中所示。As used herein, C220, C226 and C229 refer to the 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 corresponds to amino acid residues of polypeptides that are aligned with the amino acid residues defined for immunoglobulins, as shown in www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html.

在某些实施方案中，本文所述的抗体包含铰链区中的四个链间二硫键，所述链间二硫键可被还原，从而断裂，并显示可与药物-连接体上的马来酰亚胺部分(例如本文所述的药物-连接体上的马来酰亚胺部分)缀合的反应性巯基部分。In certain embodiments, the antibodies described herein comprise four interchain disulfide bonds in the hinge region, which can be reduced, thereby breaking and revealing reactive thiol moieties that can be conjugated to a maleimide moiety on a drug-linker, such as a maleimide moiety on a drug-linker described herein.

在一个实施例中，本发明提供了一种制备本文所述ADC的方法，包括以下步骤:In one embodiment, the present invention provides a method for preparing the ADC described herein, comprising the following steps:

a)提供包含抗体的溶液；a) providing a solution comprising an antibody;

b)将a)的溶液与还原剂接触；b) contacting the solution of a) with a reducing agent;

c)将b)的溶液与包含如本文所述的药物-连接体或其盐的溶液接触，以制备所述ADC。c) contacting the solution of b) with a solution comprising a drug-linker or a salt thereof as described herein to prepare the ADC.

在一个实施例中，还原剂是三(2羧乙基)膦(TCEP)。In one embodiment, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

组合物Composition

在另一方面，本申请提供了如本文所述的抗体-药物缀合物(ADC)的组合物。这种组合物可包含多个本文所述的ADC，其中每个ADC包含本文所述的药物-连接体，其中x独立地为1、2、3、4、5、6、7、8、9或10。换言之，所述组合物中的每个抗体分子可以与1、2、3、4、5、6、7、8、9或10个药物-连接体缀合。因此，所述组合物的特征在于“药物-抗体”比(DAR)在约1至约10的范围内。测定DAR的方法是技术人员熟知的，包括使用反相色谱或HPLC-MS的方法。On the other hand, 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. Therefore, 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 technicians, including methods using reverse phase chromatography or HPLC-MS.

例如，在任意实施方案中，本文所述的ADC组合物具有约1至约10或其间任何子范围的DAR，例如:约1至2、约1至3、约1至4、约1至5、约1至6、约1至7、约1至8、约1至9、约1至10、约2至3、约2至4、约2至5、约2至6，约2至7，约2至8，约2至9，约2至10，约3至4，约3至5，约3至6，约3至7，约3至8，约3至9，约3至10，约4至5，约4至6，约4至7，约4至8，约4至9，约4至10，约5至6，约5至7，约5至8，约5至9、约5至10、约6至7、约6至8、约6至9、约6至10、约7至8、约7至9、约7至10、约8至9、约8至10或约9至10。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.

在某些实施方案中，本文所述ADC组合物的DAR为约3至9，例如约3.0至3.5、约3.0至4.0、约3.0至4.5、约3.0至5.0、约3.0至6.0、约3.5至4.0、约3.5至4.5、约3.5至5.0、约3.5至5.5、约3.5至6.0、约3.5至6.5至6约4.0至4.5，约4.0至5.0，约4.0至5.5，约4.0至6.0，约4.0至6.5，约4.0至7.0，约4.0至8.0，约4.5至5.0，约4.5至5.5，约4.5至6.0，约4.5至6.5，约4.5至7.0，约4.5至7.5约5.0至8.0，约5.5至6.0，约5.5至6.5，约5.5至7.0，约5.5至7.5，约5.5至8.0，约6.0至6.5，约6.0至7.0，约6.0至7.5，约6.0至8.5，约6.5至7.0，约6.5至7.5，约6.5至7.5，约6.5至8.5，约7.0至7.5。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.

药物组合物Pharmaceutical composition

在另一个方面，本申请提供一种药物组合物，其含有前文任一项所述的一种或多种抗体-药物缀合物，以及一种或多种药用辅料。In another aspect, the present application provides a pharmaceutical composition comprising one or more antibody-drug conjugates as described in any one of the preceding items, and one or more pharmaceutical excipients.

药用辅料包括，例如，药用载体和/或赋形剂。Pharmaceutical excipients include, for example, pharmaceutically acceptable carriers and/or excipients.

在一些实施方式中，所述药物组合物的平均DAR值(药物抗体偶联比)为1-10，例如：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，或9～10，优选为3～9，例如，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或7.5～9.0。In some embodiments, the average DAR value (drug-antibody coupling ratio) of the drug 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.

本文所述抗体-药物缀合物通常与药学上可接受的胃肠外媒介一起以单位可注射形式配制，供胃肠外使用，例如推注、静脉注射、肿瘤内注射等。任选地，以冻干剂或溶液剂的形式将具有期望纯度的抗体-药物缀合物与药学上可接受的稀释剂、载体、赋型剂或稳定剂混合(Remington’s Pharmaceutical Sciences(1980)16th edition,Osol,A.Ed.)。可以通过对于要治疗的个体适宜的任何路径施用本文所述抗体-药物缀合物或含有所述抗体-药物缀合物的药物组合物。The antibody-drug conjugates described herein are generally formulated in a unit injectable form together with a pharmaceutically acceptable parenteral vehicle for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc. Optionally, the antibody-drug conjugate having the desired purity is mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer in the form of a lyophilized agent or solution (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.). The antibody-drug conjugates described herein or pharmaceutical compositions containing the antibody-drug conjugates can be administered by any route suitable for the individual to be treated.

本文所述的ADC和药物组合物可以配制成医学领域已知的任何剂型，例如片剂、丸剂、悬浮液、乳剂、溶液、凝胶、胶囊、粉末、颗粒、酏剂、锭剂、栓剂、注射剂(包括注射剂、注射用无菌粉末和注射用浓溶液)、吸入剂、喷雾剂等。优选的剂型取决于预期的给药方式和治疗用途。本发明的药物组合物在生产和储存条件下应该是无菌和稳定的。优选的剂型是注射剂。这种注射剂可以是无菌注射溶液。例如，无菌注射溶液可通过以下方法制备:将必要剂量的本发明抗体掺入合适的溶剂中，并任选地加入其它所需成分(包括但不限于pH调节剂、表面活性剂、佐剂、离子强度增强剂等，渗透剂、防腐剂、稀释剂或其任意组合)，然后过滤灭菌。此外，为了便于储存和使用，无菌注射液可以制备成无菌冻干粉末(例如，通过真空干燥或冷冻干燥)。这种无菌冻干粉末可以在使用前分散在合适的载体中，例如无菌无热原水中。The ADC 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, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. The preferred dosage form is an injection. This injection can be a sterile injection solution. For example, a sterile injection 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., permeants, preservatives, diluents or any combination thereof) are optionally added, and then filtered and sterilized. In addition, for ease of storage and use, the sterile injection can be prepared into a sterile lyophilized powder (for example, by vacuum drying or freeze drying). This sterile lyophilized powder can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

此外，本文所述的ADC可以以单位剂量形式存在于药物组合物中，以便于通过本领域已知的任何合适方法给药，包括但不限于口服、口服、舌下、眼用、局部、肠胃外、直肠、鞘内、胞质内、腹股沟、膀胱内、局部(例如，粉末、软膏或滴剂)或鼻内途径。然而，对于许多治疗用途，优选的给药途径/方式是非肠道给药(例如，静脉内、皮下、腹膜内、肌肉内)。技术人员将理解，给药的途径和/或方式将根据预期目的而变化。在一些优选的实施方案中，本文所述的ADC和药物组合物通过静脉输注或注射给药。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 of administration/mode is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). The skilled person will understand that the route and/or mode of administration will vary according to the intended purpose. In some preferred embodiments, the ADC and pharmaceutical compositions described herein are administered by intravenous infusion or injection.

在某些实施方案中，所述药物组合物还可以包含其它药物活性剂。在某些实施方案中，所述其它的药物活性剂是具有抗肿瘤活性的药物。在某些实施方案中，所述其它药物活性剂选自EGFR抑制剂、HER2抑制剂、HER3抑制剂、HER4抑制剂、IGFR-1抑制剂、mTOR抑制剂、PI3激酶抑制剂、c-met或VEGF抑制剂、化疗药物或其任意组合。在某些实施方案中，本文所述的ADC和其它药物活性剂作为单独的组分或作为混合的组分提供。因此，本文所述的ADC和其它药物活性剂可以同时、分别或依次给药。In certain embodiments, the pharmaceutical composition may also include 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 drugs or any combination thereof. In certain embodiments, the ADC described herein and other pharmaceutically active agents are provided as separate components or as mixed components. Therefore, the ADC described herein and other pharmaceutically active agents can be administered simultaneously, separately or sequentially.

使用方法How to use

本文所述的抗体-药物缀合物、本文所述的药物-连接体或其药物组合物可用于治疗各种疾病或病症，例如HER3高表达的癌症，包括实体瘤或血液系统恶性肿瘤，例如结肠癌、胃癌、乳腺癌、肺癌(例如非小细胞肺癌，特别是肺腺癌)或淋巴瘤。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.

因此，本申请提供了如前述任一实施方案中所述的抗体-药物缀合物(ADC)、药物-连接体或包含其的药物组合物在制备用于治疗HER3高表达癌症的药物中的用途。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 cancers with high HER3 expression.

同时，本申请还提供了一种治疗HER3高表达癌症的方法，该方法包括向有此需要的受试者施用治疗有效量的如前述实施方案中任一项所述的抗体-药物缀合物(ADC)、药物-连接体或包含其的药物组合物的步骤。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), a drug-linker, or a pharmaceutical composition comprising the same as described in any of the aforementioned embodiments.

在某些实施方案中，所述抗体-药物缀合物(ADC)、药物-连接体或药物组合物足以(例如，在受试者中):In certain embodiments, the antibody-drug conjugate (ADC), drug-linker, or pharmaceutical composition is sufficient (e.g., in a subject):

(1)抑制细胞(如肿瘤细胞)的增殖；(1) Inhibit the proliferation of cells (such as tumor cells);

(2)抑制肿瘤生长；(2) Inhibit tumor growth;

(3)诱导和/或增加抗体依赖性细胞毒性活性；(3) Induce and/or increase antibody-dependent cellular cytotoxicity activity;

(4)抑制HER3介导的信号转导；(4) Inhibit HER3-mediated signal transduction;

(5)预防和/或治疗HER3介导的疾病/障碍；或者(5) preventing and/or treating HER3-mediated diseases/disorders; or

(6)上述(1)-(5)的任何组合。 (6) Any combination of the above (1) to (5).

在某些实施方案中，HER3介导的疾病/病症是肿瘤，例如表达HER3的肿瘤。在某些实施方案中，肿瘤选自乳腺癌、胃癌、肺癌(例如非小细胞肺癌)、结肠直肠癌、胰腺癌、头颈鳞状细胞癌、黑色素瘤、卵巢癌、前列腺癌、肝癌、肾癌、膀胱癌或其任意组合。In certain embodiments, the HER3-mediated disease/disorder is a tumor, such as 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.

应用application

本文所述抗体-药物缀合物或其药物组合物可以用于治疗多种疾病或病症，例如HER3高表达癌症，包括实体瘤或血液系统恶性肿瘤，例如结肠癌，胃癌，乳腺癌，肺癌(例如，非小细胞肺癌，具体如肺腺癌)，或淋巴癌。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, specifically lung adenocarcinoma), or lymphoma.

因此，本申请提供前文任一项所述的抗体-药物缀合物、药物-连接体、或含有其的药物组合物在制备治疗HER3高表达癌症的药物中的用途。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.

同时，本申请还提供一种治疗HER3高表达癌症的方法，其包括向由此需要的受试者施用治疗有效量的前文任一项所述的抗体-药物缀合物、药物-连接体、或含有其的药物组合物的步骤。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.

定义definition

除非在下文中另有定义，本文中所使用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。提及本文中使用的技术意图指在本领域中通常所理解的技术，包括那些对本领域技术人员显而易见的技术的变化或等效技术的替换。并且，本文中所用的基因组学、核酸化学、分子生物学等实验室操作步骤均为相应领域内广泛使用的常规步骤。虽然相信以下术语对于本领域技术人员很好理解，但仍然阐述以下定义以更好地解释本发明。Unless otherwise defined below, the meanings of all technical terms and scientific terms used herein are intended to be the same as those commonly understood by those skilled in the art. Reference to the technology used herein is intended to refer to the technology commonly understood in the art, including those changes in technology that are obvious to those skilled in the art or the replacement of equivalent technology. In addition, laboratory operation steps such as genomics, nucleic acid chemistry, molecular biology, etc. used herein are all conventional steps 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.

术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循本领域已知的各种编号系统。The term "antibody" refers to an immunoglobulin molecule that is 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 κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (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 with high variability (called complementary determining regions (CDRs)), interspersed with more conservative 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 antigen binding sites, respectively. The allocation of amino acids in each region or domain can follow various numbering systems known in the art.

在一些实施方案中，术语“抗体”进一步包括重链恒定区包含C端赖氨酸，或缺失C端赖氨酸或C端甘氨酸-赖氨酸二肽的方案。该术语还包括可变区的N-末端氨基酸环化形成焦谷氨酸的方案。因此，在本文公开的包含抗体的组合物中，所含的各种抗体可独立地包含C端赖氨酸、C端赖氨酸缺失、C端甘氨酸-赖氨酸缺失和/或包含N端谷氨酰胺或谷氨酸，或N-末端氨基酸环化为焦谷氨酸。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 N-terminal amino acid cyclized to form pyroglutamic acid. Therefore, in the composition comprising antibodies disclosed herein, the various antibodies contained 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.

术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDRs，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)、IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)或AbM编号系统(Martin ACR,Cheetham JC,Rees AR(1989)Modelling antibody hypervariable loops:A combined algorithm.Proc Natl Acad Sci USA 86:9268-9272)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。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. There are three CDRs in the variable region of the heavy chain and light chain, respectively, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as 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; CDR2, CDR3). (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 easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

在本发明中，抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定，例如通过Kabat、Chothia、IMGT或AbM编号系统确定。在某些实施方案中，抗体或其抗原结合片段含有的CDR通过Chothia编号系统定义。In the present invention, the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as by Kabat, Chothia, IMGT or AbM numbering systems. In certain embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined by the Chothia numbering system.

www.bioinf.org.uk中Andrew C.R.Martin团队公开了以下的通用规则，可用于定义抗体序列中的CDR，所述CDR包括这样的氨基酸，它们能与包含和所述抗体相结合的抗原的表位的氨基酸特异性相互作用。在极少数的例子中，这些通常恒定的特征不会出现。然而，Cys残基是最保守的特征。

Andrew CRMartin's group at www.bioinf.org.uk has published the following general rules that can be used to define CDRs in antibody sequences that include amino acids that specifically interact with amino acids in the epitope of the antigen to which the antibody binds. In rare cases, these normally constant features do not appear. However, Cys residues are the most conserved features.

VH的整个氨基酸序列通常根据Kabat编号，而可变区的三个CDR可根据前述编号方案中的任一种来定义。在具体的实施方案中，VH中的氨基酸位置的编号可以是从氨基酸位置1开始并且继续按顺序到序列的末端或根据Kabat编号。除非另有说明，本文VH和VL中的氨基酸位置根据顺序编号定义。The entire amino acid sequence of VH is generally numbered according to Kabat, while the three CDRs of the variable region may be defined according to any of the aforementioned numbering schemes. In a specific embodiment, the numbering of the amino acid positions in VH may be starting from amino acid position 1 and continuing sequentially to the end of the sequence or according to Kabat numbering. Unless otherwise indicated, the amino acid positions in VH and VL herein are defined according to sequential numbering.

重链恒定区中的氨基酸位置的编号可以是从氨基酸位置1开始并且继续按顺序到序列的末端或根据Eu编号。IgGl重链恒定区氨基酸序列有330个氨基酸，依次编号为1至330。对应的序列根据Eu编号从位置号118开始到位置号447结束。除非另有说明，本文中的重链和轻链中的氨基酸位置是根据顺序编号定义的。The numbering of amino acid positions in the heavy chain constant region can be starting from amino acid position 1 and continuing in sequence 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 number 118 and ends at position number 447 according to Eu numbering. Unless otherwise stated, amino acid positions in the heavy and light chains herein are defined according to sequential numbering.

术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。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.

术语抗体的“抗原结合片段”是指抗体的片段的多肽，例如全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab片段、Fab'片段、F(ab')2片段、F(ab')3片段、Fd、Fv、scFv、di-scFv、(scFv)2、二硫键稳定的Fv蛋白(“dsFv”)、单结构域抗体(sdAb，纳米抗体)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。The term "antigen-binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, such as a polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". 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 (sdAb, nanobodies), and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’)2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。The term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab') 2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab'fragment" means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd fragment (consisting of VH and CH1 domains) of the heavy chain.

术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDRs赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDRs)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。The term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment 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., a 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 a complete binding site.

术语“Fc”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。The term "Fc" refers to an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain of an antibody bound via a disulfide bond. The Fc fragment of an antibody has a variety of different functions but does not participate in antigen binding.

术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS(SEQ ID NO:55)氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4(SEQ ID NO:56)的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。在某些实施方案中，VH和VL结构域可以以任何合适的排列彼此相对定位。例如，包含NH2-VH-VH-COOH、NH2-VL-VL-COOH的scFv。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 may be positioned relative to each other in any suitable arrangement. For example, a scFv comprising NH 2 -VH-VH-COOH, NH 2 -VL-VL-COOH.

术语“单域抗体(single-domain antibody,sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力(Holt,L.等人,生物技术趋势(Trends in Biotechnology),21(11):484-490，2003)。单域抗体也称为纳米抗体(nanobody)。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.

上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。 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.

在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。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.

可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。Antibody antigen-binding fragments (e.g., the antibody fragments 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 antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.

术语“单克隆抗体”和“mAb”具有相同的含义且可互换使用可互换，其是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即，除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。此外，修饰语“单克隆”仅表明该抗体的特征为从高度同源的抗体群中获得，不能理解为需要通过任何特定方法来制备所述抗体。The terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably, and refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, and generally contain at least two or more different antibodies, which generally 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 group, and it should not be understood that the antibody needs to be prepared by any specific method.

术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性。例如，术语“嵌合抗体”可包括这样的抗体，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链可变区来自第二抗体(例如人抗体)。例如，通过免疫全人转基因小鼠产生的抗体可以称为嵌合抗体，其由全人源的可变区和鼠源的恒定区组成。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 still retains binding activity to the target antigen. For example, the term "chimeric antibody" may include antibodies in which the heavy chain and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy chain 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 can be called a chimeric antibody, which consists of a fully human variable region and a murine constant region.

术语“鼠源抗体”是指通过下述方法获得的抗体：融合免疫接种过的小鼠的B细胞与骨髓瘤细胞，筛选出既能无限增殖又能分泌抗体的鼠杂交融合细胞，继而进行筛选、抗体制备和抗体纯化；或者是指，由抗原侵入小鼠体内后B细胞分化增殖而形成浆细胞所分泌产生的抗体。The term "murine antibody" refers to antibodies obtained by the following method: fusing B cells of immunized mice with myeloma cells, screening out mouse hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, and then performing screening, antibody preparation and antibody purification; or refers to antibodies secreted by plasma cells formed by differentiation and proliferation of B cells after antigens invade the mouse body.

术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。供体抗体可以是有预期性质(例如，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力和/或增强免疫应答的能力)的小鼠、大鼠、兔或非人灵长类动物(例如，食蟹猴)抗体。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).

术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。The term "identity" is used to refer to the matching 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 being 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 a total of 6 positions match). Typically, two sequences are compared when they are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by 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 MoI 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.

术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。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 a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, for example, 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).

本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。The compilation of the twenty conventional amino acids involved in this article follows 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.

术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。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, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents 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, which can stabilize 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 their degradation products (such as lactalbumin hydrolysate), etc.

如本文中所使用的，术语“药学上可接受的盐”包括酸加成盐和碱式盐。As used herein, the term "pharmaceutically acceptable salts" includes acid addition salts and basic salts.

示例性的酸加成盐包括乙酸盐、铵、抗坏血酸盐、苯甲酸盐、苯磺酸盐、硫酸氢盐、硼酸盐、丁酸盐、柠檬酸盐、樟脑酸盐、樟脑磺酸盐、富马酸盐、盐酸盐、氢溴酸盐、氢碘酸盐、乳酸盐、马来酸盐、甲烷磺酸盐(也称为甲磺酸盐)、萘磺酸盐、硝酸盐、草酸盐、磷酸盐、丙酸盐、水杨酸盐、琥珀酸盐、硫酸盐、酒石酸盐、硫氰酸盐、甲苯磺酸盐(也称为甲苯磺酸盐)等。另外，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,D.C.on their website)。这些公开内容通过引用并入本文。在一个实施方案中，酸式盐是铵盐或二铵盐。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, D.C. on their website). The disclosures of these are incorporated herein by reference. In one embodiment, the acid salt is an ammonium salt or a diammonium salt.

示例性的碱性盐包括铵盐，碱金属盐如钠盐、锂盐和钾盐，碱土金属盐如钙盐和镁盐，与有机碱(例如有机胺)形成的盐如二环己胺、叔丁胺、胆碱，以及与精氨酸、赖氨酸等氨基酸形成的盐。碱性含氮基团可以用诸如低级烷基卤化物(例如甲基、乙基或丁基氯化物、溴化物或碘化物)、硫酸二烷基酯(例如硫酸二甲酯、硫酸二乙酯和硫酸二丁酯)、长链卤化物(例如，癸基、月桂基或硬脂基的氯化物、溴化物或碘化物)、芳烷基卤化物(例如，苄基或苯乙基溴)等进行季铵化。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., decyl, lauryl or stearyl chloride, bromide or iodide), aralkyl halides (e.g., benzyl or phenethyl bromide), and the like.

所有这些酸式盐和碱式盐旨在作为本公开范围内的药学上可接受的盐，并且出于本公开的目的，所有酸式盐和碱式盐被认为与相应化合物的游离形式是等价的。All such acid salts and base salts are intended as pharmaceutically acceptable salts within the scope of the disclosure and all acid salts and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the disclosure.

术语“包括”、“包含”、“具有”、“含有”或“涉及”及其在本文中的其它变体形式为包含性的(inclusive)或开放式的，且不排除其它未列举的元素或方法步骤。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.

术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如，肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其它治疗等等。ADC的治疗有效量可能因以下因素而异：待治疗疾病的严重程度，患者自身免疫系统的一般状态，患者的一般状况，例如年龄，体重和性别，药物的给药方式以及同时进行的其他治疗等。The term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient who already has the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc. The therapeutically effective amount of 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 simultaneously, etc.

术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括但不限于，减轻症状、减轻疾病的程度、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。另外，“治疗”还可以指与不接受治疗的预期存活相比延长存活。The term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, alleviation of symptoms, alleviation of the extent of the disease, stabilization (i.e., no longer worsening) of the state of the disease, delay or slowing of the progression of the disease, improvement or alleviation of the state of the disease, and relief of symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" may also refer to prolonged survival compared to the expected survival without treatment.

术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，所述受试者(例如人)患有肿瘤，或者具有患有上述疾病的风险。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.

术语“癌症”和“肿瘤”可互换使用，其是指以体内异常细胞的不受控生长为特征的一大类疾病。不受管制的细胞分裂可能导致恶性肿瘤或侵入邻近组织的细胞的形成，并可能通过淋巴系统或血流到达身体的远端部位(转移)。癌症包括良性和恶性癌症以及休眠肿瘤或微转移。癌症也包括血液肿瘤，尤其是血液系统恶性肿瘤。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 (metastasize) via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes blood tumors, especially hematologic malignancies.

术语“血液系统恶性肿瘤”包括淋巴瘤，白血病，骨髓瘤或淋巴恶性肿瘤，以及脾癌和淋巴结肿瘤。示例性淋巴瘤包括B细胞淋巴瘤和T细胞淋巴瘤。B细胞淋巴瘤，包括例如霍奇金淋巴瘤。T细胞淋巴瘤，包括例如皮肤T细胞淋巴瘤。血液系统恶性肿瘤还包括白血病，例如继发性白血病或急性淋巴细胞性白血病。血液系统恶性肿瘤还包括骨髓瘤(例如多发性骨髓瘤)及其它血液和/或B细胞或T细胞相关的癌症。The term "hematologic malignancies" includes lymphomas, leukemias, myelomas or lymphoid malignancies, as well as spleen cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphomas and T-cell lymphomas. B-cell lymphomas include, for example, Hodgkin's lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphomas. Hematologic malignancies also include leukemias, such as secondary leukemias or acute lymphocytic leukemias. Hematologic malignancies also include myeloma (e.g., multiple myeloma) and other blood and/or B-cell or T-cell related cancers.

术语“烷基”表示直链或支链烃基去掉1个氢原子得到的基团，例如“C1-20烷基”、“C1- 10烷基”、“C1-6烷基”、“C1-4烷基”、“C1-3烷基”等，具体实例包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、异戊基、2-甲基丁基、新戊基、1-乙基丙基、正己基、异己基、3-甲基戊基、2-甲基戊基、1-甲基戊基、3,3-二甲基丁基、2,2-二甲基丁基、1,1-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、1,2-二甲基丙基等。The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight or branched hydrocarbon group, for example, " C1-20 alkyl", " C1-10 alkyl", " C1-6 alkyl", " C1-4 alkyl", " C1-3 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.

术语“亚烷基”表示直链或支链烃基去掉2个氢原子得到的基团，例如“C1-20亚烷基”、“C1-10亚烷基”、“C3-10亚烷基”、“C5-8亚烷基”、“C1-6亚烷基”、“C1-4亚烷基”、“C1-3 亚烷基”等，具体实例包括但不限于：亚甲基、亚乙基、1,3-亚丙基、1,4-亚丁基、1,5-亚戊基或1,6-亚己基等。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.

术语“亚烯基”是指含有至少一个碳碳双键的直链或支链的烃基失去两个氢原子得到的二价基团，包括例如“C2-20亚烯基”、“C3-10亚烯基”、“C5-8亚烯基”等。其实例包括但不限于：亚乙烯基、1-亚丙烯基、2-亚丙烯基、1-亚丁烯基、2-亚丁烯基、1,3-亚丁二烯基、1-亚戊烯基、2-亚戊烯基、3-亚戊烯基、1,3-亚戊二烯基、1,4-亚戊二烯基、1-亚己烯基、2-亚己烯基、3-亚己烯基、1,4-亚己二烯基等。The term "alkenylene" refers to a divalent group obtained by losing two hydrogen atoms from a straight or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C 2-20 alkenylene", "C 3-10 alkenylene", "C 5-8 alkenylene", etc. Examples include, but are not limited to, vinylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 1,3-butadienylene, 1-pentenylene, 2-pentenylene, 3-pentenylene, 1,3-pentadienylene, 1,4-pentadienylene, 1-hexenylene, 2-hexenylene, 3-hexenylene, 1,4-hexadienylene, etc.

术语“亚炔基”是指含有至少一个碳碳三键的直链或支链烃基失去两个氢原子得到的二价基团。包括例如“C2-20亚炔基”、“C3-10亚炔基”、“C5-8亚炔基”等。其实例包括但不限于：亚乙炔基、1-亚丙炔基、2-亚丙炔基、1-亚丁炔基、2-亚丁炔基、1,3-亚丁二炔基、1-亚戊炔基、2-亚戊炔基、3-亚戊炔基、1,3-亚戊二炔基、1,4-亚戊二炔基、1-亚己炔基、2-亚己炔基、3-亚己炔基、1,4-亚己二炔基等。The term "alkynylene" refers to a divalent group obtained by losing two hydrogen atoms from a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond. Examples include, for example, "C 2-20 alkynylene", "C 3-10 alkynylene", "C 5-8 alkynylene", etc. Examples include, but are not limited to, ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 1,3-butadiynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 1,3-pentadiynylene, 1,4-pentadiynylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 1,4-hexadiynylene, etc.

术语“脂杂环”是指含至少一个选自N、O和S的环成员的饱和或部分饱和的环状结构。具体实例包括但不限于5-6元脂杂环、5-6元含氮脂杂环、5-6元含氧脂杂环等，例如四氢呋喃、吡咯烷、哌啶、四氢吡喃等。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.

术语“杂芳环”是指含至少一个选自N、O和S的环成员的芳香环状结构。具体实例包括但不限于5-6元芳杂环、5-6元含氮芳杂环、5-6元含氧芳杂环等，例如呋喃、噻吩、吡咯、噻唑、异噻唑、噻二唑、噁唑、异噁唑、噁二唑、咪唑、吡唑、1,2,3-三唑、1,2,4-三唑、1,2,3-噁二唑、1,2,4-噁二唑、1,2,5-噁二唑、1,3,4-噁二唑、吡啶、嘧啶、哒嗪、吡嗪、1,2,3-三嗪、1,3,5-三嗪、1,2,4,5-四嗪等。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.

术语“芳香族环系”是指包含至少一个芳环(例如苯环等)或杂芳环(例如嘧啶环等)的单环或多环体系，两个或更多个芳环和/或杂芳环可以形成稠合环或通过单键连接(例如二嘧啶基苯基等)，所述芳香族环系可以是二价或更高价态(例如三价或四价)，例如5-20元芳香族环系。The term "aromatic ring system" refers to a monocyclic or polycyclic system comprising at least one aromatic ring (e.g., a benzene ring, etc.) or a 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.

术语“连接体”指连接生物活性分子(例如细胞毒性药物片段)和靶向部位(例如抗体或其抗原结合片段)的结构片段。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 an antigen-binding fragment thereof).

术语“取代”指所指定的化合物或结构片段上的一个或多个(例如1个、2个、3个、4个或5个)氢被取代基所代替，条件是未超过所指定的原子在当前情况下的正常原子价并且所述取代形成稳定的化合物。取代基和/或变量的组合仅仅当这种组合形成稳定的化合物时才是允许的。例如，所述取代基各自独立地由一个或多个以下结构构成：-O-、-S-、-NR’-、卤素、-CN、-OH、-NH2、-NO2、-CN、＝O、C1-C6(亚)烷基、C1-C6卤代(亚)烷基、C1-C6烷氧基、C2-C6(亚)烯基、C2-C6(亚)炔基、C3-C8(亚)环烷基、3-8元(亚)杂环基、C6-C10(亚)芳基和5-10元(亚)杂芳基等。其中R’为氢或C1-C6烷基。The term "substituted" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogens on the specified compound or structural fragment by a substituent, provided that the normal atomic valence of the specified atom in the current situation is not exceeded and the substitution forms a stable compound. Combinations of substituents and/or variables are permitted only when 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, -NH 2 , -NO 2 , -CN, =O, C 1 -C 6 (ene) alkyl, C 1 -C 6 halo (ene) alkyl, C 1 -C 6 alkoxy, C 2 -C 6 (ene) alkenyl, C 2 -C 6 (ene) alkynyl, C 3 -C 8 (ene) cycloalkyl, 3-8 member (ene) heterocyclyl, C 6 -C 10 (ene) aryl and 5-10 member (ene) heteroaryl, etc. wherein R' is hydrogen or C 1 -C 6 alkyl.

如果官能团或结构片段被描述为“取代或未取代”，则该官能团或结构片段可(1)未被取代或(2)被取代。该官能团被取代时，取代该官能团的取代基各自独立地由一个或多个以下结构构成：-O-、-S-、-NR’-、卤素、-CN、-OH、-NH2、-NO2、-CN、＝O、C1-C6(亚)烷基、C1-C6卤代(亚)烷基、C1-C6烷氧基、C2-C6(亚)烯基、C2-C6(亚)炔基、C3-C8(亚)环烷基、3-8元(亚)杂环基、C6-C10(亚)芳基和5-10元(亚)杂芳基等。其中R’为氢或C1-C6烷基。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 - C10 aryl and 5-10 membered heteroaryl, etc., wherein R' is hydrogen or C1 - C6 alkyl.

如本文中所使用的，术语“大约(about)”或“近似(approximately)”与数值变量结合使用时通常意味着变量的值在实验误差范围内(例如，在平均值的95％置信区间内)或在±10％或更宽范围内。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.

应当注意，如果所描述的结构和该结构的名称之间存在差异，则所描述的结构将被赋予更大的权重。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.

本申请涉及用于治疗HER3阳性癌症的抗体-药物缀合物，并且示例性地公开了具有如通式Ab-[M-L-E-D]x所示的结构并且使用全人抗体22B6D2-hIgG1作为靶向部分的抗体-药物缀合物。结果表明，该偶联物具有较好的药物抗体比，对HER3阳性细胞具有良好的结合活性，对HER3阳性的癌症，如结肠癌、胃癌、乳腺癌、肺癌(如非小细胞肺癌，特别是肺腺癌)具有很好的靶向杀伤作用。因此，本申请提供了用于治疗高表达HER3的癌症的抗体-药物缀合物、包含该抗体-药物缀合物的药物组合物及其在治疗高表达HER3的癌症中的应用。The present application relates to an antibody-drug conjugate for treating HER3-positive cancers, and exemplarily discloses an antibody-drug conjugate having a structure as shown in the general formula Ab-[M-L-E-D]x and using the fully human antibody 22B6D2-hIgG1 as a targeting portion. 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 highly express HER3, a pharmaceutical composition comprising the antibody-drug conjugate, and its use in treating cancers that highly express HER3.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

此处所说明的附图用来提供对本发明的进一步理解，构成本申请的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：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:

图1显示了抗人HER3抗体偶联药物与MDA-MB-453细胞的亲和力的检测结果。FIG1 shows the results of the affinity test of the anti-human HER3 antibody-drug conjugate to MDA-MB-453 cells.

图2显示了抗人HER3抗体偶联药物与NCI-N87细胞的亲和力的检测结果。FIG2 shows the results of the affinity test of the anti-human HER3 antibody-drug conjugate to NCI-N87 cells.

图3显示了抗人HER3抗体偶联药物在MDA-MB-453细胞的内吞活性测试结果Figure 3 shows the results of the endocytic activity test of anti-human HER3 antibody-drug conjugates in MDA-MB-453 cells

图4显示了抗人HER3抗体偶联药物在HCC1569细胞的内吞活性测试结果。FIG4 shows the results of the endocytic activity test of the anti-human HER3 antibody-drug conjugate in HCC1569 cells.

图5显示了抗人HER3抗体偶联药物体外对MDA-MB-453细胞杀伤活性检测结果。FIG5 shows the results of the in vitro detection of the killing activity of anti-human HER3 antibody-drug conjugates on MDA-MB-453 cells.

图6显示了抗人HER3抗体偶联药物的体外对293T-HER3细胞杀伤活性检测结果。FIG6 shows the results of in vitro detection of the killing activity of anti-human HER3 antibody-drug conjugates on 293T-HER3 cells.

图7A显示了抗人HER3抗体偶联药物的体内药效检测结果。 FIG7A shows the in vivo efficacy test results of the anti-human HER3 antibody-drug conjugate.

图7B显示了抗人HER3抗体偶联药物的体内药效检测中的小鼠体重变化情况。FIG7B shows the changes in mouse body weight during the in vivo efficacy test of the anti-human HER3 antibody-drug conjugate.

具体实施方式DETAILED DESCRIPTION

下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，绝不作为对本发明及其应用或使用的任何限制。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。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, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit 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 creative work are within the scope of protection of the present invention.

本发明涉及的序列的信息描述于下面的表中：








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








本文中所使用的缩写具有以下含义：
The abbreviations used in this document have the following meanings:

以下的实施例中记载的化合物的结构通过核磁共振(1H NMR)或质谱(MS)来确定。The structures of the compounds described in the following examples were confirmed by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

核磁共振(1H NMR)的测定使用Bruker 400MHz核磁共振仪；氘代试剂为六氘代二甲基亚砜(DMSO-d6)；内标物质为四甲基硅烷(TMS)。Nuclear magnetic resonance ( 1 H NMR) was measured using a Bruker 400 MHz nuclear magnetic resonance instrument; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard substance was tetramethylsilane (TMS).

实施例中使用的核磁共振(NMR)图谱中的缩写示于以下。The abbreviations in the nuclear magnetic resonance (NMR) spectra used in the Examples are shown below.

s：单峰(singlet)、d：二重峰(doublet)、t：三重峰(triplet)、q：四重峰(quartet)、m：多重峰(multiplet)、br：宽峰(broad)、J：偶合常数、Hz：赫兹、DMSO-d6：氘化二甲基亚砜。δ值用ppm值表示。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.

质谱(MS)的测定使用Agilent(ESI)质谱仪，型号为Agilent 6120B。Mass spectrometry (MS) was measured using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

实施例一 N-((S)-10-苄基-1-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六烷-16-基)-6-(2,5-二氧代-2,5-二氢-1-H-吡咯-1-基)己酰胺(A-01)
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-tetraazahexadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1-H-pyrrol-1-yl)hexanamide (A-01)

将化合物A-01-1(0.40g，640.59μmol，其合成参考专利申请CN 111936169A)、依喜替康甲磺酸盐(0.37g，704.65μmol)溶于DMF(8mL)中，加入HATU(0.32g，832.77μmol)和DIPEA(0.25g，1.92mmol)，25℃反应4小时。减压除去DIPEA并加水冷冻干燥以除去大部分DMF得到粗品，粗品用制备高效液相色谱纯化(条件如下)得标题化合物273mg。Compound A-01-1 (0.40 g, 640.59 μmol, its synthesis reference 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 the mixture was reacted at 25°C for 4 hours. DIPEA was removed under reduced pressure and freeze-dried with water to remove most of the DMF to obtain a crude product, which was purified by preparative HPLC (conditions as follows) to obtain 273 mg of the title compound.

色谱柱：Waters XBridge Prep C18 OBD 45mm×450mm×8.0μmColumn: Waters XBridge Prep C18 OBD 45mm×450mm×8.0μm

流动相A：乙腈；流动相B：水(0.05％三氟乙酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

结构表征数据如下：The structural characterization data are as follows:

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

实施例二 N-((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺和N-((1R,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺(1-8-A和1-8-B)
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)

步骤一：1-氯-3-溴-2-甲基-5-硝基苯的合成(1-8-2) Step 1: Synthesis of 1-chloro-3-bromo-2-methyl-5-nitrobenzene (1-8-2)

25℃下，将化合物1-8-1(5.00g，29.14mmol)溶于正庚烷(25mL)中，加入浓硫酸(25mL)，加热至50℃，50℃下分批次加入NBS(6.22g，34.97mmol)，保持50℃反应2小时，用薄层色谱检测反应(乙酸乙酯:石油醚＝1:10)。将反应液冷却至室温，而后滴加入冰水中，甲苯萃取，有机相合并，分别经亚硫酸钠溶液，水，和饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩，粗品用制备高效液相色谱纯化(条件如下)，制备液冷冻干燥得标题化合物4.88g。At 25°C, compound 1-8-1 (5.00 g, 29.14 mmol) was dissolved in n-heptane (25 mL), 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 mixture was kept at 50°C for 2 hours. The reaction was detected 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, extracted with toluene, and the organic phases were combined, 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), and the preparative solution was freeze-dried to obtain 4.88 g of the title compound.

色谱柱：C18 ODS 45mm×450mm×8.0μmChromatographic column: C18 ODS 45mm×450mm×8.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

步骤二：3-氯-5-溴-4-甲基苯胺的合成(1-8-3)Step 2: Synthesis of 3-chloro-5-bromo-4-methylaniline (1-8-3)

25℃下，将化合物1-8-2(4.88g，19.48mmol)溶于乙酸乙酯(100mL)中，加入铂碳(2.00g，19.48mmol，含量5％)，氢气置换后在氢气保护下60℃反应4h，用高效液相质谱联用色谱监测反应。反应液经过滤、浓缩，得标题化合物粗品3.68g，未经进一步纯化直接用于下一步反应。At 25°C, compound 1-8-2 (4.88 g, 19.48 mmol) was dissolved in ethyl acetate (100 mL), and platinum carbon (2.00 g, 19.48 mmol, content 5%) was added. After hydrogen replacement, the mixture was reacted at 60°C for 4 h under hydrogen protection, and the reaction was monitored by HPLC-MS/MS. The reaction solution was filtered and concentrated to obtain 3.68 g of crude title compound, which was directly used in the next step without further purification.

步骤三:N-(3-氯-5-溴-4-甲基苯基)乙酰胺的合成(1-8-4)Step 3: Synthesis of N-(3-chloro-5-bromo-4-methylphenyl)acetamide (1-8-4)

20℃下，将化合物1-8-3(3.63g，14.82mmol)溶于乙酸乙酯(70mL)，加入三乙胺(4.50g，44.45mmol)和醋酸酐(2.27g，22.23mmol)，保持20℃反应20h，用高效液相质谱联用色谱监测反应。向反应液加入水，乙酸乙酯萃取，合并有机相，经无水硫酸钠干燥，减压浓缩得粗品，粗品经乙酸乙酯:石油醚＝1:5混合溶剂打浆得到标题化合物2.86g。At 20°C, compound 1-8-3 (3.63 g, 14.82 mmol) was dissolved in ethyl acetate (70 mL), triethylamine (4.50 g, 44.45 mmol) and acetic anhydride (2.27 g, 22.23 mmol) were added, and the reaction was maintained at 20°C for 20 h, and the reaction was monitored by HPLC-MS. Water was added to the reaction solution, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was slurried with a mixed solvent of ethyl acetate: petroleum ether = 1:5 to obtain 2.86 g of the title compound.

步骤四：(Z)-4-(5-乙酰胺基-3-氯-2-甲基苯基)丁-3-烯酸的合成(1-8-5)Step 4: Synthesis of (Z)-4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid (1-8-5)

20℃下，将化合物1-8-4(1.80g，6.86mmol)溶于THF(20mL)和水(5mL)中，加入乙烯基乙酸(708.31mg，8.23mmol)，DIPEA(1.95g，15.08mmol)，三(邻甲基苯基)磷(62.60mg，0.20mmol)，反应体系经氮气置换后加热至70℃反应5h，用高效液相质谱联用色谱监测反应。向反应液中加入1N氢氧化钠溶液调pH＝8，加入乙酸乙酯萃取，水相经1N盐酸调pH＝3，乙酸乙酯萃取，合并有机相，经无水硫酸钠干燥，减压浓缩，得标题化合物0.82g，直接用于下一步反应。At 20°C, compound 1-8-4 (1.80 g, 6.86 mmol) was dissolved in THF (20 mL) and water (5 mL), and vinylacetic acid (708.31 mg, 8.23 mmol), DIPEA (1.95 g, 15.08 mmol), tri(o-methylphenyl)phosphine (62.60 mg, 0.20 mmol) were added. The reaction system was replaced with nitrogen and heated to 70°C for 5 h. The reaction was monitored by HPLC-MS. 1N sodium hydroxide solution was added to the reaction solution to adjust pH=8, and ethyl acetate was added for extraction. The aqueous phase was adjusted to pH=3 with 1N hydrochloric acid, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 0.82 g of the title compound, which was directly used in the next step.

步骤五：4-(5-乙酰胺基-3-氯-2-甲基苯基)丁酸的合成(1-8-6)Step 5: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)butyric acid (1-8-6)

20℃下，将化合物1-8-5(2.60g，9.71mmol)溶于THF(50mL)中，加入Pd/C(0.52g，含量10％)，体系经氢气置换后在氢气球保护下40℃反应2h，用高效液相质谱联用色谱监测反应。将反应液过滤，滤液浓缩，得标题化合物2.43g，未经进一步纯化直接用于下一步反应。At 20°C, compound 1-8-5 (2.60 g, 9.71 mmol) was dissolved in THF (50 mL), Pd/C (0.52 g, content 10%) was added, the system was replaced with hydrogen and reacted at 40°C for 2 h under the protection of a hydrogen balloon, and 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 directly used in the next step without further purification.

步骤六：N-(3-氯-4-甲基-8-氧代-5,6,7,8-四氢萘-1-基)乙酰胺的合成(1-8-7)Step 6: Synthesis of N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-7)

将化合物1-8-6(2.43g，9.01mmol)溶于三氟乙酸(10mL)中，降温至5℃，滴加入三氟乙酸酐(3.78g，18.02mmol，2.50mL)，保持5℃反应4h，用高效液相质谱联用色谱监测反应。将反应液加入水中，用10N氢氧化钠溶液调pH＝9，加入乙酸乙酯萃取，合并有机相，经无水硫酸钠干燥，减压浓缩，经硅胶柱纯化(乙酸乙酯:石油醚＝0～20％)得标题化合物1.53g。Compound 1-8-6 (2.43 g, 9.01 mmol) was dissolved in trifluoroacetic acid (10 mL), cooled to 5°C, trifluoroacetic anhydride (3.78 g, 18.02 mmol, 2.50 mL) was added dropwise, and the reaction was maintained at 5°C for 4 h. The reaction was monitored by HPLC-MS. The reaction solution was added to water, adjusted to pH = 9 with 10N sodium hydroxide solution, extracted with ethyl acetate, combined the organic phases, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column (ethyl acetate: petroleum ether = 0-20%) to obtain 1.53 g of the title compound.

步骤七:(Z)-N-(3-氯-7-(羟基亚氨基)-4-甲基-8-氧代-5,6,7,8-四氢萘-1-基)乙酰胺的合成(1-8-8)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)

5℃下，将叔丁醇钾(1.50g，13.37mmol)溶于THF(16mL)和叔丁醇(4mL)中，滴加入化合物1-8-7(1.53g，6.08mmol)的THF溶液(16mL)，10分钟后滴加入亚硝酸戊酯(1.14g，9.73mmol)，保持5℃反应1h，用高效液相质谱联用色谱监测反应。用1N盐酸将反应液调pH＝5，乙酸乙酯萃取，合并有机相经无水硫酸钠干燥，减压浓缩，粗品经甲基叔丁基醚打浆得标题化合物1.20g。At 5°C, potassium tert-butoxide (1.50 g, 13.37 mmol) was dissolved in THF (16 mL) and tert-butanol (4 mL), and 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 the reaction was monitored by HPLC-MS. The reaction solution was adjusted to pH = 5 with 1N hydrochloric acid, extracted with ethyl acetate, the combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was beaten with methyl tert-butyl ether to obtain 1.20 g of the title compound.

步骤八:N-(7-氨基-3-氯-4-甲基-8-氧代-5,6,7,8-四氢萘-1-基)乙酰胺的合成(1-8-9)Step 8: Synthesis of N-(7-amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-9)

20℃下，将化合物1-8-8(0.50g，1.78mmol)溶于甲醇(8mL)和2N盐酸(8mL)中，加入Pd/C(0.15g，含量10％)，体系经氢气置换后在氢气球保护下保持5℃反应2h，用高效液相质谱联用色谱监测反应。反应液经过滤、浓缩，得标题化合物的盐酸盐0.52g，未经进一步纯化直接用于下一步反应。At 20°C, compound 1-8-8 (0.50 g, 1.78 mmol) was dissolved in methanol (8 mL) and 2N hydrochloric acid (8 mL), Pd/C (0.15 g, content 10%) was added, the system was replaced with hydrogen and kept at 5°C for 2 h under the protection of a hydrogen balloon, and the reaction was monitored by HPLC-MS/MS. The reaction solution was filtered and concentrated to obtain 0.52 g of the hydrochloride of the title compound, which was directly used in the next step without further purification.

步骤九:N,N’-(3-氯-4-甲基-8-氧代-5,6,7,8-四氢萘-1,7-二基)二乙酰胺的合成(1-8-10)Step 9: Synthesis of N,N'-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (1-8-10)

20℃下，将化合物1-8-9(0.52g，1.70mmol)溶于吡啶(5mL)中，加入醋酸酐(2mL)，保持20℃反应2h，用高效液相质谱联用色谱监测反应。将反应液加入水中，乙酸乙酯萃取，有机相经水洗，合并，经无水硫酸钠干燥，减压浓缩，经硅胶柱纯化(乙酸乙酯:石油醚＝0～30％)得标题化合物0.22g。At 20°C, compound 1-8-9 (0.52 g, 1.70 mmol) was dissolved in pyridine (5 mL), acetic anhydride (2 mL) was added, and 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, extracted with ethyl acetate, the organic phase was washed with water, combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column (ethyl acetate: petroleum ether = 0-30%) to obtain 0.22 g of the title compound.

步骤十：N-(8-氨基-6-氯-5-甲基-1-氧代-1,2,3,4-四氢萘-2-基)乙酰胺的合成(1-8-11)Step 10: Synthesis of N-(8-amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-8-11)

20℃下，将化合物1-8-10(450.97mg，1.46mmol)溶于甲醇(16mL)中，加入2N盐酸(16mL)，加热至60℃反应2h，用高效液相质谱联用色谱监测反应。向冷却后的反应液中加入饱和碳酸氢钠溶液调pH＝8，乙酸乙酯萃取，合并有机相，经无水硫酸钠干燥，减压浓缩，得标题化合物230.00mg，未经进一步纯化直接用于下一步反应。At 20°C, compound 1-8-10 (450.97 mg, 1.46 mmol) was dissolved in methanol (16 mL), 2N hydrochloric acid (16 mL) was added, and the mixture was heated to 60°C for 2 h, and the reaction was monitored by HPLC-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.

步骤十一：N-((9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)乙酰胺的合成(1-8-12)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)

将化合物1-8-11(230.00mg，0.78mmol)溶于甲苯(10mL)中，加入(S)-4-乙基-4- 羟基-7,8-二氢-1H-吡喃并[3,4-f]吲哚嗪-3,6,10(4H)-三酮(230.00mg，0.87mmol)，对甲基苯磺酸(26.73mg，0.16mmol)，加热至140℃反应5h，用高效液相质谱联用色谱监测反应。将反应液浓缩，粗品经硅胶柱纯化(甲醇:二氯甲烷＝0～10％)得标题化合物150.00mg。Compound 1-8-11 (230.00 mg, 0.78 mmol) was dissolved in toluene (10 mL), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (230.00 mg, 0.87 mmol) and p-toluenesulfonic acid (26.73 mg, 0.16 mmol) were added, and the mixture was heated to 140°C for 5 h, and the reaction was monitored by HPLC-MS/MS. The reaction solution was concentrated, and the crude product was purified by silica gel column (methanol: dichloromethane = 0-10%) to obtain 150.00 mg of the title compound.

步骤十二:(9S)-1-氨基-5-氯-9-乙基-9-羟基-4-甲基-1,2,3,9,12,15-六氢-10H,13H-苯并吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-10,13-二酮的合成(1-2)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)

将化合物1-8-12(40.00mg，0.081mmol)加入浓盐酸(1mL)中，加热至100℃反应5h，用高效液相质谱联用色谱监测反应。反应液过滤，滤液用制备高效液相色谱纯化(条件如下)，制备液冷冻干燥得标题化合物1-2的盐酸盐。1-2的盐酸盐在下述纯化条件下分离得到两个异构体，依据保留时间命名为1-2-A(三氟乙酸盐5.00mg，保留时间9.85min)和1-2-B(三氟乙酸盐7.00mg，保留时间10.62min)。Compound 1-8-12 (40.00 mg, 0.081 mmol) was added to concentrated hydrochloric acid (1 mL), heated to 100 ° C for 5 h, and the reaction was monitored by HPLC-MS. The reaction solution was filtered, and the filtrate was purified by preparative HPLC (conditions as follows), and the preparative solution was freeze-dried to obtain the hydrochloride of the title compound 1-2. The hydrochloride of 1-2 was separated under the following purification conditions to obtain two isomers, which were named 1-2-A (trifluoroacetate 5.00 mg, retention time 9.85 min) and 1-2-B (trifluoroacetate 7.00 mg, retention time 10.62 min) according to the retention time.

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％三氟乙酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

结构表征数据如下：The structural characterization data are as follows:

1-2-A：1-2-A:

1H 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). 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).

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

1-2-B：1-2-B:

1H 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). 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).

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

步骤十三:2-((叔丁基二苯基硅基)氧基)-N-((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)乙酰胺和2-((叔丁基二苯基硅基)氧基)-N-((1R,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)乙酰胺的合成(1-8-13-A和1-8-13-B)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 ((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-8-13-A and 1-8-13-B)

25℃下，将化合物1-2的盐酸盐(40.00mg，81.91μmol)溶于N,N-二甲基甲酰胺(1mL)中，依次加入2-((叔丁基二苯基硅基)氧基)乙酸(30.91mg，98.29μmol)，HATU(62.25mg，163.81μmol)和N,N-二异丙基乙胺(42.34mg，327.63μmol)，保持25℃反应0.5小时，用高效液相质谱联用色谱监测反应。反应完成后向反应液中加入水，用二氯甲烷/甲醇(v/v＝10/1)萃取，有机相合并，经无水硫酸钠干燥后减压浓缩，粗品经制备薄层色谱纯化(二氯甲烷:甲醇＝20:1)分离得到两个异构体，依据Rf值将两个异构体命名为1-8-13-A(15.00mg，Rf值为0.3)和1-8-13-B(12.00mg，Rf值为0.35)。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 HPLC-MS/MS. 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 two isomers, which are named 1-8-13-A (15.00 mg, Rf value is 0.3) and 1-8-13-B (12.00 mg, Rf value is 0.35) according to their Rf values.

步骤十四:N-((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺和N-((1R,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺的合成(1-8-A和1-8-B)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)

25℃下，在两个反应瓶中分别将1-8-13-A(15.00mg)和1-8-13-B(12.00mg)溶于四氢呋喃(1mL)中，滴加入四丁基氟化铵(1M四氢呋喃溶液)/冰醋酸混合液(v/v＝13/1)(50μL)，保持25℃反应0.5小时，用高效液相质谱联用色谱监测反应。反应完成后将反应液分别用制备高效液相色谱纯化，制备液分别冻干对应得到标题化合物1-8-A(6.94mg)和1-8-B(4.00mg)。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 bottles, and a mixture of tetrabutylammonium fluoride (1M tetrahydrofuran solution)/glacial acetic acid (v/v=13/1) (50 μL) was added dropwise, and the reaction was maintained at 25°C for 0.5 hours, and the reaction was monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative HPLC, and the preparative solution was lyophilized to obtain the title compounds 1-8-A (6.94 mg) and 1-8-B (4.00 mg).

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

1-8-A结构表征数据如下:The structural characterization data of 1-8-A are as follows:

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＝19.0Hz,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).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＝19.0 Hz,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).

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

1-8-B结构表征数据如下:The structural characterization data of 1-8-B are as follows:

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＝19.0Hz,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).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＝19.0 Hz,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).

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

实施例三N-((10S)-10-苄基-1-(((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4'；6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六烷-16-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺和N-((10S)-10-苄基-1-(((1R,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4'；6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六烷-16-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(A-07-A和A-07-B)
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-tetraazahexadecane-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-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-07-A and A-07-B)

步骤一：(S)-10-苄基-23-(2-(甲磺酰基)嘧啶-5-基)-6,9,12,15,18-五氧代-3-氧杂-5,8,11,14,17-五氮杂二十三烷-22-炔羧酸的合成(A-07-3) 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)

25℃下，将化合物A-07-2(30.00mg，0.07mmol)溶于DMF(0.2mL)中，加入2,5-二氧吡咯烷-1-基-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸酯(A-07-1，28.00mg，0.08mmol)，30℃反应1h，用高效液相质谱联用色谱监测反应。反应液直接用制备高效液相色谱纯化(条件如下)，制备液冷冻干燥得标题化合物20.00mg。At 25°C, compound A-07-2 (30.00 mg, 0.07 mmol) was dissolved in DMF (0.2 mL), 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, and the mixture was reacted at 30°C for 1 h, and 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.

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

结构表征数据如下：The structural characterization data are as follows:

ESI-MS(m/z):691.0[M+H2O]+.ESI-MS(m/z):691.0[M+H 2 O] + .

步骤二：N-((10S)-10-苄基-1-(((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4'；6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六烷-16-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺和N-((10S)-10-苄基-1-(((1R,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4'；6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六烷-16-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺的合成(A-07-A和A-07-B)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-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide and 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-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide 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-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-07-A and A-07-B)

25℃下，将1-2的盐酸盐(30.00mg，61.43μmol)溶于N,N-二甲基甲酰胺(1mL)中，依次加入A-07-3(49.66mg，73.72μmol)、HATU(35.01mg，92.14μmol)和N,N-二异丙基乙胺(23.82mg，184.29μmol)，保持25℃反应0.5小时，用高效液相质谱联用色谱监测反应。反应完成后将反应液用制备高效液相色谱纯化(条件如下)，制备液冷冻干燥得标题化合物A-07。A-07在下述纯化条件下分离得到两个异构体，依据保留时间命名为A-07-A(11.04mg，保留时间7.5min)和A-07-B(19.42mg，保留时间8.0min)。At 25°C, the hydrochloride of 1-2 (30.00 mg, 61.43 μmol) was dissolved in N, N-dimethylformamide (1 mL), and A-07-3 (49.66 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol) and N, N-diisopropylethylamine (23.82 mg, 184.29 μmol) were added in sequence, and the reaction was maintained at 25°C for 0.5 hours, and the reaction was monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative HPLC (conditions as follows), and the preparative solution was freeze-dried to obtain the title compound A-07. A-07 was separated under the following purification conditions to obtain two isomers, which were named A-07-A (11.04 mg, retention time 7.5 min) and A-07-B (19.42 mg, retention time 8.0 min) according to the retention time.

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

结构表征数据如下： The structural characterization data are as follows:

A-07-A：A-07-A:

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

A-07-B：A-07-B:

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

实施例四(S)-7-乙基-7-羟基-14-(2-(异丙胺基)乙基)-10,13-二氢-11H-[1,3]二氧戊环并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(2-2)
Example 4 (S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)

步骤一：3-(异丙胺基)-1-(6-硝基苯并[d][1,3]二恶英-5-基)丙-1-酮的合成(2-2-2)Step 1: Synthesis of 3-(isopropylamino)-1-(6-nitrobenzo[d][1,3]dioxin-5-yl)propan-1-one (2-2-2)

0℃下将化合物2-2-1(1.90g，8.08mmol)慢慢加入硝酸(8mL)中，缓慢升至25℃反应1小时。反应液直接经反相柱层析(乙腈/0.5％甲酸水溶液)纯化，得标题化合物的甲酸盐1.50g。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 formate salt of the title compound.

结构表征数据如下：The structural characterization data are as follows:

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

步骤二：1-(6-氨基苯并[d][1,3]二恶英-5-基)-3-(异丙胺基)丙-1-酮的合成(2-2-3)Step 2: Synthesis of 1-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-(isopropylamino)propan-1-one (2-2-3)

将化合物2-2-2的甲酸盐(1.25g，4.46mmol)加入四氢呋喃(20mL)中，加入10％钯碳(125.00mg)，氢气置换三次后25℃反应16小时。反应液经硅藻土过滤，滤液减压浓缩干，得标题化合物粗品895.00mg，未经纯化直接用于下一步反应。The formate salt of compound 2-2-2 (1.25 g, 4.46 mmol) was added to tetrahydrofuran (20 mL), and 10% palladium carbon (125.00 mg) was added. After hydrogen replacement three times, the mixture was reacted at 25° C. for 16 hours. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to dryness under reduced pressure to obtain 895.00 mg of the crude title compound, which was directly used in the next step without purification.

结构表征数据如下：The structural characterization data are as follows:

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

步骤三：((S)-7-乙基-7-羟基-14-(2-(异丙胺基)乙基)-10,13-二氢-11H-[1,3]二氧戊环并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮的合成(2-2)Step 3: Synthesis of ((S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)

将化合物2-2-3(23.00mg，0.09mmol)和(4S)-4-乙基-4-羟基-7,8-二氢-1H-吡喃并[3,4-f]吲哚嗪-3,6,10(4H)-三酮(21.00mg，0.09mmol)用甲苯(4mL)溶解，加入对甲苯磺酸(1.40mg，0.009mmol)，140℃反应12小时。减压抽干溶剂，得标题化合物的粗品，粗品经HPLC(流动相A：乙腈，流动相B：0.05％的甲酸水溶液)纯化，制备液中加入3M盐酸3滴后冷冻干燥，得标题化合物的盐酸盐8.70mg。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 removed 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), and 3 drops of 3M hydrochloric acid were added to the prepared solution, followed by freeze drying to obtain 8.70 mg of the hydrochloride of the title compound.

结构表征数据如下：The structural characterization data are as follows:

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

1H-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). 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).

实施例五4-((S)-2-(4-氨基丁基)-35-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-九氧杂-3,9-二氮杂三十五烷酰胺基)苄基((S)-4-乙基-11-(2-(N-异丙基-N-甲基磺酰胺基)乙基)-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-4-基)碳酸酯的合成(B-01)
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)

步骤一：(S)-4-乙基-11-(2-(N-异丙基-N-甲磺酰胺基)乙基)-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-4-基(4-((S)-2-(4-(((4-甲氧基苯基)二苯基甲基)氨基)丁基)-35-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-九氧基-3,9-二氮杂三十五烷酰胺基)苄基)碳酸酯的合成(B-01-2)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-diazapentacontriacontamido)benzyl)carbonate (B-01-2)

室温下，将化合物B-01-1(413.40mg，0.251mmol，其合成参考专利CN111295389B)溶于二甲基亚砜和水(2.0mL：0.5mL)中，加入溴化亚铜(72.95mg，0.503mmol)和6-(2-(甲基磺酰基)嘧啶-5-基)-N-(丙-2-炔-1-基)-己-5-炔酰胺(95.10mg，0.302mmol)，搅拌反应1h后过滤，滤液经制备高效液相色谱纯化(条件如下)，得标题化合物30.00mg。At room temperature, compound B-01-1 (413.40 mg, 0.251 mmol, its synthesis reference patent CN111295389B) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), and cuprous bromide (72.95 mg, 0.503 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (95.10 mg, 0.302 mmol) were added. The reaction was stirred for 1 hour and then filtered. The filtrate was purified by preparative HPLC (conditions as follows) to obtain 30.00 mg of the title compound.

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μm Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水
Mobile phase A: acetonitrile; Mobile phase B: water

结构表征数据如下：The structural characterization data are as follows:

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

步骤二：4-((S)-2-(4-氨基丁基)-35-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-九氧杂-3,9-二氮杂三十五烷酰胺基)苄基((S)-4-乙基-11-(2-(N-异丙基-N-甲基磺酰胺基)乙基)-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-4-基)碳酸酯的合成(B-01)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)

将化合物B-01-2(30.00mg，0.02mmol)溶于二氯甲烷(1.0mL)反应液中加入三氟乙酸(0.2mL)，室温反应30min。反应液减压浓缩后经制备高效液相色谱纯化(条件如下)，得标题化合物的三氟乙酸盐20.00mg。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 solution, and the reaction was carried out at room temperature for 30 min. The reaction solution was concentrated under reduced pressure and purified by preparative high performance liquid chromatography (conditions as follows) to obtain 20.00 mg of trifluoroacetate salt of the title compound.

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％三氟乙酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

结构表征数据如下：ESI-MS(m/z):816.0[M/2+H]+.The structural characterization data are as follows: ESI-MS (m/z): 816.0 [M/2+H] + .

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＝8.56Hz,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). 1 H 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).

实施例六(2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-乙基-7-羟基-8,11-二氧-8,10,11,13-四氢-7H-[1,3]二氧戊环并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)(异丙基)氨甲酰基)氧基)甲基)-2-(2-(2-(2-(2-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)乙氧基)乙氧基)乙酰胺基))苯氧基)-3,4,5-三羟基四氢-2H-吡喃-2-羧酸(B-03)
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]dioxolane[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)

步骤一：(2S,3R,4S,5S,6S)-2-(4-(羟甲基)-2-硝基苯氧基)-6-(甲氧羰基)四氢-2H-吡喃-3,4,5-三乙酸酯的合成(B-03-3)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)

将化合物(2R,3R,4S,5S,6S)-2-溴-6-(甲氧羰基)四氢-2H-吡喃-3,4,5-三乙酸三酯(B-03-1，12.32g，31.02mmol)和4-羟基-3-硝基苄醇(化合物B-03-2，5.00g，29.56mmol)溶于乙腈(200mL)中，搅拌下加入氧化银(27.40g，118.25mmol)，氮气置换后避光室温反应12小时。用高效液相质谱联用色谱监测反应；反应液经硅藻土抽滤，滤液减压浓缩后经硅胶柱层析纯化(石油醚:乙酸乙酯＝1:3)，得标题化合物12.80g。Compound (2R, 3R, 4S, 5S, 6S) -2-bromo-6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triacetic acid triester (B-03-1, 12.32 g, 31.02 mmol) and 4-hydroxy-3-nitrobenzyl alcohol (compound B-03-2, 5.00 g, 29.56 mmol) were dissolved in acetonitrile (200 mL), and silver oxide (27.40 g, 118.25 mmol) was added under stirring. After nitrogen replacement, the mixture was reacted at room temperature in the dark for 12 hours. The reaction was monitored by HPLC-MS/MS; the reaction solution was filtered through diatomaceous earth, 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.

结构表征数据如下：The structural characterization data are as follows:

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

步骤二：(2S,3R,4S,5S,6S)-2-(2-氨基-4-(羟甲基)苯氧基)-6-(甲氧羰基)四氢-2H-吡喃-3,4,5-三乙酸酯的合成(B-03-4)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)

将化合物B-03-3(2.20g，4.53mmol)溶于乙酸乙酯和四氢呋喃(各50mL)中，加入PtO2(0.20g)，然后用氢气球置换反应体系三次，并在氢气氛围下反应2小时。用高效液相质谱联用色谱监测反应；反应液直接过滤，用乙酸乙酯淋洗滤饼，滤液减压蒸干得标题化合物粗品2.02g，直接用于下一步反应。Compound B-03-3 (2.20 g, 4.53 mmol) was dissolved in ethyl acetate and tetrahydrofuran (50 mL each), PtO 2 (0.20 g) was added, and then the reaction system was replaced with a hydrogen balloon three times, and the reaction was carried out under a hydrogen atmosphere for 2 hours. The reaction was monitored by HPLC-MS/MS; the reaction solution was directly filtered, the filter cake was washed 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 directly used in the next step.

结构表征数据如下：The structural characterization data are as follows:

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

步骤三：(2S,3R,4S,5S,6S)-2-(2-(2-(2-(2-(2-(9H-芴-9-基甲氧基羰基)氨基)乙氧基)乙氧基)乙酰胺基)-4-(羟甲基)苯氧基)-6-(甲氧羰基)四氢-2H-吡喃-3,4,5-三乙酸酯的合成(B-03-5)Step 3: Synthesis of (2S,3R,4S,5S,6S)-2-(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)

将化合物B-03-4(456.00mg，1.00mmol)和[2-[2-(Fmoc-氨基)乙氧基]乙氧基]乙酸(385.91mg，1.00mmol)溶于二氯甲烷(10mL)，搅拌下加入2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(495.22mg，2.00mmol)，搅拌反应2小时。用高效液相质谱联用色谱监测反应；反应液经减压浓缩后经硅胶柱层析纯化(甲醇:二氯甲烷＝1:20)，得标题化合物507.00mg。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), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (495.22 mg, 2.00 mmol) was added under stirring, and 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.

结构表征数据如下：The structural characterization data are as follows:

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

步骤四：(2S,3R,4S,5S,6S)-2-(2-(2-(2-(2-(2-(9H-芴-9-基甲氧基羰基)氨基)乙氧基)乙氧基)乙酰胺基)-4-((((4-硝基苯氧基)羰基)氧基)甲基)苯氧基)-6-(甲氧基羰基)四氢-2H-吡喃-3,4,5-三乙酸酯的合成(B-03-6)Step 4: Synthesis of (2S,3R,4S,5S,6S)-2-(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)

将化合物B-03-5(507.00mg，616.18μmol)和二异丙基乙胺(238.91mg，1.85mmol)溶于二氯甲烷(20mL)中，然后将对硝基苯基氯甲酸酯(372.60mg，1.85mmol)溶于二氯甲烷(1mL)中，并缓慢滴入反应液中，加毕，室温反应15h。用高效液相质谱联用色谱监测反应；反应液经减压浓缩后经硅胶柱层析纯化(甲醇:二氯甲烷＝1:20)，得标题化合物496.00mg。Compound B-03-5 (507.00 mg, 616.18 μmol) and diisopropylethylamine (238.91 mg, 1.85 mmol) were dissolved in dichloromethane (20 mL), and then p-nitrophenyl chloroformate (372.60 mg, 1.85 mmol) was dissolved in dichloromethane (1 mL) and slowly added dropwise to the reaction solution. After addition, the reaction was allowed to react at room temperature for 15 h. 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 496.00 mg of the title compound.

结构表征数据如下：The structural characterization data are as follows:

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

步骤五：(2S,3R,4S,5S,6S)-2-(2-(2-(2-(2-(2-(9H-芴-9-基甲氧基羰基)氨基)乙氧基)乙氧基)乙酰胺基)-4-((((2-((S)-7-乙基-7-羟基-8,11-二氧基-8,10,11,13-四氢-7H-[1,3]二氧戊环并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)(异丙基)氨甲酰基)氧基)甲基)苯氧基)-6-(甲氧羰基)四氢-2H-吡喃-3,4,5-三乙酸酯的合成(B-03-7)Step 5: Synthesis of (2S,3R,4S,5S,6S)-2-(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]dioxolane[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)

将化合物B-03-6(165.51mg，0.17mmol)、化合物2-2(40.00mg，0.084mmol)和1-羟基苯并三唑(33.96mg，0.25mmol)溶于DMF(4mL)，滴入二异丙基乙胺(32.48mg，0.25mmol)，搅拌反应12h，用高效液相质谱联用色谱监测反应。加入水和乙酸乙酯搅拌，静止分液，有机相用饱和食盐水洗涤后干燥，经减压浓缩，得标题化合物粗品100.00mg，直接进行下一步反应。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, and the mixture was stirred for 12 h, and the reaction was monitored by HPLC-MS. Water and ethyl acetate were added and stirred, and the mixture was separated at rest. 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.

结构表征数据如下：The structural characterization data are as follows:

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

步骤六：(2S,3S,4S,5R,6S)-6-(2-(2-(2-(氨基乙氧基)乙氧基)乙酰胺基)-4-((((2-((S)-7-乙基-7-羟基-8,11-二氧基-8,10,11,13-四氢-7H-[1,3]二氧戊环并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)(异丙基)氨甲酰基)氧基)甲基)苯氧基)-3,4,5-三羟基四氢-2H-吡喃-2-羧酸的合成(B-03-8)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]dioxolane[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)

将化合物B-03-7(100.00mg，0.08mmol)溶于MeOH(5mL)，滴入1滴二氯甲烷，滴入氢氧化锂一水合物(15.82mg，0.377mmol)的水溶液(1mL)，搅拌反应2小时。用高效液相质谱联用色谱监测反应；滴入3N盐酸水溶液调节反应液pH＝4，减压浓缩后用制备高效液相色谱纯化(条件如下)，制备液冷冻干燥得标题化合物27.00mg。Compound B-03-7 (100.00 mg, 0.08 mmol) was dissolved in MeOH (5 mL), 1 drop of dichloromethane was added, and an aqueous solution (1 mL) of lithium hydroxide monohydrate (15.82 mg, 0.377 mmol) was added, and the mixture was stirred for 2 hours. The reaction was monitored by HPLC-MS/MS; 3N aqueous hydrochloric acid was added to adjust the pH of the reaction solution to 4, and the solution was concentrated under reduced pressure and purified by preparative HPLC (conditions as follows), and the preparative solution was freeze-dried to obtain 27.00 mg of the title compound.

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

结构表征数据如下：The structural characterization data are as follows:

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

步骤七：(2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-乙基-7-羟基-8,11-二氧-8,10,11,13-四氢-7H-[1,3]二氧戊环并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)(异丙基)氨甲酰基)氧基)甲基)-2-(2-(2-(2-(2-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)乙氧基)乙氧基)乙酰胺基))苯氧基)-3,4,5-三羟基四氢-2H-吡喃-2-羧酸的合成(B-03)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]dioxolane[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)

将化合物B-03-8(27.00mg，0.03mmol)和2,5-二氧吡咯烷-1-基6-(2-(甲磺酸磺酰基)嘧啶-5-基)己-5-炔酸酯(A-07-1)，11.26mg，0.03mmol)溶于DMF(1mL)，搅拌下滴入二异丙基乙胺(3.62mg，0.03mmol)，室温反应4小时，用高效液相质谱联用色谱监测反应。反应液用制备高效液相色谱纯化(条件如下)，制备液冷冻干燥得标题化合物11.70mg。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), and diisopropylethylamine (3.62 mg, 0.03 mmol) was added dropwise under stirring. The mixture was reacted at room temperature for 4 hours, and the reaction was monitored by HPLC-MS. The reaction solution was purified by preparative HPLC (conditions are as follows), and the preparative solution was freeze-dried to obtain 11.70 mg of the title compound.

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

结构表征数据如下：The structural characterization data are as follows:

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

实施例七N-((1S,9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4:6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺和N-((1R,9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4:6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺(1-11-A和1-11-B)
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)

步骤一：3-溴-4-氯-5-氟苯胺的合成(1-5-02)Step 1: Synthesis of 3-bromo-4-chloro-5-fluoroaniline (1-5-02)

将化合物1-5-01(2.00g，10.53mmol)溶于N,N-二甲基甲酰胺(30mL)中，然后缓慢加入N-氯代丁二酰亚胺(1.69g，12.63mmol)，加毕，室温反应16小时，用高效液相质谱联用色谱检测反应。反应液经减压浓缩得粗品，粗品经快硅胶柱纯化(乙酸乙酯:石油醚＝0-25％)得标题化合物0.95g。Compound 1-5-01 (2.00 g, 10.53 mmol) was dissolved in N,N-dimethylformamide (30 mL), and then N-chlorosuccinimide (1.69 g, 12.63 mmol) was slowly added. After the addition was completed, the mixture was reacted at room temperature for 16 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by flash silica gel column (ethyl acetate: petroleum ether = 0-25%) to obtain 0.95 g of the title compound.

结构表征数据如下：The structural characterization data are as follows:

1H 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). 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).

步骤二：N-(3-溴-4-氯-5-氟苯基)乙酰胺的合成(1-5-03)Step 2: Synthesis of N-(3-bromo-4-chloro-5-fluorophenyl)acetamide (1-5-03)

将化合物1-5-02(0.95g，4.23mmol)溶于乙酸乙酯(20mL)中，氮气保护下加入乙酸酐(648.13mg，6.35mmol)，加毕，升温至50℃反应15小时，用高效液相质谱联用色谱检测反应。反应液用甲醇(5mL)淬灭后，直接经减压蒸干得粗品，粗品经快速硅胶柱纯化(乙酸乙酯:石油醚＝0-40％)得标题化合物1.01g。Compound 1-5-02 (0.95 g, 4.23 mmol) was dissolved in ethyl acetate (20 mL), and acetic anhydride (648.13 mg, 6.35 mmol) was added under nitrogen protection. After the addition was completed, the temperature was raised to 50°C for 15 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was quenched with methanol (5 mL), it was directly evaporated to dryness under reduced pressure to obtain a crude product, which was purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-40%) to obtain 1.01 g of the title compound.

结构表征数据如下：The structural characterization data are as follows:

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

步骤三：(E)-4-(5-乙酰胺基-2-氯-3-氟苯基)-3-丁烯酸的合成(1-5-04)Step 3: Synthesis of (E)-4-(5-acetamido-2-chloro-3-fluorophenyl)-3-butenoic acid (1-5-04)

将化合物1-5-03和3-丁烯酸(387.65mg，4.50mmol)溶于1,4二氧六环(24mL)和水(8mL)的混合溶剂中，然后加入N,N-二异丙基乙胺(1.45g，11.26mmol)，三(邻甲基苯基)磷(114.21mg，375.24μmol)和醋酸钯(42.12mg，187.62μmol)，加毕，反应体系用氮气置换三次，并在氮气氛围下升温至100℃反应16小时，用高效液相质谱联用色谱检测反应。反应液冷却至室温后，加入1N的氢氧化钠水溶液(60mL)和乙酸乙酯(50mL)振荡分层。分出下层水相后，用4mol/L盐酸水溶液调节pH至3左右，然后用乙酸乙酯萃取，合并有机相用饱和食盐水洗涤，无水硫酸钠干燥，过滤，滤液经减压蒸干，得到标题化合物的粗品1.00g。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), and then N,N-diisopropylethylamine (1.45 g, 11.26 mmol), tri(o-methylphenyl)phosphine (114.21 mg, 375.24 μmol) and palladium acetate (42.12 mg, 187.62 μmol) were added. After the addition, the reaction system was replaced with nitrogen three times, and the temperature was raised to 100 ° C under a nitrogen atmosphere for 16 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was cooled to room temperature, 1N sodium hydroxide aqueous solution (60 mL) and ethyl acetate (50 mL) were added to shake and layer. After separating the lower aqueous phase, adjust the pH to about 3 with 4 mol/L hydrochloric acid aqueous solution, then extract with ethyl acetate, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and evaporate the filtrate to dryness under reduced pressure to obtain 1.00 g of a crude product of the title compound.

结构表征数据如下：The structural characterization data are as follows:

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

步骤四：4-(5-乙酰胺基-2-氯-3-氟苯基)丁酸的合成(1-5-05)Step 4: Synthesis of 4-(5-acetamido-2-chloro-3-fluorophenyl)butyric acid (1-5-05)

将化合物1-5-04的粗品(1.00g，3.68mmol)溶于四氢呋喃(15mL)，然后加入10％钯碳(0.10g)，加毕，然后用氢气球置换反应体系三次，并在氢气氛围下反应4小时，用高效液相质谱联用色谱检测反应。将反应液过滤，滤液减压浓缩干，得标题化合物的粗品1.00g。The crude product of compound 1-5-04 (1.00 g, 3.68 mmol) was dissolved in tetrahydrofuran (15 mL), and then 10% palladium carbon (0.10 g) was added. After the addition was complete, the reaction system was replaced three times with a hydrogen balloon, and the reaction was carried out under a hydrogen atmosphere for 4 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain 1.00 g of the crude product of the title compound.

结构表征数据如下：The structural characterization data are as follows:

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

步骤五：N-(4-氯-3-氟-8-氧代-5,6,7,8-四氢萘-1-基)乙酰胺的合成(1-5-06)Step 5: Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-06)

将化合物1-5-05的粗品(1.00g，3.65mmol)溶于三氟乙酸(5mL)中，降温至5℃后，缓慢加入三氟乙酸酐(3.84g，18.27mmol，2.54mL)，加毕，保持5℃反应2小时，用高效液相质谱联用色谱检测反应。反应液缓慢倒入水中，然后用乙酸乙酯萃取，合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥有机相，然后过滤，滤液经减压蒸干得粗品，粗品经快速硅胶柱纯化，得标题化合物0.43g。The crude product of compound 1-5-05 (1.00 g, 3.65 mmol) was dissolved in trifluoroacetic acid (5 mL), cooled to 5 ° C, and trifluoroacetic anhydride (3.84 g, 18.27 mmol, 2.54 mL) was slowly added. After the addition was completed, the reaction was maintained at 5 ° C for 2 hours, and 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 rapid silica gel column to obtain 0.43 g of the title compound.

结构表征数据如下：The structural characterization data are as follows:

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

步骤六：N-(4-氯-3-氟-7-(羟基亚氨基)-8-氧代-5,6,7,8-四氢萘-1-基)乙酰胺的合成(1-5-07)Step 6: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-07)

将四氢呋喃(16mL)和叔丁醇(4mL)加入反应瓶中，冰浴降温至5℃后，加入叔丁醇钾(415.18mg，3.70mmol)，然后将化合物1-5-06(0.43mg，1.68mmol)溶于四氢呋喃(1mL)中，并缓慢滴加入反应液，10分钟后再加入亚硝酸异戊酯(315.24mg，2.69mmol)，加毕，保持5℃反应1小时，用高效液相质谱联用色谱检测反应。反应液用饱和氯化铵水溶液淬灭后，用乙酸乙酯萃取，合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥有机相，然后过滤，滤液经减压浓缩，得标题化合物的粗品455.00mg。Tetrahydrofuran (16 mL) and tert-butanol (4 mL) were added to the reaction bottle, cooled to 5 ° C in an ice bath, and potassium tert-butoxide (415.18 mg, 3.70 mmol) was added, and then 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 completed, the reaction was maintained at 5 ° C for 1 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was quenched with saturated aqueous ammonium chloride solution, it was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, the organic phases were dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to obtain 455.00 mg of the crude product of the title compound.

结构表征数据如下：The structural characterization data are as follows:

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

步骤七：N-(7-氨基-4-氯-3-氟-8-氧代-5,6,7,8-四氢萘-1-基)乙酰胺的合成(1-5-08)Step 7: Synthesis of N-(7-amino-4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-08)

将化合物1-5-07的粗品(0.40g，1.41mmol)溶于甲醇(10mL)中，然后加入3mol/L的盐酸水溶液(1mL)和10％钯碳(40.00mg)，加毕，用氢气置换反应体系三次，并在氢气氛围下室温反应1小时，用高效液相质谱联用色谱检测反应。将反应液过滤，滤液经减压浓缩干，得标题化合物的盐酸盐粗品0.43g。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 carbon (40.00 mg) were added. After the addition, the reaction system was replaced with hydrogen three times, and the reaction was carried out at room temperature for 1 hour under a hydrogen atmosphere. 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 of the title compound.

结构表征数据如下：The structural characterization data are as follows:

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

步骤八：(9H-芴-9-基甲基)(8-乙酰胺-5-氯-6-氟-1-氧代-1,2,3,4-四氢萘-2-基)氨基甲酸酯的合成(1-5-09)Step 8: Synthesis of (9H-fluoren-9-ylmethyl)(8-acetamide-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (1-5-09)

将化合物1-5-08的盐酸盐粗品(0.43g，1.19mmol)溶于1,4-二氧六环(15mL)中，然后加入碳酸氢钠(400.35mg，4.77mmol)、水(5mL)和9-芴甲基-N-琥珀酰亚胺基碳酸酯(481.81mg，1.43mmol)，加毕，室温下搅拌反应2小时，用高效液相质谱联用色谱检测反应。将反应液倒入水中，然后用乙酸乙酯萃取，合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥有机相，过滤，滤液经减压浓缩得粗品。粗品经C18反相柱纯化(乙腈：0.05％甲酸水＝20％-100％)，得标题化合物301.00mg。The crude hydrochloride of compound 1-5-08 (0.43 g, 1.19 mmol) was dissolved in 1,4-dioxane (15 mL), and then sodium bicarbonate (400.35 mg, 4.77 mmol), water (5 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (481.81 mg, 1.43 mmol) were added. After the addition was completed, the reaction was stirred at room temperature for 2 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was poured into water, and then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by C18 reverse phase column (acetonitrile: 0.05% formic acid water = 20%-100%) to obtain 301.00 mg of the title compound.

结构表征数据如下：The structural characterization data are as follows:

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

步骤九：(9H-芴-9-基甲基)(8-氨基-5-氯-6-氟-1-氧代-1,2,3,4-四氢萘-2-基)氨基甲酸酯的合成(1-5-10)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)

将化合物1-5-09(300.00mg，608.61μmol)溶于二氧六环(5mL)中，加入12mol/L的浓盐酸(1mL)，加毕，升温至60℃反应2小时，用高效液相质谱联用色谱检测反应。将反应液倒入水中，然后用乙酸乙酯萃取，合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥有机相，过滤，滤液经减压浓缩得粗品。粗品经快速硅胶柱纯化(乙酸乙酯:石油醚＝0-50％)，得标题化合物198.00mg。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 the addition, the temperature was raised to 60°C for 2 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was poured into water, and then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-50%) to obtain 198.00 mg of the title compound.

结构表征数据如下：The structural characterization data are as follows:

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

步骤十：(9H-芴-9-基甲基)((9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧基-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4:6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基甲酸酯的合成(1-5-11)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)

将(S)-4-乙基-4-羟基-7,8-二氢-1H-吡喃并[3,4-f]吲哚嗪-3,6,10(4H)-三酮(138.72mg，526.96μmol)和化合物1-5-10(198.00mg，439.13μmol)加入甲苯(10mL)中，然后再加入对甲苯磺酸(75.53mg，439.13μmol)，加毕，升温至140℃反应4小时，反应液直接140℃下减压蒸干得粗品，粗品经快速硅胶柱纯化(甲醇:二氯甲烷＝0-5％)，得标题化合物256.00mg。(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 reacted for 4 hours. The reaction solution was directly evaporated to dryness under reduced pressure at 140 ° C to obtain a crude product. The crude product was purified by rapid silica gel column (methanol: dichloromethane = 0-5%) to obtain 256.00 mg of the title compound.

结构表征数据如下：The structural characterization data are as follows:

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

步骤十一：(1S,9S)-1-氨基-4-氯-9-乙基-5-氟-9-羟基-1,2,3,9,12,15-六氢-10H,13H-苯并[de]吡喃并[3',4'：6,7]吲哚嗪并[1,2-b]喹啉-10,13-二酮和(1R,9S)-1-氨基-4-氯-9-乙基-5-氟-9-羟基-1,2,3,9,12,15-六氢-10H,13H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-10,13-二酮的合成(1-5-A和1-5-B)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)

将化合物1-5-11(201.18mg，296.67μmol)溶于N,N-二甲基甲酰胺(4mL)中，然后加入二乙胺(108.49mg，1.48mmol)，加毕，室温反应0.5小时，用高效液相质谱联用色谱检测反应。反应液经减压蒸出乙二胺后，用1mol/L盐酸水溶液调pH至2-3后，反应液直接用制备高效液相色谱纯化，得标题化合物1-5-A(44.00mg)，1-5-B(43.00mg)。Compound 1-5-11 (201.18 mg, 296.67 μmol) was dissolved in N,N-dimethylformamide (4 mL), and then diethylamine (108.49 mg, 1.48 mmol) was added. After the addition was completed, the mixture was reacted at room temperature for 0.5 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the ethylenediamine was evaporated under reduced pressure, the pH of the reaction solution was adjusted to 2-3 with 1 mol/L hydrochloric acid aqueous solution, and the reaction solution was directly purified by preparative high performance liquid chromatography to obtain the title compounds 1-5-A (44.00 mg) and 1-5-B (43.00 mg).

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

1-5-A(6min LCMS出峰靠前，保留时间：1.276min)1-5-A (6min LCMS peak at the front, retention time: 1.276min)

结构表征数据如下：The structural characterization data are as follows:

1H 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＝19.3Hz,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). 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).

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

1-5-B(6min LCMS出峰靠后，保留时间：1.300min)结构表征数据如下：The structural characterization data of 1-5-B (6min LCMS peak later, retention time: 1.300min) are as follows:

1H 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). 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).

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

6min LCMS条件：6min LCMS conditions:

色谱柱：Waters SunFire C18 OBD 4.6mm×50mm×5.0μmColumn: Waters SunFire C18 OBD 4.6mm×50mm×5.0μm

流动相A：0.05％乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid)

步骤十二：N-((S)-10-苄基-1-(((1S,9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六烷-16-基)-6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺和N-((S)-10-苄基-1-((1R,9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六烷-16-基)-6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺的合成(1-5-12-A和1-5-12-B)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-tetraazahexadecane-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)

将单一构型的化合物1-5-A(36.00mg，79.70μmol)和化合物A-07-3(64.43mg，95.64μmol)溶于N,N-二甲基甲酰胺(2mL)中，然后加入4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(46.98mg，159.40μmol)和三乙胺(24.19mg，239.10μmol)，加毕，室温反应1小时，用高效液相质谱联用色谱检测反应。反应液直接经高效液相色谱纯化，得到单一构型的标题化合物1-5-12-A(51.00mg)。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), and then 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added. After addition, the mixture was reacted at room temperature for 1 hour, and the reaction was detected by HPLC-MS. The reaction solution was directly purified by HPLC to obtain the single-configuration title compound 1-5-12-A (51.00 mg).

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

结构表征数据如下：The structural characterization data are as follows:

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

将单一构型的化合物1-5-B(36.00mg，79.70μmol)和化合物A-07-3(64.43mg，95.64μmol)溶于N,N-二甲基甲酰胺(2mL)中，然后加入4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(46.98mg，159.40μmol)和三乙胺(24.19mg，239.10μmol)，加毕，室温反应1小时，用高效液相质谱联用色谱检测反应。反应液直接经高效液相色谱纯化，得到单一构型的标题化合物1-5-12-B(52.00mg)。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), and then 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added. After addition, the mixture was reacted at room temperature for 1 hour, and the reaction was detected by HPLC-MS. The reaction solution was directly purified by HPLC to obtain the single-configuration title compound 1-5-12-B (52.00 mg).

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

结构表征数据如下：The structural characterization data are as follows:

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

步骤十三：N-((1S,9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4:6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺和N-((1R,9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4:6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺的合成(1-11-A和1-11-B)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)

称取化合物1-5-12-A(40.00mg，35.99μmol)溶于二氯甲烷(2mL)和甲醇(1mL)的混合溶剂中，然后加入4mol/L盐酸的乙酸乙酯溶液(1mL)，加毕，室温反应0.5小时，用高效液相质谱联用色谱检测反应。反应液直接经减压浓缩干得粗品，粗品经高效液相色谱纯化，得单一构型的标题化合物1-11-A(4.75mg)。Weigh compound 1-5-12-A (40.00 mg, 35.99 μmol) and dissolve it in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL), then add 4 mol/L hydrochloric acid in ethyl acetate (1 mL), react at room temperature for 0.5 hours, and detect the reaction by HPLC-MS/MS. The reaction solution is directly concentrated under reduced pressure to obtain a crude product, which is purified by HPLC to obtain the title compound 1-11-A (4.75 mg) of a single configuration.

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

结构表征数据如下：The structural characterization data are as follows:

1H 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). 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).

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

称取化合物1-5-12-B(40.00mg，35.99μmol)溶于二氯甲烷(2mL)和甲醇(1mL)的混合溶剂中，然后加入4mol/L盐酸乙酸乙酯(1mL)，加毕，室温反应0.5小时，用高效液相质谱联用色谱检测反应。反应液直接经减压浓缩干得粗品，粗品经高效液相色谱纯化，得单一构型的标题化合物1-11-B(8.24mg)。Weigh compound 1-5-12-B (40.00 mg, 35.99 μmol) and dissolve it in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL), then add 4 mol/L hydrochloric acid ethyl acetate (1 mL), react at room temperature for 0.5 hours, and detect the reaction by HPLC-MS/MS chromatography. The reaction solution is directly concentrated under reduced pressure to obtain a crude product, which is purified by HPLC to obtain the title compound 1-11-B (8.24 mg) of a single configuration.

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

结构表征数据如下：The structural characterization data are as follows:

1H 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). 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).

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

实施例八 N-((10S,19S)-23-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)-10-苄基-1-(((1R,9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4:6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15,18-六氧代-3-氧杂-5,8,11,14,17-五氮杂二十三烷-19-基)-2,5,8,11,14,17,20,23,26,29,32,35-十二氧杂三十八烷-38-酰胺或N-((10S,19S)-23-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)-10-苄基-1-(((1S,9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4:6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15,18-六氧代-3-氧杂-5,8,11,14,17-五氮杂二十三烷-19-基)-2,5,8,11,14,17,20,23,26,29,32,35-十二氧杂三十八烷-38-酰胺(A-17-A)
Example 8 N-((10S,19S)-23-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamide)-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-dodecatrioctadecane-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]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 (A-17-A)

步骤一：(2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-十二氧杂三十八烷-38-酰胺基)-6-(N-(9H-芴-9-基甲氧基羰基)氨基)己酸的合成(A-17-03)Step 1: Synthesis of (2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38-octadecane-38-amido)-6-(N-(9H-fluoren-9-ylmethoxycarbonyl)amino)hexanoic acid (A-17-03)

将化合物A-17-02的盐酸盐(389.68mg，962.45μmol)溶于二氯甲烷(8mL)中，加入DIPEA(518.28mg，4.01mmol，713.88μL)和化合物A-17-01(550.00mg，802.04μmol)，25℃反应1.5小时。用稀盐酸调节反应液pH至中性，减压抽干溶剂。浓缩物用制备高效液相色谱纯化，得标题化合物A-17-03(450.00mg)。The hydrochloride of compound A-17-02 (389.68 mg, 962.45 μmol) was dissolved in dichloromethane (8 mL), and DIPEA (518.28 mg, 4.01 mmol, 713.88 μL) and compound A-17-01 (550.00 mg, 802.04 μmol) were added, and the mixture was 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 drained under reduced pressure. The concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-03 (450.00 mg).

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

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

步骤二：2-((2-((2S)-2-(2-(2-((2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-十二氧杂三十八烷-38-酰胺基)-6-(N-(9H-芴-9-基甲氧基羰基)氨基)己酰胺基)乙酰胺基)乙酰胺基)-3-苯基丙酰胺基)乙酰胺基)甲氧基)乙酸的合成(A-17-04)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)

将化合物A-17-03(50.00mg，118.09μmol)用DMF(2.5mL)溶解，加入HATU(49.39mg，129.89μmol)、化合物A-07-2(133.07mg，141.70μmol)和DIPEA(45.78mg，354.26μmol，63.06μL)，25℃反应1小时。减压抽干溶剂，浓缩物用制备高效液相色谱纯化，得标题化合物A-17-04(40.00mg)。Compound A-17-03 (50.00 mg, 118.09 μmol) was dissolved in DMF (2.5 mL), HATU (49.39 mg, 129.89 μmol), compound A-07-2 (133.07 mg, 141.70 μmol) and DIPEA (45.78 mg, 354.26 μmol, 63.06 μL) were added, and the mixture was reacted at 25° C. for 1 hour. The solvent was removed under reduced pressure, and the concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-04 (40.00 mg).

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

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

步骤三：(9H-芴-9-基甲基)((40S)-40-(((10S)-10-苄基-1-(((9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧基-3-氧杂-5,8,11,14-四氮杂十六烷-16-基)氨甲酰基)-38-氧代-2,5,8,11,14,17,20,23,26,29,35-十二氧杂-39-氮杂-44-基)氨基甲酸酯的合成(A-17-05)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 (a-17-05) 1,6,9,12,15-pentaoxy-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)carbamoyl)-38-oxo-2,5,8,11,14,17,20,23,26,29,35-dodeca-39-aza-44-yl)carbamate

将化合物A-17-04(29.86mg，59.50μmol)用DMF(3mL)溶解，加入HATU(27.15mg，71.40μmol)、化合物1-5-A(80.00mg，59.50μmol)和DIPEA(38.45mg，297.51μmol，52.96μL)，25℃反应1小时。减压抽干溶剂，浓缩物用制备高效液相色谱纯化，得标题化合物A-17-05(50.00mg)。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, and the mixture was reacted at 25° C. for 1 hour. The solvent was removed under reduced pressure, and the concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-05 (50.00 mg).

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

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

步骤四：N-((10S,19S)-23-氨基-10-苄基-1-(((9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4:6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15,18-六氧代-3-氧杂-5,8,11,14,17-五氮杂二十三烷-19-基)-2,5,8,11,14,17,20,23,26,29,32,35-十二氧杂三十八烷-38-酰胺的合成(A-17-06)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)

将化合物A-17-05(20.00mg，11.22μmol)用DMF(2.5mL)和二乙胺(0.5mL)溶解，25℃反应2小时。减压抽干溶剂，浓缩物用制备高效液相色谱纯化，得标题化合物A-17-06的甲酸盐(10.00mg)。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 removed under reduced pressure and the concentrate was purified by preparative HPLC to obtain the formate salt of the title compound A-17-06 (10.00 mg).

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

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

步骤五：N-((10S,19S)-23-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)-10-苄基-1-(((1R,9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4:6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15,18-六氧代-3-氧杂-5,8,11,14,17-五氮杂二十三烷-19-基)-2,5,8,11,14,17,20,23,26,29,32,35-十二氧杂三十八烷-38-酰胺或N-((10S,19S)-23-(6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)-10-苄基-1-(((1S,9S)-4-氯-9-乙基-5-氟-9-羟基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4:6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15,18-六氧代-3-氧杂-5,8,11,14,17-五氮杂二十三烷-19-基)-2,5,8,11,14,17,20,23,26,29,32,35-十二氧杂三十八烷-38-酰胺的合成(A-17-A)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-ynylamide)-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-dodecaoctatridecane-38-amide (A-17-A)

将化合物A-17-06的甲酸盐(7.00mg，4.49μmol)和化合物A-07-1(3.28mg，8.97μmol)用DMF(1mL)溶解，加入DIPEA(1.74mg，13.46μmol，2.40μL)，25℃反应2小时。减压抽干溶剂，浓缩物用制备高效液相色谱纯化，得标题化合物A-17-A(5.60mg)。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, and the mixture was reacted at 25° C. for 2 hours. The solvent was removed under reduced pressure, and the concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-A (5.60 mg).

色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μmColumn: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

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

实施例九 N-((S)-10-苄基-1-(((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H，12H-苯并[de]吡喃[3',4':6,7]吲哚利嗪[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧基-5,8,11,14-四氮杂十六烷-16-基)-6-(2-(甲基磺基)嘧啶-5-基)己-5-炔酰胺(A-05)
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]indolizine[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxyl-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-05)

氮气保护下，将2,5-二氧吡咯烷-1-基-6-(2-(甲磺酰基)嘧啶-5-基)己基-5-炔酸酯(A-07-1,0.66g,1.80mmol)和A-07-2(0.75g,1.77mmol)加入DMF(19mL)中，升温至35℃反应16小时，向该体系中加入(1S,9S)-1-氨基-5-氯-9-乙基-9-羟基-4-甲基-1,2,3,9,12,15-六氢-10H,13H-苯并[de]吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-10,13-二酮(1-4(即1-2-A),1.00g,1.77mmol)，冰水降温至5～15℃，加入DMTMM(0.98g,3.53mmol)，再滴入DIPEA(1.14g,8.84mmol)，25℃反应16小时。将反应液倒入DCM(600mL)、IPA(60mL)、水(100mL)混合液中搅拌10分钟，分出DCM相，盐水(100ml)洗，浓缩得到粗品，用制备高效液相色谱纯化后冷冻干燥得A-05(即为实施例三的A-07-A)化合物0.98g。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), cooled to 5-15°C with ice water, added DMTMM (0.98 g, 3.53 mmol), then added dropwise DIPEA (1.14 g, 8.84 mmol), reacted at 25°C for 16 hours. The reaction solution was poured into a mixture of DCM (600 mL), IPA (60 mL), and water (100 mL) and stirred for 10 minutes, the DCM phase was separated, washed with brine (100 ml), and concentrated to obtain a crude product, which was purified by preparative high performance liquid chromatography and freeze-dried to obtain 0.98 g of compound A-05 (i.e. A-07-A in Example 3).

A-05分离纯化方法如下：A-05 separation and purification method is as follows:

色谱柱：Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A：乙腈；流动相B：水(0.05％甲酸)

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

A-05结构表征数据如下：A-05 structural characterization data are as follows:

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

1H 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,2H),8.08-8.04(m,1H),7.30(s,1H),7.26-7.15(m,5H),6.55(s,1H),5.56-5.55(m,1H),5.48-5.35(m,2H),5.25-5.10(m,2H),4.64(d,J＝6.4Hz,2H),4.45-4.44(m,1H),4.06-3.98(m,2H),3.77-3.52(m,6H),3.41(s,3H),3.25-3.12(m,2H),3.03-3.00(m,1H),2.83-2.72(m,1H),2.58-2.56(m,2H),2.48(s,3H),2.33-2.30(m,2H),2.21-2.13(m,2H),1.91-1.76(m,4H),0.87(t,J＝7.2Hz,3H). 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).

采用以下HPLC条件检测实施例三制备的化合物A-07-A和A-07-B；以及实施例三制备的化合物A-07-A和实施例九制备的化合物A-05：The following HPLC conditions were used to detect the compounds A-07-A and A-07-B prepared in Example 3; as well as the compound A-07-A prepared in Example 3 and the compound A-05 prepared in Example 9:

仪器：Agilent 1260高效液相色谱VWD检测器Instrument: Agilent 1260 HPLC with VWD detector

色谱柱：Waters Xbridge C18 4.6*100mm*3.5μmChromatographic column: Waters Xbridge C18 4.6*100mm*3.5μm

流动相A：0.01M磷酸铵水溶液/乙腈＝90/10；流动相B：乙腈
Mobile phase A: 0.01M ammonium phosphate aqueous solution/acetonitrile = 90/10; Mobile phase B: acetonitrile

结果所示，化合物A-07-A以及化合物A-07-B的保留时间分别为6.6min以及6.8min，说明上述HPLC条件对异构体具有较好的分离度；The results show that the retention times of compound A-07-A and compound A-07-B are 6.6 min and 6.8 min, respectively, indicating that the above HPLC conditions have good separation of isomers;

结果所示，实施例三制备的化合物A-07-A以及实施例九制备的化合物A-05在HPLC中显示为一个峰，保留时间为6.6min，说明化合物A-07-A即为化合物A-05。The results show that compound A-07-A prepared in Example 3 and compound A-05 prepared in Example 9 show one peak in HPLC with a retention time of 6.6 min, indicating that compound A-07-A is compound A-05.

实施例十 N-((7S,10S,13S)-1-(((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-1-基)氨基)-7,10-二甲基-1,6,9,12-四氧-3-氧-5,8,11-三氮十四烷-13-基)-6-(2-(甲基磺基)嘧啶-5-基)己-5-酰胺(A-26)
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]indolizine[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxo-5,8,11-triazatetradecane-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-amide (A-26)

步骤一：Step 1:

将化合物A-26-1(657mg,1.22mmol)和化合物1-4(500mg,1.11mmol)溶于N,N-二甲基甲酰胺(10mL)中，随后加入HATU(630.67mg,1.66mmol)和N,N-二异丙基乙胺(428mg,3.32mmol)，室温搅拌1小时。反应完毕后，反应液直接用制备高效液相色谱纯化后冷冻干燥得A-26-2化合物700mg。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), followed by the addition of HATU (630.67 mg, 1.66 mmol) and N, N-diisopropylethylamine (428 mg, 3.32 mmol), and stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 700 mg of compound A-26-2.

其制备方法如下：The preparation method is as follows:

色谱柱：Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

步骤二：Step 2:

将化合物A-26-2(500mg,0.513mmol)溶于N,N-二甲基甲酰胺(2mL)中，加入二乙胺(75.05mg,1.03mmol)，室温反应1小时。反应结束后，反应液直接用制备高效液相色谱纯化后冷冻干燥得A-26-3化合物307mg。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, and 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.

其制备方法如下：The preparation method is as follows:

色谱柱：Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

步骤三： Step 3:

将A-26-3(170mg,0.226mmol)和化合物A-07-1(90.83mg,0.249mmol)、溶解在N,N-二甲基甲酰胺(10mL)，加入N,N-二异丙基乙胺(29.21mg,0.226mmol)。反应液在室温条件下搅拌16小时。反应液直接用制备高效液相色谱纯化后冷冻干燥得A-26化合物50.56mg。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 solution was stirred at room temperature for 16 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 50.56 mg of compound A-26.

其结构表征数据如下：Its structural characterization data are as follows:

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

其分离纯化方法如下：The separation and purification method is as follows:

色谱柱：Waters SunFire Prep C18 OBD(5μm*19mm*150mm)Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

流动相A：乙腈；流动相B：水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

1H NMR(400MHz,DMSO)δ9.11(s,2H),8.68(t,J＝6.4Hz,1H),8.49(d,J＝8.8Hz,1H),8.16(s,1H),8.10(d,J＝7.2Hz,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),2.57-2.56(m,2H),2.35-2.27(m,2H),2.22-2.12(m,2H),1.91-1.75(m,4H),1.23-1.09(m,9H),0.87(t,J＝7.2Hz,3H). 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).

实施例十一 4-((S)-2-(4-氨基丁基)-35-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己基-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-4,8-二氧代-6,12,15,18,24,27,30,33-壬氧基-3,9-二氮杂五氮杂三酰氨基)苄基((1S,9R)-5-氯-9-乙基-1-(2-羟基乙酰胺基)-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-9-基)碳酸酯(B-04)
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)

步骤一:2-(((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-1-基)氨基)-2-氧代乙酸乙酯(B-04-1)的制备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)

将(1S,9S)-1-氨基-5-氯-9-乙基-9-羟基-4-甲基-1,2,3,9,12,15-六氢-10H,13H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-10,13-二酮(2g,3.65mmol)溶于DMF(50mL),滴入DIPEA(1.18g,9.12mmol,1.59mL),冰浴冷却搅拌下滴入乙酰氧基乙酰氯(548.12mg,4.01mmol,431.59μL)，继续搅拌反应1小时。将反应液加入0.1M稀盐酸水溶液中析出固体，过滤。将滤饼溶于二氯甲烷和甲醇中，无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(甲醇/二氯甲烷＝0％～5％)再次浓缩得标题化合物(1.7g，3.077mmol)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]indolizine[1,2-b]quinoline-10,13-dione (2g, 3.65mmol) in DMF (50mL), add DIPEA (1.18g, 9.12mmol, 1.59mL), add acetoxyacetyl chloride (548.12mg, 4.01mmol, 431.59μL) under ice-cooling and stirring, and continue stirring and reacting for 1 hour. Add the reaction solution into 0.1M dilute hydrochloric acid aqueous solution to precipitate solids, and filter. 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 (methanol/dichloromethane = 0% to 5%) and concentrated again to obtain the title compound (1.7 g, 3.077 mmol)

其结构表征数据如下：Its structural characterization data are as follows:

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

步骤二:2-(((1S,9S)-9-(((4-((S)-35-叠氮基-2-(4-(4-甲氧基苯基)二苯基甲基)氨基)丁基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-壬氧基-3,9-二氮杂五苯并三酰氨基)苄基)氧基)羰基)氧基-5-氯-9-乙基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲唑嗪[1,2-b]喹啉-1-基)氨基)-2-氧代乙酸乙酯(B-04-2)的制备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)

将2-(((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-1-基)氨基)-2-氧代乙酸乙酯(500mg,0.905mmol)和DMAP(885.33mg,7.25mmol)溶于干燥二氯甲烷(5mL)中，氮气保护下冷却至0℃，滴入三光气(268.81mg,0.905mmol)的二氯甲烷溶液(5mL)，保温搅拌反应0.5小时。缓慢滴入(S)-2-(32-叠氮基-5-氧代-3,9,12,15,18,21,24,27,30-壬氧基-6-氮杂三硝基氨基)-N-(4-(羟甲基)苯基)-6-(((4-甲氧基苯基)二苯基甲基)氨基)己酰胺(1.44g,1.36mmol)的二氯甲烷溶液，自然恢复室温反应4小时。加水淬灭反应，用二氯甲烷萃取3次(100ml x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)得标题化合物(498mg，0.304mmol)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 mixture was stirred and reacted for 0.5 hour. Slowly drop a dichloromethane solution of (S)-2-(32-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) and allow to react at room temperature for 4 hours. Add water to quench the reaction, extract with dichloromethane 3 times (100 ml x 3), combine the organic phases, wash with saturated brine, and then dry and concentrate. Purify on a silica gel column (MeOH/DCM = 0% to 5%) to obtain the title compound (498 mg, 0.304 mmol)

其结构表征数据如下：Its structural characterization data are as follows:

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

步骤三:4-((S)-35-叠氮基-2-(4-((4-甲氧基苯基)二苯甲基)氨基)丁基)-4,8-二氧代-6,12,15,18,24,27,30,33-壬氧基-3,9-二氮杂五氮杂三酰氨基)苄基((1S,9S)-5-氯-9-乙基-1-(2-羟基乙酰胺基)-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]氮茚并[1,2-b]喹啉-9-基)碳酸酯(B-04-3)的制备Step 3: Preparation of 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 (B-04-3)

将2-(((1S,9S)-9-(((4-((S)-35-叠氮基-2-(4-(4-甲氧基苯基)二苯基甲基)氨基)丁基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-壬氧基-3,9-二氮杂五苯并三酰氨基)苄基)氧基)羰基)氧基-5-氯-9-乙基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲唑嗪[1,2-b]喹啉-1-基)氨基)-2-氧代乙酸乙酯(200mg,0.122mmol)溶于THF(3mL)和MeOH(3mL)中，搅拌下滴入碳酸钠(25.88mg,0.224mmol)水溶液(1mL)，滴加完毕后继续搅拌1小时。向反应液中滴入稀盐酸中和反应，减压浓缩后直接进行下一步。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 0,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidin[1,2-b]quinolin-1-yl)amino)-2-oxoacetic acid ethyl ester (200mg, 0.122mmol) was dissolved in THF (3mL) and MeOH (3mL), and sodium carbonate (25.88mg, 0.224mmol) aqueous solution (1mL) was added dropwise under stirring. After the addition was completed, stirring was continued for 1 hour. Dilute hydrochloric acid was added dropwise to the reaction solution to neutralize the reaction, and the next step was directly carried out after reduced pressure concentration.

其结构表征数据如下：Its structural characterization data are as follows:

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

步骤四:4-((S)-2-(4-氨基丁基)-35-叠氮基-4,8-二氧代-6,12,15,18,21,24,27,30,33-壬氧基-3,9-二氮杂五苯并三酰氨基)苄基((1S,9S)-5-氯-9-乙基-1-(2-羟基乙酰胺基)-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-9-基)碳酸酯(B-04-4)的制备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)

将4-((S)-35-叠氮基-2-(4-((4-甲氧基苯基)二苯甲基)氨基)丁基)-4,8-二氧代-6,12,15,18,24,27,30,33-壬氧基-3,9-二氮杂五氮杂三酰氨基)苄基((1S,9S)-5-氯-9-乙基-1-(2-羟基乙酰胺基)-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]氮茚并[1,2-b]喹啉-9-基)碳酸酯(190mg,119.04μmol)溶于二氯甲烷(5mL)，加入三氟乙酸(0.5mL)后继续反应1小时。向反应液中滴入饱和碳酸氢钠水溶液中和后分液，将有机相浓缩得粗品。经反相柱色谱(乙腈/1％甲酸水溶液＝0％～50％)纯化后冷冻干燥得标题化合物(95mg,69.35μmol)。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 then separated, and the organic phase was concentrated to obtain a crude product. The crude product 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).

其结构表征数据如下：Its structural characterization data are as follows:

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

步骤五:4-((S)-2-(4-氨基丁基)-35-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己基-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-4,8-二氧代-6,12,15,18,24,27,30,33-壬氧基-3,9-二氮杂五氮杂三酰氨基)苄基((1S,9R)-5-氯-9-乙基-1-(2-羟基乙酰胺基)-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-9-基)碳酸酯(B-04)的制备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)

将4-((S)-2-(4-氨基丁基)-35-叠氮基-4,8-二氧代-6,12,15,18,21,24,27,30,33-壬氧基-3,9-二氮杂五苯并三酰氨基)苄基((1S,9S)-5-氯-9-乙基-1-(2-羟基乙酰胺基)-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-9-基)碳酸酯(90mg,0.066mmol)和6-(2-(甲基磺酰基)嘧啶-5-基)-N-(丙-2-炔-1-基)己-5-炔酰胺(24.07mg,0.079mmol)溶于DMSO(2mL)和水(0.2mL)中，加入溴化亚铜(9.42mg,0.066mmol)后继续搅拌2小时。将反应液直接过滤，将反应液直接过滤浓缩的粗品，经制备高效液相色谱纯化后冷冻干燥得标题化合物(42.2mg，24.69μmol)。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] indolizine [1,2-b] quinolin-9-yl) carbonate (90 mg, 0.066 mmol) and 6-(2-(methylsulfonyl) pyrimidin-5-yl)-N-(prop-2-yn-1-yl) hex-5-ynamide (24.07 mg, 0.079 mmol) were dissolved in DMSO (2 mL) and water (0.2 mL), and cuprous bromide (9.42 mg, 0.066 mmol) was added and stirred for 2 hours. The reaction solution was directly filtered, and the concentrated crude product was purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (42.2 mg, 24.69 μmol).

其结构表征数据如下：Its structural characterization data are as follows:

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

其制备高效液相色谱方法如下：The preparation high performance liquid chromatography method is as follows:

色谱柱:SunFire Prep C18 OBD 19mm×150mm×5.0μmChromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

流动相A:乙腈；流动相B:水(0.05％甲酸)
Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

实施例十二、抗体的制备Example 12. Preparation of Antibodies

前期通过免疫H2L2小鼠(由和铂医药提供，该小鼠产生的抗体是由全人源的可变区和鼠源的恒定区组成的嵌合抗体)，通过杂交瘤筛选获得人鼠嵌合抗体22B6D2(重链可变区，SEQ ID NO:15；轻链可变区，SEQ ID NO:16)、47A3E3(重链可变区，SEQ ID NO:32；轻链可变区，SEQ ID NO:33)和100H7D3(重链可变区，SEQ ID NO:46；轻链链可变区，SEQ ID NO:47)，将上述重链可变区序列分别与人IgG1重链恒定区(SEQ ID NO:50)组合、上述轻链可变区序列分别与人IgG1轻链恒定区(SEQ ID NO:51)组合，形成3个完整的全人源抗体(见表1)，并进行密码子优化后构建到pTT5质粒中，将每一个抗体重链和轻链对应的pTT5质粒同时转染到CHO-S细胞中，采用蛋白A对上清中的表达抗体进行纯化，从而获得相应抗体。In the early stage, by immunizing H2L2 mice (provided by Hebo Pharmaceuticals, the antibody produced by this mouse is a chimeric antibody composed of a fully human variable region and a mouse constant region), 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. , SEQ ID NO:47), 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), to form three complete fully human antibodies (see Table 1), and after codon optimization, they were constructed into the pTT5 plasmid, and 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.

HER3对照抗体来源专利申请CN200680049887中的U1-59，进行密码子优化后，抗体重、轻链核苷酸序列分别合成克隆到pTT5载体，按上述方法表达纯化。 The HER3 control antibody was derived from U1-59 in patent application CN200680049887. After codon optimization, the heavy and light chain nucleotide sequences of the antibody were synthesized and cloned into the pTT5 vector, and expressed and purified according to the above method.

表1：22B6D2-hIgG1、47A3E3-hIgG1、100H7D3-hIgG1的序列信息
Table 1: Sequence information of 22B6D2-hIgG1, 47A3E3-hIgG1, 100H7D3-hIgG1

实施例十三、包含细胞生物活性分子和连接体的化合物与抗体的偶联Example 13: Conjugation of a compound comprising a cell bioactive molecule and a linker with an antibody

以下实施例所制备得到的抗体-药物缀合物中所涉及的抗体22B6，47A3，100H7即分别为前述的22B6D2-hIgG1、47A3E3-hIgG1、100H7D3-hIgG1抗体。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.

样品的偶联制备如下：The sample coupling preparation is as follows:

分别取0.46ml 22B6，47A3，100H7，U1-59，hIgG1抗体(均调整浓度至11.0mg/mL)，用0.1M依地酸二钠的溶液(pH 7.7)稀释，然后用1M Na2HPO4溶液调pH至7.7，加入10mM TCEP(三(2-羧乙基)膦)溶液混匀，室温放置90min。向上述溶液体系加入4.0-10倍物质的量的溶解在二甲基亚砜的“药物-连接体”化合物，混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的10mM组氨酸缓冲溶液，然后添加蔗糖和吐温20，混匀，得到抗体-药物缀合物(即ADC)，见表2。0.46 ml of 22B6, 47A3, 100H7, U1-59, and hIgG1 antibodies (all adjusted to 11.0 mg/mL) were taken respectively, diluted with 0.1 M edetate disodium solution (pH 7.7), then adjusted to pH 7.7 with 1 M Na 2 HPO 4 solution, added with 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution, mixed, and left at room temperature for 90 min. 4.0-10 times the amount of the substance of the "drug-linker" compound dissolved in dimethyl sulfoxide was added to the above solution system, mixed, and left at room temperature for 2 h. After completion, the buffer was replaced with a 10 mM histidine buffer solution of pH 6.0 using a NAP-5 gel column (Cytiva), and then sucrose and Tween 20 were added and mixed to obtain an antibody-drug conjugate (i.e., ADC), as shown in Table 2.

ADC药物/抗体比值(DAR值)的测定如下：The ADC drug/antibody ratio (DAR value) is determined as follows:

LC-MS测定ADC样品分子量，计算药物/抗体比DAR值LC-MS determination of ADC sample molecular weight and calculation of drug/antibody ratio DAR value

对ADC样品进行LC-MS分子量分析。ADC samples were subjected to LC-MS molecular weight analysis.

色谱测定条件：Chromatographic conditions:

液相色谱柱：Thermo MAbPac RP 3.0*100mm； Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

流动相A：0.1％FA/H2O；流动相B：0.1％FA/ACN；Mobile phase A: 0.1% FA/H 2 O; Mobile phase B: 0.1% FA/ACN;

流速：0.25ml/min；样品室温度：8℃；柱温：60℃；进样量：2μl；
Flow rate: 0.25 ml/min; Sample chamber temperature: 8 °C; Column temperature: 60 °C; Injection volume: 2 μl;

质谱测定条件：Mass spectrometry conditions:

质谱型号：AB Sciex Triple TOF 5600+；Mass spectrometer model: AB Sciex Triple TOF 5600+;

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

m/z 600-4000；Time bins to sum 40。m/z 600-4000; Time bins to sum 40.

表2 ADC编号以及DAR

Table 2 ADC number and DAR

1.LC-MS测定ADC 22B6-A-07-A-1分子量，计算药物/抗体比DAR值。对ADC 22B6-A-07-A-1进行LC-MS分子量分析见表3。
1. LC-MS determination of the molecular weight of ADC 22B6-A-07-A-1 and calculation of the drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-A-07-A-1 is shown in Table 3.

表3：ADC 22B6-A-07-A-1实测分子量
Table 3: Measured molecular weight of ADC 22B6-A-07-A-1

表4：ADC 22B6-A-07-A-1的DAR值
Table 4: DAR values for ADC 22B6-A-07-A-1

计算得ADC 22B6-A-07-A-1的药物/抗体比值为DAR＝7.99见表4。The calculated drug/antibody ratio of ADC 22B6-A-07-A-1 is DAR=7.99 (see Table 4).

2.LC-MS测定ADC 22B6-A-07-A-2分子量，计算药物/抗体比DAR值。对ADC 22B6-A-07-A-2进行LC-MS分子量分析见表5。
2. LC-MS determination of the molecular weight of ADC 22B6-A-07-A-2 and calculation of the drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-A-07-A-2 is shown in Table 5.

表5：ADC 22B6-A-07-A-1实测分子量
Table 5: Measured molecular weight of ADC 22B6-A-07-A-1

表6：ADC 22B6-A-07-A-2的DAR值
Table 6: DAR values for ADC 22B6-A-07-A-2

计算得ADC 22B6-A-07-A-2的药物/抗体比值为DAR＝3.56见表6。The calculated drug/antibody ratio of ADC 22B6-A-07-A-2 is DAR=3.56 (see Table 6).

3.LC-MS测定ADC 47A3-A-07-A分子量，计算药物/抗体比DAR值。对ADC 47A3-A-07-A进行LC-MS分子量分析见表7。
3. LC-MS determination of the molecular weight of ADC 47A3-A-07-A and calculation of the drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 47A3-A-07-A is shown in Table 7.

表7：ADC 47A3-A-07-A实测分子量
Table 7: ADC 47A3-A-07-A measured molecular weight

表8：ADC 47A3-A-07-A的DAR值

Table 8: DAR values for ADC 47A3-A-07-A

计算得ADC 47A3-A-07-A的药物/抗体比值为DAR＝6.27见表8。The calculated drug/antibody ratio of ADC 47A3-A-07-A is DAR=6.27, see Table 8.

4.LC-MS测定ADC 100H7-A-07-A分子量，计算药物/抗体比DAR值。对ADC 100H7-A-07-A进行LC-MS分子量分析见表9。
4. LC-MS determination of the molecular weight of ADC 100H7-A-07-A and calculation of the drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 100H7-A-07-A is shown in Table 9.

表9：ADC 100H7-A-07-A实测分子量
Table 9: ADC 100H7-A-07-A measured molecular weight

表10：ADC 100H7-A-07-A的DAR值
Table 10: DAR values for ADC 100H7-A-07-A

计算得ADC 100H7-A-07-A的药物/抗体比值为DAR＝8.01见表10。The calculated drug/antibody ratio of ADC 100H7-A-07-A is DAR=8.01 (see Table 10).

5.LC-MS测定ADC U1-59-A-07-A分子量，计算药物/抗体比DAR值。对ADC U1-59-A-07-A进行LC-MS分子量分析见表11。
5. LC-MS determination of ADC U1-59-A-07-A molecular weight and calculation of drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC U1-59-A-07-A is shown in Table 11.

表11：ADC U1-59-A-07-A实测分子量
Table 11: ADC U1-59-A-07-A measured molecular weight

表12：ADC U1-59-A-07-A的DAR值
Table 12: DAR values for ADC U1-59-A-07-A

计算得ADC U1-59-A-07-A的药物/抗体比值为DAR＝8.02见表12。The calculated drug/antibody ratio of ADC U1-59-A-07-A is DAR=8.02, see Table 12.

6.LC-MS测定ADC hIgG1-A-07-A分子量，计算药物/抗体比DAR值。对ADC hIgG1-A-07-A进行LC-MS分子量分析见表13。
6. LC-MS determination of ADC hIgG1-A-07-A molecular weight and calculation of drug/antibody ratio DAR value The LC-MS molecular weight analysis of ADC hIgG1-A-07-A is shown in Table 13.

表13：ADC hIgG1-A-07-A实测分子量
Table 13: Measured molecular weight of ADC hIgG1-A-07-A

表14：ADC hIgG1-A-07-A的DAR值

Table 14: DAR values of ADC hIgG1-A-07-A

计算得ADC hIgG1-A-07-A(8)的药物/抗体比值为DAR＝8.03见表14。The drug/antibody ratio of ADC hIgG1-A-07-A(8) was calculated to be DAR=8.03 (see Table 14).

7.LC-MS测定ADC 22B6-A-01分子量，计算药物/抗体比DAR值。对ADC 22B6-A-01进行LC-MS分子量分析见表15。
7. LC-MS determination of ADC 22B6-A-01 molecular weight and calculation of drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-A-01 is shown in Table 15.

表15：ADC 22B6-A-01实测分子量
Table 15: Measured molecular weight of ADC 22B6-A-01

表16：ADC 22B6-A-01的DAR值
Table 16: DAR values for ADC 22B6-A-01

计算得ADC 22B6-A-01的药物/抗体比值为DAR＝7.93见表16。The calculated drug/antibody ratio of ADC 22B6-A-01 is DAR=7.93, see Table 16.

8.LC-MS测定ADC U1-59-A-01分子量，计算药物/抗体比DAR值。对ADC U1-59-A-01进行LC-MS分子量分析见表17。
8. LC-MS determination of ADC U1-59-A-01 molecular weight and calculation of drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC U1-59-A-01 is shown in Table 17.

表17：ADC U1-59-A-01实测分子量
Table 17: ADC U1-59-A-01 measured molecular weight

表18：ADC U1-59-A-01的DAR值
Table 18: DAR values for ADC U1-59-A-01

计算得ADC U1-59-A-01的药物/抗体比值为DAR＝7.89见表18。The calculated drug/antibody ratio of ADC U1-59-A-01 is DAR=7.89, see Table 18.

9.LC-MS测定ADC 22B6-B-03-1分子量，计算药物/抗体比DAR值。对ADC 22B6-B-03-1进行LC-MS分子量分析见表19。
9. LC-MS determination of the molecular weight of ADC 22B6-B-03-1 and calculation of the drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-B-03-1 is shown in Table 19.

表19：ADC 22B6-B-03-1实测分子量
Table 19: Measured molecular weight of ADC 22B6-B-03-1

表20：ADC 22B6-B-03-1的DAR值
Table 20: DAR values for ADC 22B6-B-03-1

计算得ADC 22B6-B-03-1的药物/抗体比值为DAR＝8.0见表20。The calculated drug/antibody ratio of ADC 22B6-B-03-1 is DAR=8.0, see Table 20.

10.LC-MS测定ADC 22B6-B-03-2分子量，计算药物/抗体比DAR值。对ADC 22B6-B-03-2进行LC-MS分子量分析见表21。
10. LC-MS determination of the molecular weight of ADC 22B6-B-03-2 and calculation of the drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-B-03-2 is shown in Table 21.

表21：ADC 22B6-B-03-2实测分子量
Table 21: Measured molecular weight of ADC 22B6-B-03-2

表22：ADC 22B6-B-03-2的DAR值

Table 22: DAR values for ADC 22B6-B-03-2

计算得ADC 22B6-B-03-2的药物/抗体比值为DAR＝3.14，见表22。The calculated drug/antibody ratio of ADC 22B6-B-03-2 is DAR=3.14, see Table 22.

11.LC-MS测定ADC hIgG1-B-03-1分子量，计算药物/抗体比DAR值。对ADC hIgG1-B-03-1进行LC-MS分子量分析见表23。
11. LC-MS determination of the molecular weight of ADC hIgG1-B-03-1 and calculation of the drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC hIgG1-B-03-1 is shown in Table 23.

表23：ADC hIgG1-B-03-1实测分子量
Table 23: Measured molecular weight of ADC hIgG1-B-03-1

表24：ADC hIgG1-B-03-1的DAR值
Table 24: DAR values of ADC hIgG1-B-03-1

计算得ADC hIgG1-B-03-1的药物/抗体比值为DAR＝7.90见表24。The calculated drug/antibody ratio of ADC hIgG1-B-03-1 is DAR=7.90, see Table 24.

12.LC-MS测定ADC hIgG1-B-03-2分子量，计算药物/抗体比DAR值。对ADC hIgG1-B-03-2进行LC-MS分子量分析见表25。
12. LC-MS determination of the molecular weight of ADC hIgG1-B-03-2 and calculation of the drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC hIgG1-B-03-2 is shown in Table 25.

表25：ADC hIgG1-B-03-2实测分子量
Table 25: Measured molecular weight of ADC hIgG1-B-03-2

表26：ADC hIgG1-B-03-2的DAR值
Table 26: DAR values of ADC hIgG1-B-03-2

计算得ADC hIgG1-B-03-2的药物/抗体比值为DAR＝4.20见表26。The calculated drug/antibody ratio of ADC hIgG1-B-03-2 is DAR=4.20, see Table 26.

13.LC-MS测定ADC 22B6-B-01分子量，计算药物/抗体比DAR值。对ADC 22B6-B-01进行LC-MS分子量分析见表27。
13. LC-MS determination of the molecular weight of ADC 22B6-B-01 and calculation of the drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-B-01 is shown in Table 27.

表27：ADC 22B6-B-01实测分子量

Table 27: ADC 22B6-B-01 measured molecular weight

表28：ADC 22B6-B-01的DAR值
Table 28: DAR values for ADC 22B6-B-01

计算得ADC 22B6-B-01的药物/抗体比值为DAR＝8.07见表28。The calculated drug/antibody ratio of ADC 22B6-B-01 is DAR=8.07, see Table 28.

14.LC-MS测定ADC hIgG1-B-01分子量，计算药物/抗体比DAR值。对ADC hIgG1-B-01进行LC-MS分子量分析见表29。
14. LC-MS determination of the molecular weight of ADC hIgG1-B-01 and calculation of the drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC hIgG1-B-01 is shown in Table 29.

表29：ADC hIgG1-B-01实测分子量
Table 29: Measured molecular weight of ADC hIgG1-B-01

表30：ADC hIgG1-B-01的DAR值

Table 30: DAR values of ADC hIgG1-B-01

计算得ADC hIgG1-B-01的药物/抗体比值为DAR＝8.06见表30。The calculated drug/antibody ratio of ADC hIgG1-B-01 is DAR=8.06, see Table 30.

15.LC-MS测定ADC hIgG1-A-01分子量，计算药物/抗体比DAR值。对ADC hIgG1-A-01进行LC-MS分子量分析见下表。15. LC-MS determination of ADC hIgG1-A-01 molecular weight, calculation of drug/antibody ratio DAR value. The LC-MS molecular weight analysis of ADC hIgG1-A-01 is shown in the table below.

表31：ADC hIgG1-A-01的DAR值
Table 31: DAR values of ADC hIgG1-A-01

实施例十四、ADC 22B6-A-26的制备实施例Example 14. Preparation Example of ADC 22B6-A-26

取2.667ml 22B6抗体(18.75mg/mL)，用133.35uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.63，加入10mM TCEP(三(2-羧乙基)膦，184.19uL，pH 7.60)溶液混匀，室温放置1.5h。再缓慢加入10倍物质的量的溶解在二甲基亚砜的A-26(341.73uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到ADC 22B6-A-26，质谱法测定DAR值为7.97。2.667 ml of 22B6 antibody (18.75 mg/mL) was diluted with 133.35 uL 20 mM PB + 0.1 M EDTA (pH 7.60), and then the pH was adjusted to 7.63 with 1 M Na 2 HPO 4 solution, and 10 mM TCEP (tris (2-carboxyethyl) phosphine, 184.19 uL, pH 7.60) solution was added and mixed, and the mixture was placed at room temperature for 1.5 hours. Then, 10 times the amount of the substance was slowly added to the A-26 (341.73 uL, 10 mM) solution dissolved in dimethyl sulfoxide, and the mixture was mixed, and the mixture was placed at room temperature for 2 hours. After completion, the buffer was replaced with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain ADC 22B6-A-26, and the DAR value was 7.97 determined by mass spectrometry.

实施例十五、ADC hIgG1-A-26的制备实施例Example 15. Preparation Example of ADC hIgG1-A-26

取2.555ml hIgG1抗体(19.57mg/mL)，用127uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.63，加入10mM TCEP(三(2-羧乙基)膦，190.8uL，pH 7.60)溶液混匀，室温放置1.5h。再加入10倍物质的量的溶解在二甲基亚砜的A-26(354uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体-药物缀合物(即ADC hIgG1-A-26)。质谱法测定DAR值为8.06。Take 2.555ml hIgG1 antibody (19.57mg/mL), dilute with 127uL 20mM PB+0.1M EDTA (pH 7.60), then adjust the pH to 7.63 with 1M Na 2 HPO 4 solution, add 10mM TCEP (tri(2-carboxyethyl)phosphine, 190.8uL, pH 7.60) solution, mix well, and let stand at room temperature for 1.5h. Then add 10 times the amount of substance dissolved in dimethyl sulfoxide A-26 (354uL, 10mM) solution, mix well, let stand at room temperature for 2h, and after completion, use NAP-5 gel column (Cytiva) to replace the buffer with 20mM histidine buffer solution at pH 6.0 to obtain antibody-drug conjugate (i.e. ADC hIgG1-A-26). The DAR value determined by mass spectrometry is 8.06.

实施例十六a.ADC 22B6-B-04的制备实施例Example 16a. Preparation Example of ADC 22B6-B-04

取0.854ml 22B6抗体(23.43mg/mL)，用42.68uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.57，加入20mM TCEP(三(2-羧乙基)膦，73.68uL，pH 7.60)溶液混匀，室温放置1.5小时。再缓慢加入12倍物质的量的溶解在二甲基亚砜的B-04(164uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到ADC 22B6-B-04-1，质谱法测定DAR值为8.62。0.854 ml of 22B6 antibody (23.43 mg/mL) was taken and diluted with 42.68 uL 20 mM PB + 0.1 M EDTA (pH 7.60), and then the pH was adjusted to 7.57 with 1 M Na 2 HPO 4 solution, and 20 mM TCEP (tris (2-carboxyethyl) phosphine, 73.68 uL, pH 7.60) solution was added and mixed, and the mixture was placed at room temperature for 1.5 hours. Then, 12 times the amount of the substance was slowly added to the B-04 (164 uL, 10 mM) solution dissolved in dimethyl sulfoxide, and the mixture was mixed, and the mixture was placed at room temperature for 2 hours. After completion, the buffer was replaced with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain ADC 22B6-B-04-1, and the DAR value was 8.62 determined by mass spectrometry.

实施例十六b.ADC 22B6-B-04的制备实施例Example 16b. Preparation Example of ADC 22B6-B-04

取2.667ml 22B6抗体(18.75mg/mL)，用133.35uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.63，加入10mM TCEP(三(2-羧乙基)膦，184.19uL，pH 7.60)溶液混匀，室温放置1.5h。再缓慢加入10倍物质的量的溶解在二甲基亚砜的B-04(341.73uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到ADC 22B6-B-04-2，质谱法测定DAR值为6.87。2.667 ml of 22B6 antibody (18.75 mg/mL) was diluted with 133.35 uL 20 mM PB + 0.1 M EDTA (pH 7.60), and then the pH was adjusted to 7.63 with 1 M Na 2 HPO 4 solution, and 10 mM TCEP (tris (2-carboxyethyl) phosphine, 184.19 uL, pH 7.60) solution was added and mixed, and the mixture was placed at room temperature for 1.5 hours. Then, 10 times the amount of the substance was slowly added to the B-04 (341.73 uL, 10 mM) solution dissolved in dimethyl sulfoxide, and the mixture was mixed, and the mixture was placed at room temperature for 2 hours. After completion, the buffer was replaced with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain ADC 22B6-B-04-2, and the DAR value was 6.87 determined by mass spectrometry.

实施例十六c.ADC hIgG1-B-04的制备实施例Example 16c. Preparation Example of ADC hIgG1-B-04

将抗体替换为hIgG1抗体，用与上文第十六b节同样方法制备hIgG1-B-04样品，质谱法测定DAR值为7.02。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.

实施例十七.检测抗体-药物缀合物活性Example 17. Detection of Antibody-Drug Conjugate Activity

1.抗人HER3抗体及其药物偶联物细胞亲和力检测1. Cell affinity detection of anti-human HER3 antibodies and their drug conjugates

用流式仪(Beckman，型号Cytoflex)检测待测ADC与MDA-MB-453细胞或NCI-N87细胞的亲和力。The affinity of the tested ADC to MDA-MB-453 cells or NCI-N87 cells was detected by flow cytometry (Beckman, model Cytoflex).

用Tryple(厂家Gibco)溶液消化贴壁生长的MDA-MB-453和NCI-N87细胞，计数并取适量细胞，用PBS洗涤两次，重悬于1％ BSA(PBS配制)溶液中，然后将细胞转移到96孔尖底板中，50μl每孔；用1％ BSA分别稀释候选抗体及其药物偶联物，15μg/mL起始，3倍梯度稀释，然后取50μl稀释好的抗体或抗体偶联药物加入到含有细胞的尖底板中，4度孵育40min；PBS洗涤细胞两次，然后每孔加入50μl稀释好的二抗，混匀，4度孵育30min；PBS洗涤细胞两次，然后将细胞重悬于200μl PBS中，流式上机检测。数据处理：导出平均荧光信号值，然后导入GraphPad Prism 6软件，计算EC50，结果如表1和图1、图2所示。结果说明，本发明ADC具有明显的细胞亲和性。Digest the adherent MDA-MB-453 and NCI-N87 cells with Tryple (Gibco) solution, count and take appropriate amount of cells, wash twice with PBS, resuspend in 1% BSA (prepared with PBS) solution, and then transfer the cells to a 96-well conical bottom plate, 50μl per well; dilute the candidate antibody and its drug conjugate with 1% BSA, starting at 15μg/mL, 3-fold gradient dilution, and then take 50μl of the diluted antibody or antibody-conjugated drug and add it to the conical bottom plate containing cells, incubate at 4 degrees for 40min; wash the cells twice with PBS, then add 50μl of diluted secondary antibody to each well, mix well, and incubate at 4 degrees for 30min; wash the cells twice with PBS, then resuspend the cells in 200μl PBS, and detect on the flow cytometer. Data processing: export the average fluorescence signal value, and then import it into GraphPad Prism 6 software to calculate EC50. The results are shown in Table 1 and Figures 1 and 2. The results show that the ADC of the present invention has obvious cell affinity.

表1抗人HER3抗体及ADC细胞亲和力测定结果
Table 1 Anti-human HER3 antibody and ADC cell affinity determination results

2.抗人HER3抗体偶联药物内吞活性测试2. Anti-human HER3 antibody-drug conjugate endocytosis activity test

用流式仪(Beckman，型号Cytoflex)检测待测ADC或抗体在MDA-MB-453、HCC1569细胞上的内吞活性。The endocytic activity of the ADC or antibody to be tested on MDA-MB-453 and HCC1569 cells was detected using a flow cytometer (Beckman, model Cytoflex).

用Trypsin-EDTA(0.25％，上海源培)溶液消化贴壁生长的细胞并计数，用完全培养基调整细胞密度至1×105个/ml，每孔加入100μl细胞悬液到96孔板中(细胞数目为1×104个/孔)，将96孔板放置于37℃，CO2恒温培养箱孵育培养24h。取出96孔板，吸弃培养基，每孔加入50μl新鲜完全培养基；采用完全培养基梯度稀释双特异抗体及对照抗体，终浓度为0.55、1.64、4.94、14.81、44.44、133.33、400、1200ng/ml，共计8个浓度点；完全培养基稀释PHrodo试剂(Thermo)至12μg/ml(PHrodo的终浓度为3μg/ml)；将梯度稀释的待测抗体与稀释后的PHrodo试剂1:1混合均匀(30μl:30μl)，在室温避光孵育30min；取50μl待测抗体和PHrodo试剂混合物加入96孔板中，37℃、5％CO2培养24h；取出96孔板，吸弃培养基，无菌PBS洗涤1次，每孔加入100μl的Trypsin-EDTA(0.25％)消化细胞，再加入100μl完全培养基中和；将孔里细胞吹打分散后，FACS上机检测。数据处理：导出平均荧光信号值，然后导入GraphPad Prism 6软件，计算EC50，结果如表2所示和图3、图4所示，结果说明，本发明ADC具有较强的内吞活性。The adherent cells were digested with Trypsin-EDTA (0.25%, Shanghai Yuanpei) solution and counted. The cell density was adjusted to 1×10 5 cells/ml with complete culture medium. 100 μl of cell suspension was added to each well of a 96-well plate (the number of cells was 1×10 4 cells/well). The 96-well plate was placed in a 37°C, CO 2 constant temperature incubator and incubated for 24 h. The 96-well plate was removed, the culture medium was discarded, and 50 μl of fresh complete culture medium was added to each well; the bispecific antibody and the control antibody were gradiently diluted with complete culture medium, and the final concentrations were 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 to 12 μg/ml with complete culture medium (the final concentration of PHrodo was 3 μg/ml); the gradiently diluted antibody to be tested was mixed evenly with the diluted PHrodo reagent in a ratio of 1:1 (30 μl:30 μl), and incubated at room temperature in the dark for 30 min; 50 μl of the mixture of the antibody to be tested and the PHrodo reagent was added to the 96-well plate, and incubated at 37°C, 5% CO 2Cultivate 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; disperse the cells in the wells by blowing, and then detect them on FACS. Data processing: export the average fluorescence signal value, and then import it into GraphPad Prism 6 software to calculate EC 50. The results are shown in Table 2 and Figures 3 and 4. The results show that the ADC of the present invention has strong endocytosis activity.

表2抗人HER3抗体及ADC内吞活性测定结果
Table 2 Results of endocytic activity assay of anti-human HER3 antibodies and ADCs

3.抗人HER3抗体偶联药物体外细胞毒性3. In vitro cytotoxicity of anti-human HER3 antibody-drug conjugates

用TrypLE(厂家Gibco)溶液消化MDA-MB-453或者293T-HER3细胞，计数并取适量细胞，用生长培养基稀释后，5000cells/100μl/well铺到96孔板，37℃、5％CO2培养过夜；第二天用生长培养基稀释ADC，150μg/ml起始，2.5倍梯度稀释(或者7.5μg/ml起始，5倍梯度稀释)，然后取100μl稀释好的ADC加入含有细胞的96孔板中，将板子放入培养箱，37℃、5％CO2培养6天后，每孔加入20μl CCK8，37℃孵育2-5h，酶标仪读取OD450nm吸光值；将原始数据导入Graph Prism 6计算IC50值。如表3和图5、图6所示，本发明的ADC药物(例如22B6-A-26)有靶点特异的杀伤，其中肿瘤细胞上22B6-A-26与hIgG1-A-26的IC50相差9.1倍；过表达细胞(HEK293T-h HER3)上hIgG1-A-26无明显杀伤，22B6-A-26ADC药物与hIgG1-A-26的IC50差距更大。MDA-MB-453 or 293T-HER3 cells were digested with TrypLE (manufacturer Gibco) solution, and an appropriate amount of cells were counted and taken. After diluting with growth medium, 5000 cells/100 μl/well were spread on a 96-well plate and cultured at 37°C and 5% CO2 overnight; on the second day, ADC was diluted with growth medium, starting at 150 μg/ml, and 2.5-fold gradient dilution (or starting at 7.5 μg/ml, 5-fold gradient dilution), and then 100 μl of the diluted ADC was added to the 96-well plate containing cells, and the plate was placed in an incubator. After culturing at 37°C and 5% CO2 for 6 days, 20 μl of CCK8 was added to each well, incubated at 37°C for 2-5 hours, and the OD450nm absorbance value was read by 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; hIgG1-A-26 has no obvious killing on overexpressing cells (HEK293T-h HER3), and the IC50 difference between the 22B6-A-26 ADC drug and hIgG1-A-26 is even greater.

表3抗人HER3 ADC体外细胞毒性结果
Table 3 In vitro cytotoxicity results of anti-human HER3 ADC

4.不同抗体偶联药物NCI-H358模型体内药效检测4. In vivo efficacy testing of different antibody-drug conjugates in the NCI-H358 model

人非小细胞肺癌细胞NCI-H358(南京科佰)体外单层培养，培养条件为RPMI-1640培养基中加10％胎牛血清，于37℃、含5％CO2的培养箱中培养。2-3次/周用胰酶-EDTA进行消化处理传代。当细胞呈指数生长期时，取培养液进行支原体检测后，收集细胞并计数。每只小鼠右侧肩胛处皮下接种5×106个NCI-H358细胞(悬浮于0.05ml PBS+0.05ml基质胶中)。待平均肿瘤体积生长至100～200mm3时，剔除瘤体积不规则及过小或过大的小鼠，剩余小鼠根据肿瘤体积和动物体重进行随机分组，共4组，每组5只，尾静脉注射给药(i.v.)，每周给药一次(QW)，共给药两次，给药后每周2次用游标卡尺进行肿瘤测量，并按如下计算公式计算肿瘤体积：V＝0.5a×b2，其中a和b分别表示肿瘤的长径和短径，抗肿瘤药效用肿瘤生长抑制率TGI(％)评价，计算公式：TGI(％)(瘤体积)＝[1-(TVt-TV0)/(CVt-CV0)]×100％；当肿瘤出现消退时，TGI(％)(瘤体积)＝100％-(TVt-TV0)/TV0×100％。TV0为分组给药时受试药物组的平均瘤体积；TVt为给药后t天受试药物组的平均瘤体积；CV0为分组给药时溶媒组的平均瘤体积；CVt为给药后t天溶媒组的平均瘤体积。如果肿瘤相比起始体积缩小，即Vt<V0时，定义为肿瘤部分消退(PR)；如果肿瘤完全消失，定义为肿瘤完全消退(CR)。每天观察记录动物死亡情况，具体结果见表4，图7A-7B。Human non-small cell lung cancer cells NCI-H358 (Nanjing Kebai) were cultured in vitro in monolayers, with RPMI-1640 medium plus 10% fetal bovine serum, and cultured in an incubator at 37°C and 5% CO2 . Digestion and passage were performed with trypsin-EDTA 2-3 times per week. When the cells were in the exponential growth phase, the culture medium was taken for mycoplasma detection, and the cells were collected and counted. 5×10 6 NCI-H358 cells (suspended in 0.05 ml PBS + 0.05 ml Matrigel) were subcutaneously inoculated at the right scapula of each mouse. When the average tumor volume grew to 100-200 mm3 , 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 weight, with 5 mice in each group. The drugs were administered by tail vein injection (iv), once a week (QW), for a total of two times. After administration, the tumor was measured with a vernier caliper twice a week, and the tumor volume was calculated according to the following formula: V = 0.5a × b2 , wherein a and b represent the major and minor diameters of the tumor, respectively. The antitumor efficacy was evaluated by tumor growth inhibition rate TGI (%), calculated by the formula: TGI (%) (tumor volume) = [1-(T Vt -T V0 )/(C Vt -C V0 )] × 100%; when the tumor regressed, TGI (%) (tumor volume) = 100% - (T Vt -T V0 )/T V0 × 100%. T V0 is the average tumor volume of the test drug group at the time of group administration; T Vt is the average tumor volume of the test drug group t days after administration; C V0 is the average tumor volume of the vehicle group at the time of group administration; C Vt is the average tumor volume of the vehicle group t days after administration. If the tumor is smaller than the initial volume, that is, when V t <V 0 , it is defined as partial tumor regression (PR); if the tumor disappears completely, it is defined as complete tumor regression (CR). The death of animals was observed and recorded every day, and the specific results are shown in Table 4 and Figures 7A-7B.

表4抗体偶联药物对NCI-H358细胞荷瘤鼠模型的疗效分析


注：与溶媒组相比，*：P<0.05，P<0.05表示有显著差异。Table 4 Analysis of the efficacy of antibody-drug conjugates on the NCI-H358 cell tumor-bearing mouse model


Note: Compared with the vehicle group, * : P<0.05, P<0.05 indicates significant difference.

结果显示：22B6-A-26显示出明显药效，各组动物耐受较好,显示出以上药物的良好疗效和安全性。The results showed that 22B6-A-26 showed significant efficacy and was well tolerated by animals in each group, demonstrating the good efficacy and safety of the above drugs.

尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解，根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。 Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims (22)

一种抗体-药物缀合物，其具有式Ab-[M-L-E-D]x所示结构，其中：An antibody-drug conjugate having a structure shown in the formula Ab-[MLED] x , wherein: Ab是与人表皮生长因子受体3(HER3)特异性结合的抗体或其抗原结合片段；Ab is an antibody or an antigen-binding fragment thereof that specifically binds to human epidermal growth factor receptor 3 (HER3); M是与抗体或其抗原结合片段连接的接头部位，并且为其中，环A为5-6元脂杂环、或5-20元芳香族环系，所述脂杂环和芳香族环系任选地被一个或多个选自氧基(＝O)、卤素、氰基、氨基、羧基、巯基和C1-6烷基的基团取代；M1选自单键和取代或未取代的以下基团：C1-20亚烷基、C2-20亚烯基或C2-20亚炔基；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 groups selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 alkyl; M 1 is selected from a single bond and the following substituted or unsubstituted groups: C 1-20 alkylene, C 2-20 alkenylene or C 2-20 alkynylene; L是连接M和E之间的结构片段，并且选自由下述的一个或多个取代或未取代的基团组成的结构：C1-6亚烷基、-N(R’)-、羰基、-O-、Val、Cit、Phe、Lys、Lys(COCH2CH2(OCH2CH2)sOCH3)、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、Gly-Gly-Gly-Gly-Gly、 其中R’代表氢、C1-6烷基或含-(CH2CH2O)r-的烷基；r选自1-10的整数；s选自1-20的整数；L is a structural fragment connecting M and E, and is selected from a structure consisting of one or more substituted or unsubstituted groups: C 1-6 alkylene, -N(R')-, carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) s OCH 3 ), D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, wherein R' represents hydrogen, C 1-6 alkyl or alkyl containing -(CH 2 CH 2 O) r -; r is selected from integers of 1-10; s is selected from integers of 1-20; E是连接L和D的结构片段，并且为单键或选自以下取代或未取代的结构：-NH-CH2-，-NH-CH2-O-CH2-CO-， 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-CH 2 -, -NH-CH 2 -O-CH 2 -CO-, D是细胞毒性药物与E连接后得到的所述细胞毒性药物相应的片段，其中所述细胞毒性药物选自微管蛋白抑制剂、DNA嵌入剂、DNA拓扑异构酶抑制剂和RNA聚合酶抑制剂；优选地，所述微管蛋白抑制剂为奥瑞他汀类化合物或美登素类化合物；优选地，所述DNA嵌入剂为吡咯并苯二氮卓(PBD)；优选地，所述DNA拓扑异构酶抑制剂为拓扑异构酶I抑制剂(例如，喜树碱、羟基喜树碱、9-氨基喜树碱、SN-38、伊立替康、拓扑替康、贝洛替康、或卢比替康)或拓扑异构酶II抑制剂(例如，阿霉素、PNU-159682、多卡米星、柔红霉素、米托蒽醌、鬼臼毒素、或依托泊苷)；优选地，所述RNA聚合酶抑制剂为α-鹅膏草碱(α-amanitin)或其药学上可接受的盐、酯或类似物； 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选自1至10。x is selected from 1 to 10. 权利要求1所述的抗体-药物缀合物，M为其中，环A为5-20元芳香族环系，所述芳香族环系任选地被一个或多个选自卤素、氰基、氨基、羧基、巯基和C1-6烷基的基团取代；M1为取代或未取代的C2-20亚炔基；The antibody-drug conjugate of claim 1, wherein M is wherein Ring A is a 5-20 membered aromatic ring system, the aromatic ring system is optionally substituted by one or more groups selected from halogen, cyano, amino, carboxyl, thiol and C 1-6 alkyl; M 1 is a substituted or unsubstituted C 2-20 alkynylene group; 优选地，M为其中，环A为6元杂芳环，所述杂芳环任选地被一个或多个选自卤素、氰基、氨基、羧基、巯基和C1-6烷基的基团取代；M1为取代或未取代的C3-10亚炔基；Preferably, M is wherein Ring A is a 6-membered heteroaromatic ring, which is optionally substituted by one or more groups selected from halogen, cyano, amino, carboxyl, thiol and C 1-6 alkyl; M 1 is a substituted or unsubstituted C 3-10 alkynylene group; 优选地，M为 Preferably, M is 权利要求1或2所述的抗体-药物缀合物，L选自由下述的一个或多个组成的结构：Val、Cit、Phe、Lys、Lys(COCH2CH2(OCH2CH2)sOCH3)、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和Gly-Gly-Gly-Gly-Gly；其中s选自1-20的整数；The antibody-drug conjugate of claim 1 or 2, wherein L is selected from the group consisting of one or more of the following structures: Val, Cit, Phe, Lys, Lys( COCH2CH2 ( OCH2CH2 ) sOCH3 ) , 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; 优选地，L为一个或多个取代或未取代的Ala组成；Preferably, L is composed of one or more substituted or unsubstituted Ala; 优选地，L为取代的或未取代的Ala-Ala-Ala；Preferably, L is substituted or unsubstituted Ala-Ala-Ala; 优选地，L为 Preferably, L is 权利要求1-3任一项所述的抗体-药物缀合物，E为单键或选自以下取代或未取代的结构：-NH-CH2-、-NH-CH2-O-CH2-CO-、 The antibody-drug conjugate according to any one of claims 1 to 3, wherein E is a single bond or a substituted or unsubstituted structure selected from the following: -NH-CH 2 -, -NH-CH 2 -O-CH 2 -CO-, 优选地，E为-NH-CH2-O-CH2-CO或-NH-CH2-。Preferably, E is -NH-CH 2 -O-CH 2 -CO or -NH-CH 2 -. 权利要求1-4任一项所述的抗体-药物缀合物，选自以下结构：
The antibody-drug conjugate according to any one of claims 1 to 4, Select from the following structures:
权利要求1-5任一项所述的抗体-药物缀合物，所述细胞毒性药物选自式I和式II所示化合物：
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:
其中，in, 在式I中，In Formula I, R1，R2各自独立地选自C1-6烷基和卤素；R 1 and R 2 are each independently selected from C 1-6 alkyl and halogen; R3选自H和-CO-CH2OH；R 3 is selected from H and -CO-CH 2 OH; 在式II中，In Formula II, R4和R5各自独立地选自H、卤素和羟基；或者R4和R5与相连碳原子连接成5-6元含氧杂环； 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选自氢和-C1-4亚烷基-NRaRb； R6 is selected from hydrogen and -C1-4alkylene - NRaRb ; R7选自C1-6烷基和-C1-4亚烷基-NRaRb； R7 is selected from C1-6 alkyl and -C1-4 alkylene- NRaRb ; 其中Ra、Rb在每次出现时各自独立地选自H、C1-6烷基、-SO2-C1-6烷基和-CO-C1-6烷基；wherein Ra , Rb, at each occurrence, are independently selected from H, C1-6 alkyl, -SO2 - C1-6 alkyl, and -CO- C1-6 alkyl; 优选地，所述细胞毒性药物选自以下化合物：

Preferably, the cytotoxic drug is selected from the following compounds:

优选地，所述细胞毒性药物为 Preferably, the cytotoxic drug is 权利要求1-6任一项所述的抗体-药物缀合物，D为所述细胞毒性药物上的-OH、-NH2或二级胺基失掉一个H得到的一价结构；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; 优选地，D选自下述结构：
Preferably, D is selected from the following structures:
优选地，所述D为 Preferably, D is 权利要求1-7任一项所述的抗体-药物缀合物，其中所述抗体或其抗原结合片段包含:The antibody-drug conjugate of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof comprises: (1)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Chothia编号系统定义：(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:1或其变体的CDR-H1，氨基酸序列为SEQ ID NO:2或其变体的CDR-H2，氨基酸序列为SEQ ID NO:3或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:4或其变体的CDR-L1，氨基酸序列为SEQ ID NO:5或其变体的CDR-L2，氨基酸序列为SEQ ID NO:6或其变体的CDR-L3；或，(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, (1b)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:19或其变体的CDR-H1，氨基酸序列为SEQ ID NO:20或其变体的CDR-H2，氨基酸序列为SEQ ID NO:21或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:22或其变体的CDR-L1，氨基酸序列为SEQ ID NO:23或其变体的CDR-L2，氨基酸序列为SEQ ID NO:24或其变体的CDR-L3；或，(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:36或其变体的CDR-H1，氨基酸序列为SEQ ID NO:37或其变体的CDR-H2，氨基酸序列为SEQ ID NO:38或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:45或其变体的CDR-L1，氨基酸序列为SEQ ID NO:23或其变体的CDR-L2，氨基酸序列为SEQ ID NO:52或其变体的CDR-L3；(1c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:36 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:37 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO:45 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO:23 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO:52 or a variant thereof; 其中，(1a)、(1b)、(1c)任一项中所述的变体与其所源自的氨基酸序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的氨基酸序列同一性，或者所述变体与其所源自的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；条件是，所述变体的CDR的氨基酸序列与(1a)、(1b)和(1c)的VH和VL的相应CDR的氨基酸序列具有100％序列同一性，并且所述变体结合HER3；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% 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; 或者，or, (2)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号系统 定义：(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:7或其变体的CDR-H1，氨基酸序列为SEQ ID NO:8或其变体的CDR-H2，氨基酸序列为SEQ ID NO:9或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:4或其变体的CDR-L1，氨基酸序列为SEQ ID NO:5或其变体的CDR-L2，氨基酸序列为SEQ ID NO:6或其变体的CDR-L3；或，(2a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:7 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:8 or a variant thereof, and CDR-H3 having 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 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, (2b)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:25或其变体的CDR-H1，氨基酸序列为SEQ ID NO:26或其变体的CDR-H2，氨基酸序列为SEQ ID NO:21或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:22或其变体的CDR-L1，氨基酸序列为SEQ ID NO:23或其变体的CDR-L2，氨基酸序列为SEQ ID NO:24或其变体的CDR-L3；或，(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:39或其变体的CDR-H1，氨基酸序列为SEQ ID NO:40或其变体的CDR-H2，氨基酸序列为SEQ ID NO:38或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:45或其变体的CDR-L1，氨基酸序列为SEQ ID NO:23或其变体的CDR-L2，氨基酸序列为SEQ ID NO:52或其变体的CDR-L3；(2c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:39 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:40 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO:45 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO:23 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO:52 or a variant thereof; 其中，(2a)、(2b)、(2c)任一项中所述的变体与其所源自的氨基酸序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的氨基酸序列同一性，或者所述变体与其所源自的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；条件是，所述变体的CDR的氨基酸序列与(2a)、(2b)和(2c)的VH和VL的相应CDR的氨基酸序列具有100％序列同一性，并且所述变体结合HER3；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; 或者，or, (3)下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:10或其变体的CDR-H1，氨基酸序列为SEQ ID NO:11或其变体的CDR-H2，氨基酸序列为SEQ ID NO:12或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:13或其变体的CDR-L1，氨基酸序列为SEQ ID NO:14或其变体的CDR-L2，氨基酸序列为SEQ ID NO:6或其变体的CDR-L3；或，(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:27或其变体的CDR-H1，氨基酸序列为SEQ ID NO:28或其变体的CDR-H2，氨基酸序列为SEQ ID NO:29或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:30或其变体的CDR-L1，氨基酸序列为SEQ ID NO:31或其变体的CDR-L2，氨基酸序列为SEQ ID NO:24或其变体的CDR-L3；或，(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)包含如下3个CDR的重链可变区(VH)：氨基酸序列为SEQ ID NO:41或其变体的CDR-H1，氨基酸序列为SEQ ID NO:42或其变体的CDR-H2，氨基酸序列为SEQ ID NO:43或其变体的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：氨基酸序列为SEQ ID NO:44或其变体的CDR-L1，氨基酸序列为SEQ ID NO:31或其变体的CDR-L2，氨基酸序列为SEQ ID NO:52或其变体的CDR-L3；(3c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having an amino acid sequence of SEQ ID NO:41 or a variant thereof, CDR-H2 having an amino acid sequence of SEQ ID NO:42 or a variant thereof, and CDR-H3 having 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 having an amino acid sequence of SEQ ID NO:44 or a variant thereof, CDR-L2 having an amino acid sequence of SEQ ID NO:31 or a variant thereof, and CDR-L3 having an amino acid sequence of SEQ ID NO:52 or a variant thereof; 其中，(3a)、(3b)、(3c)任一项中所述的变体与其所源自的氨基酸序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的氨基酸序列同一性，或者所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；条件是，所述变体的CDR的氨基酸序列与(3a)、(3b)和(3c)的VH和VL的相应CDR的氨基酸序列具有100％序列同一性，并且所述变体结合HER3。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 with 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 with 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. 权利要求1-8任一项所述的抗体-药物缀合物，其中所述抗体或其抗原结合片段包含：The antibody-drug conjugate of any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof comprises: (a)包括SEQ ID NO：15所示氨基酸序列的VH或其变体，和/或，包括SEQ ID NO：16所示氨基酸序列的VL或其变体；(a) a VH comprising the amino acid sequence shown in SEQ ID NO: 15 or a variant thereof, and/or a VL comprising the amino acid sequence shown in SEQ ID NO: 16 or a variant thereof; (b)包括SEQ ID NO：32所示氨基酸序列的VH或其变体，和/或，包括SEQ ID NO：33所示氨基酸序列的VL或其变体；或(b) a VH comprising the amino acid sequence shown in SEQ ID NO: 32 or a variant thereof, and/or a VL comprising the amino acid sequence shown in SEQ ID NO: 33 or a variant thereof; or (c)包括SEQ ID NO：46所示氨基酸序列的VH或其变体，和/或，包括SEQ ID NO：47所示氨基酸序列的VL或其变体；(c) a VH comprising the amino acid sequence shown in SEQ ID NO: 46 or a variant thereof, and/or a VL comprising the amino acid sequence shown in SEQ ID NO: 47 or a variant thereof; 其中，所述变体与其所源自的氨基酸序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的氨基酸序列同一性，或者所述变体与其所源自的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换；条件是，所述变体的CDR的氨基酸序列与(a)、(b)和(c)的VH和VL的相应CDR的氨基酸序列具有100％序列同一性，并且所述变体结合HER3。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% 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. 权利要求1-9任一项所述的抗体-药物缀合物，其中所述抗体或其抗原结合片段进一步包含：The antibody-drug conjugate of any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof further comprises: (a)人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和(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)人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；(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; 优选地，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区，例如人IgG1重链恒定区或人IgG4重链恒定区；优选地，所述抗体或其抗原结合片段包含如SEQ ID NO：50所示的重链恒定区(CH)或其变体，所述变体与SEQ ID NO：50相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)；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; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids); 优选地，所述轻链恒定区是κ轻链恒定区；优选地，所述抗体或其抗原结合片段包含如SEQ ID NO：51所示的轻链恒定区(CL)或其变体，所述变体与SEQ ID NO：51相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)；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); 优选地，所述抗体或其抗原结合片段包含如SEQ ID NO：50所示的重链恒定区(CH)和如SEQ ID NO：51所示的轻链恒定区(CL)。Preferably, the antibody or its antigen-binding fragment 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. 权利要求1-10任一项所述的抗体-药物缀合物，其中所述抗体或其抗原结合片段包含：The antibody-drug conjugate of any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof comprises: (1)包含SEQ ID NO：15所示氨基酸序列的VH和SEQ ID NO：50所示氨基酸序列的重链恒定区(CH)的重链，和，包含SEQ ID NO：16所示氨基酸序列的VL和SEQ ID NO：51所示的氨基酸序列的轻链恒定区(CL)的轻链；(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) having an amino acid sequence 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) having an amino acid sequence as shown in SEQ ID NO: 51; (2)包含SEQ ID NO：32所示氨基酸序列的VH和SEQ ID NO：50所示氨基酸序列的重链恒定区(CH)的重链，和，包含SEQ ID NO：33所示氨基酸序列的VL和SEQ ID NO：51所示氨基酸序列的轻链恒定区(CL)的轻链；或(2) a heavy chain comprising a VH having an amino acid sequence as shown in SEQ ID NO: 32 and a heavy chain constant region (CH) having an amino acid sequence 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: 33 and a light chain constant region (CL) having an amino acid sequence as shown in SEQ ID NO: 51; or (3)包含SEQ ID NO：46所示氨基酸序列的VH和SEQ ID NO：50所示氨基酸序列的重链恒定区(CH)的重链，和，包含SEQ ID NO：47所示氨基酸序列的VL和SEQ ID NO：51所示氨基酸序列的轻链恒定区(CL)的轻链。(3) A heavy chain comprising VH with the amino acid sequence shown in SEQ ID NO: 46 and a heavy chain constant region (CH) with the amino acid sequence shown in SEQ ID NO: 50, and a light chain comprising VL with the amino acid sequence shown in SEQ ID NO: 47 and a light chain constant region (CL) with the amino acid sequence shown in SEQ ID NO: 51. 权利要求1-11任一项所述的抗体-药物缀合物，所述缀合物为：
The antibody-drug conjugate according to any one of claims 1 to 11, wherein the conjugate is:
其中，抗体-药物缀合物中的HA选自：Wherein, the HA in the antibody-drug conjugate is selected from: (1)包含如SEQ ID NO:15所示的VH和如SEQ ID NO:16所示的VL的抗体或其抗原结合片段，例如包含SEQ ID NO：15所示的VH和SEQ ID NO：50所示的CH，以及SEQ ID NO：16所示的VL和SEQ ID NO：51所示的CL的抗体或其抗原结合片段；(1) an antibody or an antigen-binding fragment thereof comprising the VH shown in SEQ ID NO: 15 and the VL shown in SEQ ID NO: 16, for example, an antibody or an antigen-binding fragment thereof comprising the VH shown in SEQ ID NO: 15 and the CH shown in SEQ ID NO: 50, and the VL shown in SEQ ID NO: 16 and the CL shown in SEQ ID NO: 51; (2)包含如SEQ ID NO:32所示的VH和如SEQ ID NO:33所示的VL的抗体或其抗原结合片段，例如包含SEQ ID NO：32所示的VH和SEQ ID NO：50所示的CH，以及SEQ ID NO：33所示的VL和SEQ ID NO：51所示的CL的抗体或其抗原结合片段；和(2) an antibody or an 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 an 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; and (3)包含如SEQ ID NO:46所示的VH和如SEQ ID NO:47所示的VL的抗体或其抗原结合片段，例如含SEQ ID NO：46所示的VH和SEQ ID NO：50所示的CH，以及SEQ ID NO：47所示的VL和SEQ ID NO：51所示的CL的抗体或其抗原结合片段；(3) an antibody or an 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 an 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为3～8。x is 3 to 8. 权利要求12所述的抗体-药物缀合物，其中，x为3-8的整数。The antibody-drug conjugate of claim 12, wherein x is an integer from 3 to 8. 权利要求12或13所述的抗体-药物缀合物，所述抗体-药物缀合物为：
The antibody-drug conjugate of claim 12 or 13, wherein the antibody-drug conjugate is:
其中，所述HA代表包括SEQ ID NO：17所示的HC和SEQ ID NO：18所示的LC的抗体或其抗原结合片段；Wherein, 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为3～8。x is 3 to 8. 权利要求14所述的抗体-药物缀合物，其中，x为3-8的整数。 The antibody-drug conjugate of claim 14, wherein x is an integer from 3 to 8. 权利要求1-15任一项所述的抗体-药物缀合物，其中：The antibody-drug conjugate of any one of claims 1 to 15, wherein: (i)重链C末端缺少赖氨酸残基；(i) The heavy chain lacks a lysine residue at the C-terminus; (ii)重链N末端是谷氨酰胺、谷氨酸或焦谷氨酸；或者，(ii) the N-terminus of the heavy chain is glutamine, glutamic acid or pyroglutamic acid; or, (iii)重链C末端缺少赖氨酸残基并且重链N末端是谷氨酰胺、谷氨酸或焦谷氨酸。(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. 权利要求1-16任一项所述的抗体-药物缀合物，在式Ab-[M-L-E-D]x中，Ab通过半胱氨酸残基与式中剩余部分缀合。The antibody-drug conjugate according to any one of claims 1 to 16, wherein in the formula Ab-[MLED] x , Ab is conjugated to the remaining part of the formula via a cysteine residue. 一种药物组合物，其含有权利要求1-17任一项所述的一种或多种抗体-药物缀合物，以及一种或多种药用辅料。A pharmaceutical composition comprising one or more antibody-drug conjugates according to any one of claims 1 to 17, and one or more pharmaceutical excipients. 权利要求18所述的药物组合物，其平均DAR值(药物抗体偶联比)为1-10，例如：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，或9～10，优选为3～9，例如，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或7.5～9.0。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, 4-1 0, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 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. 权利要求1-17任一项所述的抗体-药物缀合物或权利要求18或19所述的药物组合物在制备治疗HER3高表达癌症的药物中的用途；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 cancer with high HER3 expression; 优选地，所述HER3高表达癌症包括实体瘤或血液系统恶性肿瘤，例如结肠癌，胃癌，乳腺癌，肺癌(例如，非小细胞肺癌，具体如肺腺癌)，或淋巴癌。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. 权利要求1-17任一项所述的抗体-药物缀合物或权利要求18或19所述的药物组合物，其用于治疗HER3高表达癌症；The antibody-drug conjugate according to any one of claims 1 to 17 or the pharmaceutical composition according to claim 18 or 19, which is used to treat HER3-overexpressing cancer; 优选地，所述HER3高表达癌症包括实体瘤或血液系统恶性肿瘤，例如结肠癌，胃癌，乳腺癌，肺癌(例如，非小细胞肺癌，具体如肺腺癌)，或淋巴癌。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. 一种治疗HER3高表达癌症的方法，其包括向有此需要的受试者施用治疗有效量的权利要求1-17任一项所述的抗体-药物缀合物或权利要求18或19所述的药物组合物的步骤；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; 优选地，所述HER3高表达癌症包括实体瘤或血液系统恶性肿瘤，例如结肠癌，胃癌，乳腺癌，肺癌(例如，非小细胞肺癌，具体如肺腺癌)，或淋巴癌。 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.
PCT/CN2024/114694 2023-09-01 2024-08-27 Antibody-drug conjugate and preparation method therefor and use thereof Pending WO2025045015A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202311130165.6 2023-09-01
CN202311130165 2023-09-01
CN202411158958 2024-08-22
CN202411158958.3 2024-08-22

Publications (1)

Publication Number Publication Date
WO2025045015A1 true WO2025045015A1 (en) 2025-03-06

Family

ID=94818101

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/114694 Pending WO2025045015A1 (en) 2023-09-01 2024-08-27 Antibody-drug conjugate and preparation method therefor and use thereof

Country Status (2)

Country Link
TW (1) TW202515921A (en)
WO (1) WO2025045015A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220072144A1 (en) * 2018-09-20 2022-03-10 Daiichi Sankyo Company, Limited Treatment of her3-mutated cancer by administration of anti-her3 antibody-drug conjugate
WO2022170971A1 (en) * 2021-02-09 2022-08-18 苏州宜联生物医药有限公司 Bioactive substance conjugate, preparation method therefor and use thereof
WO2022253035A1 (en) * 2021-06-02 2022-12-08 四川科伦博泰生物医药股份有限公司 Antibody drug conjugate, and preparation method therefor and use thereof
WO2023165475A1 (en) * 2022-03-03 2023-09-07 四川科伦博泰生物医药股份有限公司 Her3 binding protein and use thereof
WO2023208216A1 (en) * 2022-04-29 2023-11-02 Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. Antibody-drug conjugates and preparation methods and use thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220072144A1 (en) * 2018-09-20 2022-03-10 Daiichi Sankyo Company, Limited Treatment of her3-mutated cancer by administration of anti-her3 antibody-drug conjugate
WO2022170971A1 (en) * 2021-02-09 2022-08-18 苏州宜联生物医药有限公司 Bioactive substance conjugate, preparation method therefor and use thereof
WO2022253035A1 (en) * 2021-06-02 2022-12-08 四川科伦博泰生物医药股份有限公司 Antibody drug conjugate, and preparation method therefor and use thereof
WO2023165475A1 (en) * 2022-03-03 2023-09-07 四川科伦博泰生物医药股份有限公司 Her3 binding protein and use thereof
WO2023208216A1 (en) * 2022-04-29 2023-11-02 Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. Antibody-drug conjugates and preparation methods and use thereof

Also Published As

Publication number Publication date
TW202515921A (en) 2025-04-16

Similar Documents

Publication Publication Date Title
JP2024521629A (en) Antibody drug conjugates, and methods for preparing same and uses thereof
US20250281631A1 (en) Antibody-drug conjugates and preparation methods and use thereof
CN103288957B (en) Antibody drug derivative for inhibiting tumor growth, preparation method and application thereof
WO2024078586A1 (en) Antibody-drug conjugate binding to human ptk7 and method for preparation and use thereof
CN109600993A (en) Anti-EGFR Antibody Drug Conjugates
TW202304929A (en) Anti-c-met antibody drug conjugates
WO2024114318A9 (en) Drug linker compound, preparation method therefor, and use thereof
CN119053607A (en) Camptothecins compound and conjugate thereof, preparation method and application thereof
CN119300868A (en) Camptothecin compounds and conjugates thereof, preparation methods and uses thereof
WO2025045015A1 (en) Antibody-drug conjugate and preparation method therefor and use thereof
JP7594708B1 (en) Antibody Drug Conjugates (ADCs) Targeting Nectin-4 and Containing an Exatecan Payload
CA3268621A1 (en) Antibody-drug conjugate binding to human ptk7 and method for preparation and use thereof
JP2025541673A (en) Heterocyclic compounds, and methods for producing and using the same
WO2025214403A1 (en) Antibody-drug conjugate, composition and use thereof and method therefor
WO2024235131A1 (en) Macrocyclic drug conjugate, and preparation method therefor and use thereof
WO2024235135A1 (en) Macrocyclic compound and preparation method and use thereof
WO2024235133A1 (en) Polycyclic drug conjugate, and preparation method therefor and use thereof
WO2025167742A1 (en) Antibody-drug conjugate, method for preparing same, and use thereof
WO2025167743A1 (en) Antibody-drug conjugate, preparation method therefor, and use thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24858551

Country of ref document: EP

Kind code of ref document: A1