WO2023088382A1 - 抗体-药物偶联物及其用途 - Google Patents

抗体-药物偶联物及其用途 Download PDF

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WO2023088382A1
WO2023088382A1 PCT/CN2022/132627 CN2022132627W WO2023088382A1 WO 2023088382 A1 WO2023088382 A1 WO 2023088382A1 CN 2022132627 W CN2022132627 W CN 2022132627W WO 2023088382 A1 WO2023088382 A1 WO 2023088382A1
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cancer
seq
antibody
amino acid
acid sequence
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French (fr)
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姚兵
惠希武
潘福君
沈明月
淡墨
刘伯宁
杨金玉
高晓
袁灿
高虎
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石药集团巨石生物制药有限公司
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    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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 invention relates to an antibody-drug conjugate of a camptothecin derivative with antitumor effect and its application.
  • Antibody-drug Conjugate is composed of three different components (antibody, linker and drug). ADC technology is to couple the antibody and drug molecules together through the linker. The specific targeted delivery of drug molecules to target tissues can reduce the systemic side effects of drugs, expand the therapeutic window of drugs and improve the therapeutic potential of antibodies.
  • camptothecin derivatives are known to exhibit antitumor effects as topoisomerase I inhibitors. People have been committed to finding high-efficiency and low-toxicity camptothecin derivatives. So far, a series of semi-synthetic and fully synthetic camptothecin derivatives have appeared and entered the clinical application or clinical trial stage.
  • Daiichi Sankyo s ADC drug DS-8201, using the cytotoxic drug (payload) DXd, has a unique mechanism of action, compared with the common chemotherapy drug irinotecan (irinotecan), the activity is 10 times higher; DXd has a strong penetration The ability of the cell membrane, which enables it to kill nearby cancer cells after killing the cancer cells that have swallowed the ADC, resulting in a "bystander effect"; the half-life in the blood is significantly shortened, which helps to reduce toxic side effects The production of linkers; the linker has high stability, and non-tumor tissues will not be affected by toxic drugs; it can be specifically cleaved by lysosomal proteases highly expressed in tumors; multiple cells can be coupled to one antibody molecule For toxic drugs, increase the drug antibody ratio (Drug Antibody Ratio, DAR). It provides a new research direction for the development of ADC drugs.
  • DAR drug Antibody Ratio
  • WO2020063673A1 and WO2020063676A1 disclose some exitecan analogs and their ligand-drug conjugates, wherein the exitecan analogs have proliferation inhibitory activity on SK-BR-3 cells and U87 cells.
  • CN111689980A discloses some exitecan analogs and their antibody-drug conjugates, but this application only records the cell activity of exitecan analogs and SN38, and according to the activity recorded, most of the compounds show that they are equivalent to SN38 For antibody-drug conjugates, the antibody used in the antibody-drug conjugate is not disclosed. It can be seen that this patent application only did a preliminary study on the activity of exitecan analogues, and did not pay attention to the effects of other aspects.
  • the toxin linked to the ADC drug is highly toxic, and after the ADC drug is formed, the therapeutic window is also narrow.
  • the ADC drug DS-8021 of Exitecan developed by Daiichi Sankyo has been successfully marketed.
  • DS-8021 uses a single antibody linked to 8 toxins.
  • the drugs designed in subsequent clinical trials targeting the Trop2 target have reduced the antibody-to-drug ratio due to safety issues.
  • the reduction in the number of conjugated drugs will also reduce the therapeutic effect of ADC drugs.
  • Prodrugs and ADCs require a variety of enzymes and targets, which results in relatively large individual differences, resulting in uneven responses of patients to prodrugs, and is prone to toxicity.
  • the development of high-efficiency and low-toxicity antibody-camptothecin derivative ADC to increase the therapeutic effect is the direction of our research.
  • the epidermal growth factor receptor (HER) family includes four structurally similar receptor molecules, ErbB1 (EGFR), ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4), which belong to receptor tyrosine kinases.
  • EGFR epidermal growth factor receptor
  • HER2 ErbB2
  • HER3 ErbB3
  • HER4 ErbB4
  • EGFR Epidermal Growth Factor Receptor
  • HER1, ErbB1 Epidermal Growth Factor Receptor
  • HER1, ErbB1 is composed of 1186 amino acid residues, a transmembrane glycoprotein with a molecular weight of 170kDa, and has tyrosine kinase activity.
  • EGFR is expressed in many epithelial tissues, including skin and hair follicles. Under normal conditions, EGFR is a monomer.
  • EGFR epidermal growth factor
  • TGF- ⁇ transforming growth factor
  • it forms a homologous/heterologous dimer, and the dimer is phosphorylated
  • it activates a variety of downstream signaling pathways, promotes cell proliferation, angiogenesis, metastasis, invasion and inhibits apoptosis.
  • Many solid tumors have been found to overexpress EGFR, such as colorectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer and esophageal cancer. Make EGFR a good target for tumor therapy.
  • anti-EGFR drugs mainly include tyrosine kinase inhibitors (TKIs), anti-EGFR monoclonal antibodies (mAbs) and antibody-drug conjugates.
  • TKIs compete with adenosine 5'triphosphate (ATP) for binding to the intracellular catalytic domain of EGFR, thereby inhibiting EGFR autophosphorylation and downstream signaling.
  • ATP adenosine 5'triphosphate
  • Due to the mutation-prone characteristics of EGFR, the competitive binding mode of EGFR-TKIs has also developed from the first-generation non-selective reversible binding (represented by gefitinib, erlotinib, and icotinib) to the second-generation non-selective combination.
  • Anti-EGFR monoclonal antibodies or bispecific antibodies block ligand-induced EGFR tyrosine kinase activation by binding to ligands to competitively bind to the extracellular domain of EGFR.
  • EGFR monoclonal antibodies that have been approved for marketing in China include Cetuximab and Nimotuzumab, clinical data show that EGFR monoclonal antibodies are only effective against KRAS wild type (KRAS wild type), and have no tumor suppressive activity against KRAS mutants.
  • Antibody-drug conjugates are endocytosed by binding to EGFR on the surface of tumor cells, releasing small molecule toxins to kill tumor cells.
  • MRG003 a subsidiary of Lepu Biotech, is the first EGFR ADC drug to enter the clinical stage in China. It consists of humanized anti-EGFR monoclonal antibody JMT101 and toxin tubulin inhibitor MMAE through degradable mc-vc-PAB ( Abbreviated as Vc) linker coupling.
  • Vc degradable mc-vc-PAB
  • MRG003 Phase Ib patients are EGFR-positive patients with advanced or metastatic colorectal cancer, squamous cell carcinoma of the head and neck, and nasopharyngeal cancer that have progressed after multiple treatments. Whether TKI-resistant patients have drug effect (phase I dose escalation and dose expansion study of anti-EGFR ADC MRG003 in patients with advanced solid tumors.doi.org/10.1016/j.annonc.2021.08.1315). In order to solve the problem of EGFR-TKI drug resistance, the development of safe and effective ADC drugs is the direction of our research.
  • conditionally activated ADCs came into being to reduce the on-target toxicity.
  • the tumor microenvironment and the normal internal environment of the human body in terms of physical and chemical properties, the most notable being its low oxygen, low pH and high pressure. It is precisely because of these characteristics that there are a large number of growth factors, cell chemokines and various proteolytic enzymes in the tumor microenvironment to produce immune inflammatory reactions, which are very conducive to tumor proliferation, invasion, adhesion, vascular Generation and anti-radiation chemotherapy.
  • pH-dependent antibody modification is to enable the constructed ADC molecules to bind antigens in healthy tissues or in the circulatory system, but to maintain the ability to bind antigens in the tumor microenvironment, thereby reducing the efficacy of ADC drugs.
  • the present invention provides antibody-drug conjugates represented by formula I, and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels thereof:
  • Ab is an antibody targeting HER2, HER3 or EGFR or an antigen-binding fragment thereof
  • R is selected from a C 1-6 alkyl group
  • n is selected from an integer of 1-8 or a decimal of 1-8.
  • n is selected from an integer of 4-8 or a decimal of 4-8.
  • n is an integer selected from 1-8, it can be 1, 2, 3, 4, 5, 6, 7, 8; when n is an integer selected from 4-8, it can be 4, 5, 6, 7, 8.
  • n refers to the average number of linker-drug molecules conjugated to each antibody unit.
  • R is selected from C 1-3 alkyl groups.
  • R is selected from methyl, ethyl, propyl, isopropyl.
  • R is selected from methyl
  • the present invention provides antibody-drug conjugates represented by formula Ia or formula Ib, and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels thereof:
  • the present invention provides antibody-drug conjugates represented by formula I-1, Ia-1 and formula Ib-1, and their pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels:
  • formula I-1 formula Ia-1 and formula Ib-1, Ab and n are as defined in formula I.
  • Another aspect of the present invention provides antibody-drug conjugates represented by formula II, and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels thereof:
  • Ab is an antibody targeting HER2, HER3 or EGFR, and n is selected from an integer of 1-8 or a decimal of 1-8.
  • n is selected from an integer of 4-8 or a decimal of 4-8.
  • n is an integer selected from 1-8, it can be 1, 2, 3, 4, 5, 6, 7, 8; when n is an integer selected from 4-8, it can be 4, 5, 6, 7, 8.
  • n refers to the average number of linker-drug molecules conjugated to each antibody unit.
  • the Ab is an antibody targeting HER2 or an antigen-binding fragment thereof, it includes a heavy chain variable region (HV) and a light chain variable region (LV), wherein the heavy chain variable region sequence includes heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining The heavy chain complementarity determining region 2 (HCDR2) and the heavy chain complementarity determining region 3 (HCDR3), the light chain variable region includes the light chain complementarity determining region 1 (LCDR1), the light chain complementarity determining region 2 (LCDR2) and the light chain complementarity determining region 3 (LCDR3 ), wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in
  • the Ab when it is an antibody targeting HER2 or an antigen-binding fragment thereof, it includes a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HV is as SEQ ID NO: 7, the amino acid sequence of LV is shown in SEQ ID NO:8.
  • HV heavy chain variable region
  • LV light chain variable region
  • the Ab when it is an antibody targeting HER2 or an antigen-binding fragment thereof, it includes a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is shown in SEQ ID NO: 9, and the amino acid sequence of LC is as shown in SEQ ID NO:9.
  • the amino acid sequence is shown in SEQ ID NO:10.
  • the Ab when the Ab is an antibody targeting HER3 or an antigen-binding fragment thereof, it includes a heavy chain variable region (HV) and a light chain variable region (LV), wherein the heavy chain
  • the variable region sequence includes heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3)
  • the light chain variable region includes light chain complementarity determining region 1 (LCDR1) , light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3)
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO: 11
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 12
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO: 12
  • the amino acid sequence is shown in SEQ ID NO:13, and/or the amino acid sequence of LCDR1 is shown in SEQ ID NO:14, the amino acid sequence of LCDR2 is
  • the Ab when it is an antibody targeting HER3 or an antigen-binding fragment thereof, it includes a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HV is as SEQ ID NO: Shown in 17, the aminoacid sequence of LV is shown in SEQ ID NO:18.
  • HV heavy chain variable region
  • LV light chain variable region
  • the Ab when it is an antibody targeting HER3 or an antigen-binding fragment thereof, it includes a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is shown in SEQ ID NO: 19, and the amino acid sequence of LC is as shown in SEQ ID NO: 19.
  • the amino acid sequence is shown in SEQ ID NO:20.
  • the Ab when the Ab is an antibody targeting EGFR or an antigen-binding fragment thereof, it includes a heavy chain variable region (HV) and a light chain variable region (LV), wherein the heavy chain
  • the variable region sequence includes heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3)
  • the light chain variable region includes light chain complementarity determining region 1 (LCDR1) , light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3)
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO: 21
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 22
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO: 22
  • the amino acid sequence is shown in SEQ ID NO:23, and/or the amino acid sequence of LCDR1 is shown in SEQ ID NO:24, the amino acid sequence of LCDR2 is
  • the Ab when the Ab is an antibody targeting EGFR or an antigen-binding fragment thereof, it includes a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid of HCDR1
  • HV heavy chain variable region
  • LV light chain variable region
  • the amino acid of HCDR1 The sequence is shown in SEQ ID NO:31 or SEQ ID NO:35 or SEQ ID NO:38, the amino acid sequence of HCDR2 is shown in SEQ ID NO:22, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:23 or SEQ ID NO: 32, and/or the amino acid sequence of LCDR1 is shown in SEQ ID NO:24, the amino acid sequence of LCDR2 is shown in SEQ ID NO:25, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:26.
  • HV heavy chain variable region
  • LV light chain variable region
  • the Ab when the Ab is an antibody targeting EGFR or an antigen-binding fragment thereof, it includes a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid of HCDR1
  • HV heavy chain variable region
  • LV light chain variable region
  • the amino acid of HCDR1 The sequence is shown in SEQ ID NO:31 or SEQ ID NO:35 or SEQ ID NO:38, the amino acid sequence of HCDR2 is shown in SEQ ID NO:22, the amino acid sequence of HCDR3 is shown in SEQ ID NO:32, and LCDR1
  • LCDR2 The amino acid sequence of LCDR2 is shown in SEQ ID NO:24, the amino acid sequence of LCDR2 is shown in SEQ ID NO:25, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:26.
  • the Ab when it is an antibody targeting EGFR or an antigen-binding fragment thereof, it includes a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HV is as SEQ ID NO: Shown in 27, the aminoacid sequence of LV is shown in SEQ ID NO:28.
  • HV heavy chain variable region
  • LV light chain variable region
  • the Ab when it is an antibody targeting EGFR or an antigen-binding fragment thereof, it includes a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HV is as SEQ ID NO: 33 or shown in SEQ ID NO:36 or SEQ ID NO:39, the amino acid sequence of LV is shown in SEQ ID NO:28.
  • HV heavy chain variable region
  • LV light chain variable region
  • the Ab when it is an antibody targeting EGFR or an antigen-binding fragment thereof, it includes a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is shown in SEQ ID NO: 29, and the amino acid sequence of LC is as shown in SEQ ID NO:29.
  • the amino acid sequence is shown in SEQ ID NO:30.
  • the Ab when it is an antibody targeting EGFR or an antigen-binding fragment thereof, it includes a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is as SEQ ID NO: 34 or SEQ ID NO: 37 or shown in SEQ ID NO:40, the amino acid sequence of LC is shown in SEQ ID NO:30.
  • HC heavy chain
  • LC light chain
  • the antibody is preferably a monoclonal antibody, and the monoclonal antibody is preferably selected from human antibodies, humanized antibodies, and chimeric antibodies.
  • the antigen-binding fragment is preferably selected from Fab', (Fab') 2 , Fab, Fv, scFv, dAb.
  • the numbering rule of antibody amino acid sequence adopts Kabat numbering rule.
  • the present invention also provides unconjugated any of the above antibodies (Ab) or antigen-binding fragments thereof.
  • the present invention provides the following antibody-drug conjugates, and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels thereof:
  • DP001 is an antibody having two heavy chains of the amino acid sequence shown in SEQ ID NO:9 and two light chains of the amino acid sequence shown in SEQ ID NO:10;
  • Patritumab is an antibody having two heavy chains of the amino acid sequence shown in SEQ ID NO:19 and two light chains of the amino acid sequence shown in SEQ ID NO:20;
  • SWY2110 is an antibody having two heavy chains with amino acid sequences shown in SEQ ID NO:29 and two light chains with amino acid sequences shown in SEQ ID NO:30;
  • SWY2111 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:34 and two light chains with the amino acid sequence shown in SEQ ID NO:30;
  • SWY2112 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:37 and two light chains with the amino acid sequence shown in SEQ ID NO:30;
  • SWY2113 is an antibody having two heavy chains with amino acid sequences shown in SEQ ID NO:40 and two light chains with amino acid sequences shown in SEQ ID NO:30;
  • n is selected from an integer of 1-8 or a decimal of 1-8.
  • n is selected from an integer of 4-8 or a decimal of 4-8.
  • n is an integer selected from 1-8, it can be 1, 2, 3, 4, 5, 6, 7, 8; when n is an integer selected from 4-8, it can be 4, 5, 6, 7, 8.
  • linker-drug compounds represented by formula III, and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels thereof:
  • linker-drug compounds represented by formula IIIa and formula IIIb, and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels thereof:
  • linker-drug compounds of the following formula and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels thereof:
  • the present invention provides a pharmaceutical composition, which comprises the antibody-drug conjugate of the present invention, and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels thereof.
  • the pharmaceutical composition further includes pharmaceutically acceptable adjuvants and carriers.
  • the present invention provides the antibody-drug conjugate of the present invention, and its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope label in the preparation of drugs for the treatment of proliferative diseases.
  • the proliferative disease is preferably a disease associated with abnormal expression of HER2, HER3 or EGFR, including cancer, and the cancer is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer , liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (such as small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, Colorectal cancer, leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic
  • the present invention provides a method for treating or preventing proliferative diseases, the method comprising administering a therapeutically effective dose of the antibody-drug conjugate of the present invention, and pharmaceutically acceptable salts, hydrates, and solvates thereof to patients in need thereof , stereoisomer or isotope label, or a pharmaceutical composition comprising the same;
  • the proliferative disease is preferably a disease associated with abnormal expression of HER2, HER3 or EGFR, including cancer.
  • the cancer is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glial cancer tumor, neuroblastoma, sarcoma, lung cancer (e.g. small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g.
  • acute lymphoblastic leukemia acute myeloid leukemia, acute promyelocytic Leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia
  • bone cancer skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (such as Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma) .
  • lymphoma such as Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma
  • the present invention provides the antibody-drug conjugates of the present invention, and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels thereof for use in the treatment of drug-resistant proliferative diseases.
  • the drug-resistant proliferative disease is a drug-resistant disease related to the abnormal expression of HER2, HER3 or EGFR, including cancer.
  • the drug-resistant cancer is preferably a drug-resistant cancer caused by HER2, HER3 or EGFR gene mutation, and the cancer is preferably breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer , liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (such as small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, Colorectal cancer, leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia) bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (such as Hodgkin's lymphoma, non-Hodgkin's lymphoma or relapsed anaplastic large cell
  • the drug resistance is preferably resistant to receptor tyrosine kinase inhibitors, more preferably resistant to first-generation, second-generation or third-generation EGFR inhibitors, even more preferably resistant to gefitinib, errand Resistance to lotinib, icotinib, afatinib, dacomitinib, osimertinib, or amotinib, especially to gefitinib, afatinib, osimertinib, or amotinib resistance to gefitinib, more preferably to gefitinib or osimertinib.
  • the present invention provides a method for treating or preventing drug-resistant proliferative diseases, the method comprising administering a therapeutically effective dose of the antibody-drug conjugate of the present invention, and a pharmaceutically acceptable salt thereof, to a patient in need. Hydrates, solvates, stereoisomers or isotope labels or pharmaceutical compositions containing them; the drug-resistant proliferative disease is a drug-resistant disease associated with abnormal expression of HER2, HER3 or EGFR , including cancer.
  • the drug-resistant cancer is preferably a drug-resistant cancer caused by HER2, HER3 or EGFR gene mutation.
  • the cancer is preferably breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma , neuroblastoma, sarcoma, lung cancer (such as small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia , chronic myeloid leukemia, chronic lymphocytic leukemia) bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (such as Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma), More preferably, it is colon cancer, rectal cancer, lung cancer or pancreatic cancer.
  • leukemia such
  • the drug resistance is preferably resistant to receptor tyrosine kinase inhibitors, more preferably resistant to first-generation, second-generation or third-generation EGFR inhibitors, even more preferably resistant to gefitinib, errand Resistance to lotinib, icotinib, afatinib, dacomitinib, osimertinib, or amotinib, especially to gefitinib, afatinib, osimertinib, or amotinib resistance to gefitinib, more preferably to gefitinib or osimertinib.
  • the present invention provides the use of the linker-drug compound of the present invention, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels in the preparation of antibody-drug conjugates.
  • the antibody-drug conjugate of the present invention can be used alone or in combination with other antitumor agents.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody-drug conjugate of the present invention, its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels and other antitumor agents.
  • the present invention provides the preparation method of the antibody-drug conjugate described in the present invention, and its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope label, which comprises the following steps
  • reaction solution is purified by ultrafiltration using an ultrafiltration centrifuge tube.
  • MES 2-(N-morpholine) ethanesulfonic acid
  • the replacement factor is greater than 1000 times,
  • unconjugated linker-drug compounds and other low molecular weight reagents are removed to obtain purified antibody-drug conjugates.
  • the antibody-drug conjugates of the present invention all exhibit better anti-inflammatory effects.
  • Cell proliferation effect with significant anti-tumor effect, for drug-resistant tumors such as drug-resistant tumors caused by HER2, HER3 or EGFR gene mutations, especially for receptor tyrosine kinase inhibitor anti-tumor agents
  • the tumor has better inhibitory activity than DS-8201, better stability, including plasma stability and buffer stability, lower toxicity, better bystander effect, and wider therapeutic window.
  • the pH-dependent modification of the antibody is carried out, and the ADC obtained by coupling the modified antibody with a linker-drug compound has comparable tumor inhibitory activity to the original ADC in terms of drug efficacy, and shows anti-tumor activity on normal tissues in terms of safety. On target toxicity is reduced, and the ADC obtained after antibody optimization further expands the therapeutic window.
  • antibody-drug conjugate refers to linking an antibody (such as a monoclonal antibody) or an antibody fragment with a biologically active cytotoxic drug through a stable chemical linker compound.
  • linker-drug compound refers to a partial structure in the "antibody-drug conjugate” consisting of a linker compound and a drug compound.
  • the linker-drug compound is connected to the antibody through conventional coupling methods in the art, including: lysine coupling, reductive disulfide bond coupling between light and heavy chains and directional coupling (Beck A, Reichert JM. Antibody-drug conjugates: Present and future; MAbs, 2014, 6:15-17; McCombs J R, Owen S C. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. The AAPS journal, 20 15 , 17:339-351).
  • the present invention preferably couples through a reductive disulfide bond between the light and heavy chains, i.e.
  • one of the disulfide bond sites between the light and heavy chains two sites between the heavy chain and two sites between the heavy chain and the light chain
  • One or more thiol groups sulfur atoms of cysteine residues formed after reduction are reacted for linkage.
  • alkyl refers to a monovalent saturated aliphatic hydrocarbon group, straight-chain or branched, containing 1-20 carbon atoms, preferably containing 1-10 carbon atoms (i.e. C 1-10 Alkyl), further preferably containing 1-8 carbon atoms (C 1-8 alkyl), more preferably containing 1-6 carbon atoms (ie C 1-6 alkyl).
  • C 1-6 alkyl means that the group is an alkyl group, and the number of carbon atoms in the carbon chain is between 1-6 (specifically 1, 2, 3, 4, 5 or 6), examples include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1 ,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, etc.
  • the term "pharmaceutically acceptable salt” or “pharmaceutically acceptable salt” refers to any salt suitable for use within the scope of sound medical judgment without undue toxicity, irritation, allergic reaction, or in contact with the tissues of mammals, especially humans. Salts commensurate with a reasonable benefit/risk ratio, such as the pharmaceutically acceptable salts of amines, carboxylic acids, and other types of compounds are well known in the art. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or alone by reacting the free base or acid with a suitable reagent.
  • isotopically labeled means that the compounds of the present invention may exist in isotopically labeled or enriched form, containing one or more atoms having an atomic mass or mass number different from that found in nature The atomic mass or mass number of the most abundant atom. Isotopes can be radioactive or non-radioactive isotopes.
  • Isotopes commonly used for isotope labeling are: Hydrogen isotopes, 2 H and 3 H; Carbon isotopes: 13 C and 14 C; Chlorine isotopes: 35 Cl and 37 Cl; Fluorine isotopes: 18 F; Iodine isotopes: 123 I and 125 I ; Nitrogen isotopes: 13 N and 15 N; Oxygen isotopes: 15 O, 17 O and 18 O and sulfur isotope 35 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. Especially 2 H and 13 C are more widely used because of their easy labeling and convenient detection.
  • Isotopically labeled compounds are generally synthesized starting from labeled starting materials and carried out in the same manner as non-isotopically labeled compounds using known synthetic techniques.
  • solvate mean a physical association of a compound of the present invention with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding.
  • solvent molecules in solvates may exist in regular and/or disordered arrangements.
  • Solvates may contain stoichiometric or non-stoichiometric amounts of solvent molecules.
  • Solvate encompasses both solution-phase and isolatable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.
  • stereoisomer refers to compounds that have the same chemical structure but differ in the way the atoms or groups are arranged in space.
  • Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis/trans) isomers, atropisomers, and the like.
  • the resulting mixture of any stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, diastereoisomers on the basis of differences in the physicochemical properties of the components, for example, by chromatography method and/or fractional crystallization.
  • Figure 1 is the detection result of DP001-JSSW-001DAR value
  • Figure 2 is the detection result of DP001-ZW-002DAR value
  • Figure 3 is the detection result of DP001-JSSW-001DAR value
  • Figure 4 is partitumab-JSSW-001 DAR value detection results
  • Figure 5 is the detection result of partitumab-ZW-002DAR value
  • Figure 6 is the detection result of SWY2110-JSSW-001DAR value
  • Figure 7 is the detection result of SWY2111-JSSW-001DAR value
  • Figure 8 is the detection result of SWY2112-JSSW-001DAR value
  • Figure 9 is the detection result of SWY2113-JSSW-001DAR value
  • Figure 10 is the detection result of partitumab-Dxd DAR value
  • Figure 11 is the detection result of SWY2110-Dxd DAR value
  • Figure 12 is the detection result of SWY2110-Vc MMAE DAR value
  • Figure 13 is the effect of DP001-ADC on the tumor volume of HER2-expressed human breast cancer JIMT-1 cell xenografts in NU/NU mice
  • Figure 14 shows the effect of patritumab-ADC on the tumor volume of human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) cell xenografts in NU/NU mice
  • Figure 15 shows the effect of SWY2110-ADC on the tumor volume of human colorectal cancer DiFi cell xenografts in NU/NU mice
  • Figure 16 shows the effect of SWY2110-ADC on the tumor volume of human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) cell xenografts in NU/NU mice
  • Figure 17 is the detection result of SWY2110-ADC's inhibitory activity on DiFi cells
  • Figure 18 is the test result of SWY2110-ADC's inhibitory activity on PC9-GR cells
  • Figure 19 is the test result of DP001-ADC bystander effect
  • Figure 20 shows the effect of SWY2110-ADC on the tumor volume of human lung adenocarcinoma PC9-AR (PC9-Del19/T790M/C797S, Osimertinib-resistant cells) in NU/NU mice
  • Figure 21 shows the effect of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, SWY2113-ADC on tumor volume of human lung adenocarcinoma NCI-H1975 cell xenograft in NU/NU mice
  • Figure 22 shows the effect of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, SWY2113-ADC on the tumor volume of human breast cancer MDA-MB-468 cell xenograft in NOD-SCID mice
  • Figure 23 shows the effect of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, SWY2113-ADC on the tumor volume of human colorectal cancer DiFi cell xenografts in NU/NU mice
  • Figure 24 shows the effect of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on the tumor volume of human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) cell xenografts in NU/NU mice
  • Figure 25 shows the effect of patritumab-ADC on the tumor volume of human lung adenocarcinoma PC9-DTC (Del19/T790M/C797S, Osimertinib-resistant cells) in NOD-SCID mice
  • Figure 26 shows the effect of patritumab-ADC on the tumor volume of human colon cancer SW620 cells transplanted in NU/NU mice
  • Figure 27 is the test result of patritumab-ADC's inhibitory activity on DiFi cells
  • the structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) or/and liquid chromatography-mass chromatography (LC-MS) or/and liquid chromatography (HPLC).
  • NMR nuclear magnetic resonance
  • LC-MS liquid chromatography-mass chromatography
  • HPLC liquid chromatography
  • the starting materials in the examples of the present invention are known and commercially available, or can be synthesized using or following methods known in the art.
  • Antibodies of the present invention can be produced by hybridoma technology first described by Kohler et al., Nature (1975), or by recombinant DNA methods (US Patent No. 4,816,567, etc.).
  • the antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:9 and two light chains with the amino acid sequence shown in SEQ ID NO:10 is defined as DP001;
  • An antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:19 and two light chains with the amino acid sequence shown in SEQ ID NO:20 is defined as patritumab;
  • the antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:29 and two light chains with the amino acid sequence shown in SEQ ID NO:30 is defined as SWY2110;
  • the antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:34 and two light chains with the amino acid sequence shown in SEQ ID NO:30 is defined as SWY2111;
  • the antibody having two heavy chains with amino acid sequences shown in SEQ ID NO:37 and two light chains with amino acid sequences shown in SEQ ID NO:30 is defined as SWY2112;
  • the antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:40 and two light chains with the amino acid sequence shown in SEQ ID NO:30 is defined as SWY2113.
  • HER2 antibody DP001 (trastuzumab)
  • DP001 HCDR1 GFNIKDTYIH (SEQ ID NO: 1)
  • DP001 HCDR2 RIYPTNGYTRYAD (SEQ ID NO:2)
  • DP001 HCDR3 WGGDGFYAMDY (SEQ ID NO:3)
  • DP001 LCDR1 RASQDVNTAVA (SEQ ID NO: 4)
  • DP001 LCDR3 QQHYTTPPT (SEQ ID NO: 6)
  • Patritumab LV SEQ ID NO: 18
  • SWY2110 HCDR2 VIWSGGNTDYNTPFTS SEQ ID NO:22
  • SWY2110 HCDR3 ALDYYDYEFAY SEQ ID NO:23
  • SWY2110 LCDR2 YASESIS SEQ ID NO:25
  • SWY2111 HCDR1 DYDVH SEQ ID NO: 31
  • SWY2111 HCDR2 VIWSGGNTDYNTPFTS SEQ ID NO:22
  • SWY2111 HCDR3 ALDDYDYEFAY SEQ ID NO:32
  • SWY2111 LCDR1 RASQSIGTNIH SEQ ID NO:24
  • SWY2111 LCDR2 YASESIS SEQ ID NO:25
  • SWY2111 LCDR3 QQNNEWPTS SEQ ID NO:26
  • SWY2111 HV SEQ ID NO: 33
  • SWY2111 LV SEQ ID NO: 28
  • SWY2112 HCDR1 EYDVH SEQ ID NO: 35
  • SWY2112 HCDR2 VIWSGGNTDYNTPFTS SEQ ID NO:22
  • SWY2112 HCDR3 ALDDYDYEFAY SEQ ID NO:32
  • SWY2112 LCDR1 RASQSIGTNIH SEQ ID NO:24
  • SWY2112 LCDR2 YASESIS SEQ ID NO:25
  • SWY2112 LV SEQ ID NO: 28
  • SWY2113 HCDR1 HYDVH SEQ ID NO:38
  • SWY2113 HCDR2 VIWSGGNTDYNTPFTS SEQ ID NO:22
  • SWY2113 LCDR2 YASESIS SEQ ID NO:25
  • the first step the preparation of compound B2-1: add B1-1 (1.0g, 2.28mmol), anhydrous tetrahydrofuran (30mL), toluene (10ml), pyridine (0.5ml), lead tetraacetate ( 1.83g, 4.12mmol), the solution turned orange, heated to reflux, and the solution became colorless after one hour, and a white solid was generated. After three hours of reaction, the solution was filtered with diatomaceous earth, and the solid was rinsed with ethyl acetate, and the filtrate Dry using a rotary evaporator. The obtained crude product was purified using column chromatography to obtain a solid (640 mg). LC-MS [M+Na]+: m/z 391.1.
  • Step 4 Preparation of Compound A4-1: Add A3-1 (291mg, 0.60mmol) to a 100ml three-neck flask and dissolve in tetrahydrofuran: ethyl acetate (4mL/2mL), add Pd/C (10%w/w , 60 mg), replaced with hydrogen for 3 times, then put on a hydrogen balloon and stirred overnight. After the reaction, the reaction solution was passed through diatomaceous earth, rinsed with methanol, and the organic layer was concentrated to obtain the product A4-1. LC-MS [M+Na]+: m/z 399.1.
  • Step 5 Preparation of Compound A6-1: Add A4-1 (50mg, 0.13mmol, 1.3eq), Exitecan mesylate (A5-1) (50mg, 0.049mmol, 1.0 eq), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (48mg, 0.13mmol, 1.3eq), N,N-di Isopropylethylamine (36mg, 0.28mmol, 3.0eq) was dissolved in N,N-dimethylformamide (2ml), stirred at room temperature overnight, diluted with ethyl acetate and treated with half-saturated citric acid Washing with water, followed by washing with brine, washing with sodium bicarbonate solution, washing with brine, drying the organic layer with anhydrous sodium sulfate, filtering, removing anhydrous sodium sulfate, concentrating, using the prep-T
  • Step 6 Preparation of compound A7-1: Add A6-1 (56mg, 0.069mmol), N,N-dimethylformamide (2ml), piperidine (12mg, 0.14mmol) into a 25ml single-necked bottle, and stir After 2 hours, the solution was concentrated to give a white solid.
  • Step 1 Add compounds C1-1 (8g, 88.86mmol) and C2-1 (22.66g, 133.33mmol) into a 250ml reaction flask, add DMAc (40mL, 5v/w), stir to dissolve, and control the temperature to 0-10°C, then DIEA (34.45g, 266.6mmol) was added dropwise, after the dropwise addition, it was allowed to warm up to about 25°C naturally, and stirred at this temperature for 16 hours. After TLC monitoring the reaction of raw materials was complete.
  • DMAc 40mL, 5v/w
  • DIEA 34.45g, 266.6mmol
  • the second step under nitrogen protection, C4-1 (6g, 16.29mmol, 1.0eq) and C3-1 (3.23g, 17.92mmol, 1.1eq) were dissolved in THF (60mL, 10V/w) and cooled to 10 At ⁇ 15°C, TsOH (281mg, 1.63mmol, 0.1eq) was added, the addition was complete, and the reaction was carried out at 14-18°C for 4h, monitored by TLC, after the reaction was completed.
  • Step 3 Under nitrogen protection, dissolve C5-1 (2.8g, 5.74mmol, 1.0eq) in DMAc (28mL, 10V/w), cool down to 14-18°C, add DBU (436.6mg, 2.87mmol) dropwise , 0.5eq), and stirred at this temperature for 1.5h, TLC monitored the complete reaction of the raw materials, cooled to 0 ⁇ 10 ° C, added PPTS (721.23mg, 2.87mmol, 0.5eq), EDCI (1.1g, 5.74mmol, 1.0 eq), HOBT (775mg, 5.74mmol, 1.0eq) and C7-1 (2.45g, 4.88mmol, 0.85eq) were added, reacted at 0-10°C for 3-4h, and monitored by TLC after the reaction of C7-1 was complete.
  • DBU 436.6mg, 2.87mmol
  • EDCI 1.1g, 5.74mmol, 1.0 eq
  • HOBT 775mg, 5.74
  • Step 4 Under nitrogen protection, dissolve C8-1 (3.1g, 4.14mmol, 1.0eq) in DCM (46.5mL, 15V/w), add DBU (314.6mg, 2.07 mmol, 0.5eq), and stirred the reaction at this temperature for 16h, TLC monitored the complete reaction of raw materials.
  • Step 5 Under nitrogen protection, dissolve C9-1 (1.7g, 3.2mmol, 1.0eq) in water (25.5mL, 15V/w) and tert-butanol (8.5mL, 5V/w), stir to dissolve , replaced with nitrogen once, added Pd/C (0.34 g, 20% w/w), replaced with hydrogen three times, and hydrogenated at room temperature for 12 hours at 25-30° C., and the reaction of raw materials was monitored by LCMS. The reaction solution was filtered, the filter cake was washed with water (25mL*2), the filtrate was filtered again, concentrated to remove tert-butanol, and the water phase was directly lyophilized to obtain 1.5 g of white powdery solid C10-1, with a yield of 100%.
  • Step 6 Under nitrogen protection, dissolve C10-1 (1.4g, 3.2mmol, 1.0eq) in acetonitrile (14mL, 10V/w) and water (28mL, 20V/w), add C11-1 (1.86g , 6.097mmol, 1.1eq), stirred to dissolve, lowered the temperature to 0-10°C, added DIEA (330.24mg, 2.56mmol) dropwise, stirred and reacted at this temperature for 16h after the dropwise addition, and LC-MS monitored the complete reaction of raw materials. Add Na 2 HPO 4 (260mg) and NaH 2 PO 4 (5.6g) into 20mL of water, stir to dissolve and prepare a buffer solution, and cool the buffer solution to 0-5°C.
  • the system of DCM ⁇ DCM: MeOH 50:1 ⁇ 30:1 ⁇ 20:1 ⁇ 15:1 was passed through the column, and a yellow solid C12-1 was obtained.
  • Step 7 Under nitrogen protection, dissolve A5-1 (547.4mg, 1.03mmol, 1.0eq) in 5% anhydrous sodium sulfate water (6.5mL) and THF (7.8mL), stir and dissolve, Cool down to 0-10°C, add NMM (104mg, 1.03mmol, 1.0eq), and react at this temperature for 1h after addition.
  • Step 1 Preparation of compound A2-2: Add A1-2 (4.98g, 30mmol) and N,N-dimethylformamide (40ml) into a 100ml three-necked flask, stir at 0°C, add sodium hydride ( 1.26g, 31.5mmol), after the reaction solution continued to stir for 30 minutes, 2-(2-bromoethoxy)tetrahydro-2H-pyran (6.59g, 31.5mmol) was added, and then reacted overnight.
  • sodium hydride 1.26g, 31.5mmol
  • Step 6 Preparation of compound A9-2: Add A8-2 (30mg, 0.036mmol), N,N-dimethylformamide (2ml), piperidine (6mg, 0.07mmol) into a 25ml single-necked bottle, and stir After 2 hours, the solution was concentrated to give yellow solid A9-2 (30 mg). LC-MS [M+H]+: m/z 624.3.
  • Step 7 Preparation of compound ZW-002: Add A9-2 (30mg, 0.048mmol), C7 (23mg, 0.048mmol), 2-(7-azobenzotriazole)-N ,N,N',N'-tetramethyluronium hexafluorophosphate (28mg, 0.072mmol), N,N-diisopropylethylamine (31mg, 0.24mmol), in N,N-dimethyl Dissolve in amide (5ml), stir at room temperature for 1 hour, dilute with ethyl acetate (30ml), wash with ammonium chloride, then wash with brine, dry the organic layer with anhydrous magnesium sulfate, filter, remove the solid, and concentrate , and purified using the preparative liquid phase to obtain the product ZW-002 (4.2 mg).
  • the first step Dissolve B1-2 (24g, 169mmol, 1.0eq) in DMSO (1200mL, 50v/v), add B2-2 (43.35g, 253mmol, 5eq) dropwise under stirring, after addition, room temperature 20-30 °C reaction 16h.
  • reaction solution was poured into ice water (250mL, 10v/v), the temperature was controlled at 0-10 degrees, and EA (300mL, 12-15v/v) was added for extraction three times, and the organic phase was extracted with water (200mL, 8- 10v/v)*2 and saturated aqueous sodium chloride solution (150mL, 6-10v/v)*1 were washed separately, the organic phase was dried over anhydrous sodium sulfate (50g), concentrated by filtration, and passed through the column to obtain yellow oil B3-2.
  • the second step: B3-2 (14g, 66.6mmol, 1.0eq) was dissolved in THF (280mL, 20v/v), and B4-2 (29.47g, 79.92mmol, 1.2eq), TsOH (1.15g, 6.66mmol , 0.1eq), the addition was completed, and the reaction was stirred at room temperature 20-30°C for 3h.
  • reaction solution was poured into a mixture of ice water (500mL, 35-40v/v), EA (500mL, 35-40v/v), and the water phase was mixed with EA (500mL, 35-40v/v)*3
  • the organic layer was washed with saturated NaHCO 3 aqueous solution (250mL, 15-20v/v), water (250mL, 15-20v/v), each washed once, the organic layer was dried by adding anhydrous sodium sulfate (80g), and concentrated into a yellow oil , column chromatography gave white solid B5-2.
  • Step 3 Dissolve B5-2 (12g, 23.16mmol, 1.0eq) in DMAc (240mL, 20v/v), add DBU (1.76g, 11.8mmol, 0.5eq) dropwise, and react at 10-15°C for 60min , add PPTS (2.91g, 11.8mmol, 0.5eq), HOBt (3.12g, 23.16mmmol, 1.0eq), B7-2 (11.6g, 23.16mmmol, 1.0eq), EDCI (4.4g, 23.16mmmol, 1.0eq ), 10-15°C, react for 3h.
  • the reaction solution was poured into ice water (300mL, 25v/v), a mixture of methyltetrahydrofuran (500mL, 40-45v/v) (temperature control 0-10 degrees), and the water phase was filled with methyltetrahydrofuran ( 300mL, 25v/v)*3 extractions, combined organic phases, washed with saturated NaHCO 3 aqueous solution (200mL, 15-20v/v), water (200mL, 15-20v/v) each time, added anhydrous sodium sulfate to the organic phase (100g) was dried, filtered to remove the desiccant, and the filtrate was concentrated in a vacuum at 20-30 degrees to a yellow oil, and column chromatography gave white solid B8-2.
  • Step 4 Dissolve B8-2 (13g, 16.68mmol, 1.0eq) in ACN (130mL, 10v/v), add DBU (3.0g, 20.01mmol, 1.2eq) dropwise, and react at room temperature 20-30°C for 60min .
  • the fifth step: B9-2 (5.5g, 9.86mmol, 1.0eq) was dissolved in THF (82.5mL, 15v/v) and water (27.5mL, 5v/v), adding 10% palladium carbon (0.55g, weight ratio 0.1), add hydrogen protection, and react at 20-30°C for 16h.
  • Post-reaction treatment 1 The reaction solution was filtered to remove palladium carbon, added water (100mL, 18-20v/v), EA (100mL, 18-20v/v) * 2 extraction (organic phase was discarded), and the aqueous phase was recorded as A solution ( B10-2).
  • Operation 2 Add solution A (B10-2) (130mL) to the reaction flask, dissolve B11-2 (3.6g, 11.83mmol, 1.2eq) in ACN (49.5mL, 9v/v), add dropwise DIEA (1.27g, 9.86 mmol, 1.0eq), add 10-15 degrees and react for 5.0h.
  • Step 6 Anhydrous sodium sulfate (0.8g, 0.5v/v), water (16mL, 10v/v), THF (19.2mL, 12v/v), add A5-1 (1.6g, 3.0mmol, 1.0eq), lower the temperature by 5-10 degrees, dropwise add N-methylmorpholine (0.304g, 3.0mmol, 1.0eq), react at 5-10 degrees for 40min, then add B12-2 (2.4g, 3.6mmol, 1.2eq ), EDCI (0.863g, 4.5mmol, 1.5eq), ethyl 2-oxime cyanoacetate (0.214g, 1.5mmol, 0.5eq), and the reaction was completed at 5-10°C for 3.0h.
  • reaction solution was poured into 150mL of ice water, extracted with EA (150mL*3), washed once with 0.1M HCl (50mL), and water (150mL) each time, the organic phase was dried with 30g of anhydrous sodium sulfate, concentrated to dryness, and then column chromatography , to obtain pale yellow solid ZW-002.
  • the first step is to restore the antibody:
  • the DP001 antibody medium obtained after transient transfection of HEK293 cells expressing the plasmid encoding the light and heavy chain genes was replaced with PBS6.0/EDTA by known techniques to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0 mL) was put into a 1.5 mL polypropylene tube (tube), and 10 mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (17 ⁇ L; 2.5 equivalents to one molecule of antibody) and 1M dihydrogen phosphate were added thereto.
  • Potassium aqueous solution (Tianjin Guangfu Science and Technology Development Co., Ltd., 50 ⁇ L). After confirming that the pH of the solution was within 7.4 ⁇ 0.1, the disulfide bond in the antibody was reduced by incubating at 37° C. for 1 hour.
  • Ultrafiltration centrifuge tubes (Merck, Regenerated Cell ⁇ Lose (30kDa MWCO), 15mL sample volume) was used to purify the reaction solution by ultrafiltration.
  • Add 25mM 2-(N-morpholine)ethanesulfonic acid (MES) as a purification buffer to the reaction solution, pH 6.5, concentrate the solution to 1-2mL, add the purification buffer again, the replacement factor is greater than 1000 times,
  • MES 2-(N-morpholine)ethanesulfonic acid
  • the fourth step is the determination of the drug-antibody conjugation ratio (DAR) of the antibody-drug conjugate:
  • the loading volume is 10 ⁇ L
  • the gradient program is 0-3 minutes 27%-27%, 3-8 minutes 27%-35%, 8-25 minutes 35%-43%, 25-26 minutes 43%-95%, 26 - 95%-95% in 31 minutes, 95%-27% in 31-32 minutes, 27%-27% in 32-40 minutes.
  • L0 unlinked light chain
  • H0 heavy chain
  • drug-linked light chain light chain linked to a drug, L1
  • heavy chain heavy chain linked to a drug
  • H2 heavy chain linked to a drug
  • H3 the hydrophobicity increases with the number of linked drugs, so the elution is in the order of L0, L1, H0, H1, H2, H3 .
  • DAR values were calculated from the peak area at 280nm. The DAR value results are shown in Figure 1.
  • the first step is to restore the antibody:
  • the DP001 antibody medium obtained after transient transfection of HEK293 cells expressing the plasmid encoding the light and heavy chain genes was replaced with PBS6.0/EDTA by known techniques to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0 mL) was put into a 1.5 mL polypropylene tube (tube), and 100 mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (6.66 ⁇ L; 10 equivalents to one molecule of antibody) and 1M hydrogen phosphate were added thereto.
  • Dipotassium aqueous solution (Tianjin Guangfu Science and Technology Development Co., Ltd., 18 ⁇ L). After confirming that the pH of the solution was within 7.0 ⁇ 0.1, the disulfide bond in the antibody was reduced by incubating at 37° C. for 3 hours.
  • Example 2 Add the compound ZW-002 10mM dimethyl sulfoxide solution (87 ⁇ L; 13 equivalents to one molecule of antibody) obtained in Example 2 to the above solution at room temperature, mix well, and react at room temperature for 30 minutes, and the drug A linker is attached to the antibody. Next, 100 mM NAC (Bailingwei Technology Co., Ltd.) aqueous solution (10.2 ⁇ L) was added, and then stirred at room temperature for 20 minutes to terminate the reaction.
  • NAC Boardailingwei Technology Co., Ltd.
  • the first step is to restore the antibody:
  • the DP001 antibody medium obtained after transient transfection of HEK293 cells expressing the plasmid encoding the light and heavy chain genes was replaced with PBS6.0/EDTA by known techniques to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0 mL) was put into a 1.5 mL polypropylene tube (tube), and 100 mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (6.66 ⁇ L; 10 equivalents to one molecule of antibody) and 1M hydrogen phosphate were added thereto.
  • Dipotassium aqueous solution (Tianjin Guangfu Science and Technology Development Co., Ltd., 18 ⁇ L). After confirming that the pH of the solution was within 7.0 ⁇ 0.1, the disulfide bond in the antibody was reduced by incubating at 37° C. for 3 hours.
  • Example 2 Add the compound JSSW-001 10mM dimethyl sulfoxide solution (87 ⁇ L; 13 equivalents to one molecule of antibody) obtained in Example 1 to the above solution at room temperature, mix well, and react at room temperature for 30 minutes, and the drug A linker is attached to the antibody. Next, 100mM NAC (Bailingwei Technology Co., Ltd.) aqueous solution (10.2 ⁇ L) was added, and then stirred at room temperature for 20 minutes to terminate the reaction.
  • NAC Boardailingwei Technology Co., Ltd.
  • the first step is to restore the antibody:
  • the patritumab antibody medium obtained after transient transfection of HEK293 cells expressing and purifying the plasmid encoding the light and heavy chain genes was replaced with PBS6.0/EDTA by known techniques to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0 mL) was put into a 1.5 mL polypropylene tube (tube), and 100 mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (6.66 ⁇ L; 10 equivalents to one molecule of antibody) and 1M hydrogen phosphate were added thereto.
  • Dipotassium aqueous solution (Tianjin Guangfu Science and Technology Development Co., Ltd., 18 ⁇ L). After confirming that the pH of the solution was within 7.0 ⁇ 0.1, the disulfide bond in the antibody was reduced by incubating at 37° C. for 3 hours.
  • Example 2 Add the compound JSSW-001 10mM dimethyl sulfoxide solution (87 ⁇ L; 13 equivalents to one molecule of antibody) obtained in Example 1 to the above solution at room temperature, mix well, and react at room temperature for 30 minutes, and the drug A linker is attached to the antibody. Next, 100 mM NAC (Bailingwei Technology Co., Ltd.) aqueous solution (10.2 ⁇ L) was added, and then stirred at room temperature for 20 minutes to terminate the reaction.
  • NAC Boardailingwei Technology Co., Ltd.
  • the first step is to restore the antibody:
  • the patritumb antibody medium obtained after transient transfection of HEK293 cells expressing the plasmids encoding the light and heavy chain genes was replaced with PBS6.0/EDTA by known techniques to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0 mL) was put into a 1.5 mL polypropylene tube (tube), and 100 mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (6.66 ⁇ L; 10 equivalents to one molecule of antibody) and 1M hydrogen phosphate were added thereto.
  • Dipotassium aqueous solution (Tianjin Guangfu Science and Technology Development Co., Ltd., 18 ⁇ L). After confirming that the pH of the solution was within 7.0 ⁇ 0.1, the disulfide bond in the antibody was reduced by incubating at 37° C. for 3 hours.
  • Example 2 Add the compound ZW-002 10mM dimethyl sulfoxide solution (87 ⁇ L; 13 equivalents to one molecule of antibody) obtained in Example 2 to the above solution at room temperature, mix well, and react at room temperature for 30 minutes, and the drug A linker is attached to the antibody. Next, 100 mM NAC (Bailingwei Technology Co., Ltd.) aqueous solution (10.2 ⁇ L) was added, and then stirred at room temperature for 20 minutes to terminate the reaction.
  • NAC Boardailingwei Technology Co., Ltd.
  • the first step is to restore the antibody:
  • the SWY2110 antibody medium obtained after transient transfection of HEK293 cells expressing the plasmids encoding the light and heavy chain genes was replaced with PBS6.0/EDTA by known techniques to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0mL) was put into a 1.5mL polypropylene tube (tube), and 10mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (66.6uL; 10 equivalents to one molecule of antibody) and 1M hydrogen phosphate were added thereto Dipotassium aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.). After confirming that the pH of the solution was within 7.0 ⁇ 0.1, the disulfide bond in the antibody was reduced by incubating at 37° C. for 3 hours.
  • Example 2 Add the compound JSSW-001 10mM dimethyl sulfoxide solution (87uL; 13 equivalents to one molecule of antibody) obtained in Example 1 to the above solution at room temperature, mix well, react at room temperature for 30 minutes, and dissolve the drug A linker is attached to the antibody. Next, 100 mM NAC (Bailingwei Technology Co., Ltd.) aqueous solution (10.2 uL) was added, followed by stirring at room temperature for 20 minutes to terminate the reaction.
  • NAC Boardailingwei Technology Co., Ltd.
  • the DAR value determination method is the same as the fourth step of Example 3, and the DAR value result is shown in FIG. 6 .
  • SWY2110 and EGFR protein were modeled and docked to obtain antigen-antibody complexes, and the complexes D, E, and H scans were performed on the amino acids within the range, and several pH-dependent antibodies whose affinity decreased at pH 7.4 and remained basically unchanged or at a lower degree of affinity decrease at pH 6.0 were obtained.
  • MDA-MB-468 cells were digested into single cells using 0.25% trypsin-EDTA, and divided into multiple parts evenly, and the number of cells in each part was more than 10 6 .
  • the first step is to restore the antibody:
  • SWY2111 antibody medium was replaced with PBS6.0/EDTA to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0mL) was put into a 1.5mL polypropylene tube (tube), and 10mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (66.6uL; 10 equivalents to one molecule of antibody) and 1M hydrogen phosphate were added thereto Dipotassium aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.). After confirming that the pH of the solution was within 7.0 ⁇ 0.1, the disulfide bond in the antibody was reduced by incubating at 37° C. for 3 hours.
  • Example 2 Add the compound JSSW-001 10mM dimethyl sulfoxide solution (87uL; 13 equivalents to one molecule of antibody) obtained in Example 1 to the above solution at room temperature, mix well, react at room temperature for 30 minutes, and dissolve the drug A linker is attached to the antibody. Next, 100 mM NAC (Bailingwei Technology Co., Ltd.) aqueous solution (10.2 uL) was added, followed by stirring at room temperature for 20 minutes to terminate the reaction.
  • NAC Boardailingwei Technology Co., Ltd.
  • the DAR value determination method is the same as the fourth step of Example 3, and the DAR value result is shown in FIG. 7 .
  • the first step is to restore the antibody:
  • SWY2112 antibody medium was replaced with PBS6.0/EDTA to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0mL) was put into a 1.5mL polypropylene tube (tube), and 10mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (66.6uL; 10 equivalents to one molecule of antibody) and 1M hydrogen phosphate were added thereto Dipotassium aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.). After confirming that the pH of the solution was within 7.0 ⁇ 0.1, the disulfide bond in the antibody was reduced by incubating at 37° C. for 3 hours.
  • Example 2 Add the compound JSSW-001 10mM dimethyl sulfoxide solution (87uL; 13 equivalents to one molecule of antibody) obtained in Example 1 to the above solution at room temperature, mix well, react at room temperature for 30 minutes, and dissolve the drug A linker is attached to the antibody. Next, 100 mM NAC (Bailingwei Technology Co., Ltd.) aqueous solution (10.2 uL) was added, followed by stirring at room temperature for 20 minutes to terminate the reaction.
  • NAC Boardailingwei Technology Co., Ltd.
  • the DAR value determination method is the same as the fourth step of Example 3, and the DAR value result is shown in FIG. 8 .
  • the first step is to restore the antibody:
  • the obtained SWY2113 antibody medium was replaced with PBS6.0/EDTA to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0mL) was put into a 1.5mL polypropylene tube (tube), and 10mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (66.6uL; 10 equivalents to one molecule of antibody) and 1M hydrogen phosphate were added thereto Dipotassium aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.). After confirming that the pH of the solution was within 7.0 ⁇ 0.1, the disulfide bond in the antibody was reduced by incubating at 37° C. for 3 hours.
  • Example 2 Add the compound JSSW-001 10mM dimethyl sulfoxide solution (87uL; 13 equivalents to one molecule of antibody) obtained in Example 1 to the above solution at room temperature, mix well, react at room temperature for 30 minutes, and dissolve the drug A linker is attached to the antibody. Next, 100 mM NAC (Bailingwei Technology Co., Ltd.) aqueous solution (10.2 uL) was added, followed by stirring at room temperature for 20 minutes to terminate the reaction.
  • NAC Boardailingwei Technology Co., Ltd.
  • the DAR value determination method is the same as the fourth step of Example 3, and the DAR value result is shown in FIG. 9 .
  • the first step is to restore the antibody:
  • the DP001 antibody medium obtained after transient transfection of HEK293 cells expressing the plasmid encoding the light and heavy chain genes was replaced with PBS6.0/EDTA by known techniques to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0 mL) was put into a 1.5 mL polypropylene tube (tube), and 10 mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (17 ⁇ L; 2.5 equivalents to one molecule of antibody) and 1M dihydrogen phosphate were added thereto.
  • Potassium aqueous solution (Tianjin Guangfu Science and Technology Development Co., Ltd., 50 ⁇ L). After confirming that the pH of the solution was within 7.4 ⁇ 0.1, the disulfide bond in the antibody was reduced by incubating at 37° C. for 1 hour.
  • the first step is to restore the antibody:
  • the plasmids encoding the light and heavy chain genes were transiently transfected into HEK293 cells by known techniques to express and purify the DP001 antibody.
  • the medium was replaced with PBS6.0/EDTA to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0 mL) was put into a 1.5 mL polypropylene tube (tube), and 100 mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (6.66 ⁇ L; 10 equivalents to one molecule of antibody) and 1M hydrogen phosphate were added thereto.
  • Dipotassium aqueous solution (Tianjin Guangfu Science and Technology Development Co., Ltd., 18 ⁇ L). After confirming that the pH of the solution was within 7.0 ⁇ 0.1, the disulfide bond in the antibody was reduced by incubating at 37° C. for 3 hours.
  • the first step is to restore the antibody:
  • the patritumab antibody medium obtained after transient transfection of HEK293 cells expressing and purifying the plasmid encoding the light and heavy chain genes was replaced with PBS6.0/EDTA by known techniques to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0 mL) was put into a 1.5 mL polypropylene tube (tube), and 100 mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (6.66 ⁇ L; 10 equivalents to one molecule of antibody) and 1M hydrogen phosphate were added thereto.
  • Dipotassium aqueous solution (Tianjin Guangfu Science and Technology Development Co., Ltd., 18 ⁇ L). After confirming that the pH of the solution was within 7.0 ⁇ 0.1, the disulfide bond in the antibody was reduced by incubating at 37° C. for 3 hours.
  • the first step is to restore the antibody:
  • the SWY2110 antibody medium obtained after transient transfection of HEK293 cells expressing the plasmids encoding the light and heavy chain genes was replaced with PBS6.0/EDTA by known techniques to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0mL) was put into a 1.5mL polypropylene tube (tube), and 10mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (66.6uL; 10 equivalents to one molecule of antibody) and 1M hydrogen phosphate were added thereto Dipotassium aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.). After confirming that the pH of the solution was within 7.0 ⁇ 0.1, the disulfide bond of the antibody was reduced by incubating at 37° C. for 3 hours.
  • the first step is to restore the antibody:
  • the SWY2110 antibody medium obtained after transient transfection of HEK293 cells expressing the plasmids encoding the light and heavy chain genes was replaced with PBS6.0/EDTA by known techniques to prepare an antibody concentration of 10 mg/mL.
  • This solution (1.0mL) was put into a 1.5mL polypropylene tube (tube), and 10mM TCEP (Bailingwei Technology Co., Ltd.) aqueous solution (17uL; 2.5 equivalents to one molecule of antibody) and 1M dihydrogen phosphate were added thereto.
  • Potassium aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.). After confirming that the pH of the solution was within 7.4 ⁇ 0.1, the disulfide bond of the antibody was reduced by incubating at 37°C for 1 hour.
  • Vc MMAE mc-vc-PAB-MMAE
  • MES 2-(N-morpholine)ethanesulfonic acid
  • the fourth step is the determination of the drug-antibody conjugation ratio (DAR) of the antibody-drug conjugate:
  • the elution mobile phase A is 20mM PB, pH7.0, 1.5M(NH4) 2 SO 4 Aqueous solution
  • the mobile phase B liquid is 20mM PB, pH7.0, 25% isopropanol solution
  • the control flow rate is 0.8mL/min
  • the column temperature is 30°C
  • the detection wavelength is 280nm
  • the gradient program is 0-20 minutes
  • the mobile phase B from 0-100%, 20-25 minutes, 100% mobile phase B, 25-30 minutes, 100% mobile phase A.
  • DAR0 is unlinked, DAR2 is linked with 2 small molecules, DAR4 is linked with 4 small molecules, DAR6 is linked with 6 small molecules, and DAR8 is linked with 8 small molecules. Therefore, elution occurs in the order DAR0, DAR2, DAR4, DAR6, DAR8.
  • the DAR value is calculated according to the peak area at 280nm, where A represents the percentage of each peak area. The DAR value results are shown in Figure 12.
  • Test 1 Antibody-drug in vitro cell activity
  • the purpose of this test is to detect the effect of the antibody-drug conjugate of the present invention on SK-BR-3 (ATCC/HTB-30) tumor cells, human colorectal cancer cells DiFi, and human lung cancer gefitinib-resistant cells PC9-GR , Inhibitory activity of human lung cancer mutant cell PC-9 (Del19-T790M-C797S) tumor cell proliferation in vitro, treated cells with different concentrations of compounds in vitro, after culture, then added Resazurin (resazurin) to read the fluorescence of ex550nm/em610nm Value, four-parameter fitting process data to get IC50 value, thus calculate the biological activity of the compound.
  • Resazurin resazurin
  • Collect cells Collect SK-BR-3/MDA-MB-468 cells when the culture flask is nearly full.
  • the compound was diluted to an initial concentration of 18 ⁇ g/ml, and then diluted three times to obtain a total of 11 gradient solutions, and the twelfth column was a blank control.
  • the output parameter C is IC 50 in ng/mL.
  • SWY2110 monoclonal antibody SWY2110mAb
  • DiFi human colorectal cancer cells
  • PC9-GR human lung tinib-resistant cells
  • the inhibition rate of cell growth was calculated by the following formula:
  • Inhibition rate (%) ( normal OD value hole - OD value administration hole ) / ( normal OD value hole - OD value blank hole ) ⁇ 100%
  • Table 2 IC50 values of DP001-ADC on SK-BR-3 cells
  • the HER2-expressing human breast cancer JIMT-1 cell NU/NU mouse xenograft model was used to investigate the anti-tumor effect of antibody-drug conjugates in vivo.
  • i.v. intravenous injection.
  • the human breast cancer cell JIMT-1 was selected to construct the tumor xenograft model in nude mice.
  • the animals were divided into 4 groups (d0) according to the tumor volume in accordance with Table 4, with 6 animals in each group.
  • a single intravenous administration the administration volume is 10mL/kg
  • the solvent control group Vehicle
  • was given 0.9% sodium chloride injection observed for 13 days after administration, and compared the three antibody-drug conjugates in the table on HER2 Inhibition of xenografts in nude mice expressing cell line human breast cancer JIMT-1.
  • RTVxnd mean relative tumor volume on day n
  • TV X n the average tumor volume of the administration group on day n
  • TV X0 Average tumor volume of the administration group on day 0
  • TV M n average tumor volume on day n in the vehicle group
  • TV M0 mean tumor volume on day 0 in the vehicle group
  • human lung adenocarcinoma PC9-GR Garnier-Gefitinib drug-resistant cells
  • the human colorectal cancer DiFi cell NU/NU mouse xenograft model was used to investigate the anti-tumor effects of two SWY2110-ADC drugs in vivo.
  • i.v. intravenous injection.
  • human colorectal cancer DiFi cells were selected to construct nude mouse tumor xenograft models.
  • the animals were divided into 3 groups according to the tumor volume (d0), vehicle group and SWY2110-JSSW according to Table 8.
  • single intravenous administration the administration volume is 10mL/kg
  • vehicle control group Vehicle was given 0.9% chlorine Hua Na injection was observed for 27 days after administration
  • the inhibitory effect of the two SWY2110-ADCs in the table on human colorectal cancer DiFi nude mice xenografts was compared.
  • human lung adenocarcinoma PC9-GR (Gefitinib drug-resistant cells) cells were used to construct nude mouse tumor xenograft models.
  • the animals were divided into 5 groups according to the tumor volume in accordance with Table 10 (d0 ), every group of 6 animals, intravenous administration, administration once a week, continuous administration for 4 weeks, administration volume is 10mL/kg, vehicle control group (Vehicle) is given 0.9% sodium chloride injection, after administration After 28 days, the experiment was completed to compare the inhibitory effects of SWY2110 monoclonal antibody and three SWY2110-ADC drugs on human lung adenocarcinoma PC-9-GR (Gefitinib-resistant cells) transplanted tumors in nude mice.
  • the anti-tumor effect of SWY2110 was poor, and the tumor inhibition rate was only 20.3%.
  • the small-molecule toxin can cause the apoptosis of the negative cells co-cultured around, that is, the bystander effect.
  • the effect of the released small-molecule toxin on the growth of HEK293-Luc was judged by adding a chemiluminescent substrate.
  • DMEM complete medium add 10% Fetal Bovine Serum and 1% Penicilin/Streptomycin to the DMEM basal medium, mix thoroughly and store at 2-8°C.
  • the cells Take the cell culture flask out of the CO2 incubator and transfer it to the ultra-clean workbench or biological safety cabinet.
  • the cells are washed with PBS, digested with trypsin, and the cell growth medium is digested. After the digestion of the cell growth medium is terminated, the cell suspension is collected, and the cell density and survival rate. Take an appropriate amount of cell suspension and add it to an appropriate amount of fresh medium (with or without screening agent), and carry out passage expansion according to the cell growth characteristics and test requirements. After passage, the cells were placed in an incubator to continue culturing.
  • SK-BR-3 and MDA-MB-468 cells with adjusted density were mixed 1:1 with HEK293-Luc cell suspension; 50 ⁇ l per well was added to a 96-well transparent flat-bottom culture plate; the positive cell group was The mixed cells of SK-BR-3 and HEK293-Luc; the negative cell group is the mixed cells of MDA-MB-468 and HEK293-Luc;
  • Table 13 Inhibition rate of antibody-drugs of the present invention on fluorescent cells.
  • Different linker-drug compounds such as: Deruxtecan, JSSW-001 and ZW-002 compounds were tested at high temperature (40°C, 60°C), high humidity (25°C/75%RH and 25°C/92.5%RH), light (illuminance 4500Lux, near-ultraviolet energy 90 ⁇ w/cm 2 ) or 0.5% BSA-PBS for research, and samples were collected at different times after being placed under different conditions, and the concentration changes of the linker-drug compound in the samples were detected by LC-MS/MS method. The test results show that the stability of JSSW-001 and ZW-002 compounds is better.
  • PC9-AR human lung adenocarcinoma PC9-Del19/T790M/C797S (Osimertinib-resistant cells, hereinafter referred to as PC9-AR) cell xenograft model in NU/NU mice.
  • human lung adenocarcinoma PC9-Del19/T790M/C797S (Osimertinib - resistant cells) cells were used to construct nude mouse tumor xenograft models.
  • i.v. intravenous injection, i.v./1 time: administration once;
  • human lung adenocarcinoma NCI-H1975 human lung adenocarcinoma cell
  • mice were divided into 5 groups according to the tumor volume in accordance with Table 16 ( d0), 6 animals in each group, each dose group was given a single dose of 1 mg/kg.
  • Intravenous administration administration once, the administration volume is 10mL/kg, the vehicle control group (Vehicle) was given 0.9% sodium chloride injection, and the test was finished 21 days after administration, comparing SWY2110-JSSW-001, SWY2111-JSSW- Inhibitory effect of 001, SWY2112-JSSW-001, SWY2113-JSSW-001 on human lung adenocarcinoma NCI-H1975 xenografted tumors in nude mice.
  • the tumor inhibition rate reached 84.1%, 83.0%, 80.9% and 74.2% (P ⁇ 0.001), showing that the four ADCs all had an inhibitory effect on human lung glands.
  • Cancer NCI-H1975 tumor cells have a significant inhibitory effect. See Figure 21 and Table 17 for details.
  • Test 11 SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, SWY2113-ADC on human breast cancer MDA-MB-468 (human breast cancer cells) cell xenograft tumor efficacy test
  • i.v. intravenous injection, i.v./1 time: administration once;
  • human breast cancer MDA-MB-468 human breast cancer cells
  • mice were divided into 5 groups according to the tumor volume according to Table 18 (d0) , 8 animals in the control group, 5 animals in the experimental group, intravenous administration, administration once, the administration volume is 10mL/kg, vehicle control group (Vehicle) is given 0.9% sodium chloride injection, finishes 28 days after administration
  • vehicle control group Vehicle
  • the experiment investigated the anti-tumor effect of 4 ADC drugs in vivo.
  • i.v. intravenous injection, i.v./qw*2 times: administered twice a week;
  • human colorectal cancer DiFi human colorectal cancer cell
  • mice were divided into 5 groups (d0) according to the tumor volume according to Table 20.
  • Group 7 animals intravenous administration, administration twice, administration volume is 10mL/kg, vehicle control group (Vehicle) is given 0.9% sodium chloride injection, ends test 21 days after administration, investigates 4 ADC drugs Antitumor effects in vivo.
  • the human lung adenocarcinoma cell PC9-GR NU/NU mouse xenograft model was used to investigate the anti-tumor effects of SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 in vivo.
  • i.v. intravenous injection, i.v./1 time: administration once;
  • human lung adenocarcinoma cell PC9-GR cells were used to construct a mouse tumor xenograft model.
  • the animals were divided into 5 groups (d0) according to the tumor volume according to Table 22, with 6 animals in each group.
  • i.v. intravenous injection
  • qw*3w administered once a week for three weeks.
  • human lung adenocarcinoma PC9-DTC (Del19/T790M/C797S, Osimertinib- resistant cells) cells were selected to construct a tumor xenograft model in nude mice.
  • the vehicle control group (Vehicle) was given 0.9% sodium chloride injection, and the test was completed 26 days after administration.
  • human colon cancer SW620 cells were selected to construct nude mouse tumor xenograft models.
  • the animals were divided into three groups (d0) according to the tumor volume according to Table 26.
  • the human colorectal cancer DiFi cell NU/NU mouse xenograft model was used to investigate the anti-tumor effects of two patritumab-ADC drugs in vivo.
  • i.v. intravenous injection.
  • human colorectal cancer DiFi cells were selected to construct nude mouse tumor xenograft models.
  • the animals were divided into 3 groups (d0) according to the tumor volume according to Table 28.
  • HGB hemoglobin
  • WBC white blood cell count
  • LYMP absolute number of lymphocytes.

Abstract

一种靶向HER2、HER3、EGFR的抗体的偶联药物，及其药用盐、水合物、溶剂合物或同位素标记的类似物，及其用途和制备方法。

Description

抗体-药物偶联物及其用途 技术领域
本发明涉及具有抗肿瘤作用的喜树碱衍生物的抗体-药物偶联物及其用途。
背景技术
抗体-药物偶联物(Antibody-drug Conjugate，ADC)由三个不同的组件(抗体、接头和药物)组成，ADC技术是通过接头(linker)将抗体和药物分子偶联在一起，利用抗体的特异性靶向运输药物分子到靶组织发挥作用，降低药物的系统性毒副作用，扩展药物治疗窗和提高抗体治疗潜能。
作为抗肿瘤性的低分子化合物，喜树碱衍生物已知可作为拓扑异构酶Ⅰ抑制剂而呈现抗肿瘤作用。人们一直致力于寻找高效、低毒的喜树碱衍生物，至今，已经有一系列半合成和全合成的喜树碱衍生物出现并进入临床应用或临床试验阶段。
第一三共的ADC药物DS-8201，使用细胞毒性药物(payload)DXd，具有独特的作用机制，与常见化疗药物伊立替康(irinotecan)相比，活性提高10倍；DXd具有很强的渗透细胞膜的能力，这使其在杀伤吞入ADC的癌细胞之后，能够杀死附近的癌细胞，产生“旁观者效应”(bystander effect)；在血液中的半衰期显著缩短，有助于减少毒副作用的产生；连接子有着很高的稳定性，非肿瘤组织不会受到毒性药物的影响；能够特异性地被肿瘤中高度表达的溶酶体蛋白酶切割；可在一个抗体分子上偶联多个细胞毒性药物，提高药物抗体比例(Drug Antibody Ratio,DAR)。为ADC药物的开发提供了新的研究方向。
Figure PCTCN2022132627-appb-000001
WO2020063673A1和WO2020063676A1公开了一些依喜替康类似物及其配体-药物偶联物，其中的依喜替康类似物对SK-BR-3细胞和U87细胞具有增殖抑制 活性。
CN111689980A公开了一些依喜替康类似物及其抗体-药物偶联物，但是该申请仅记载了依喜替康类似物和SN38的细胞活性，根据其记载的活性，大部分化合物显示相当于SN38的活性，对于抗体药物偶联物，其未公开抗体-药物偶联物所用抗体，可见该专利申请仅对依喜替康类似物的活性做了初步研究，未关注其他方面的效果。
ADC药物连接的毒素毒性强，形成ADC药物后，治疗窗口也较窄。由第一三共开发的依喜替康的ADC药物DS-8021，已成功上市。针对HER2靶点，DS-8021用单个抗体连接8个毒素的形式。但是，在后续针对Trop2靶点的临床试验中设计的药物，因为安全性的问题，降低了抗体药物比。但是偶联药物数目的减少，同时也会减少ADC药物的治疗效果。
前药和ADC，需要各种酶和靶点，这就造成了个体差异比较大，导致病人对前药的反应也参差不齐，同时易形成毒性。为了解决上述问题，开发高效低毒的抗体-喜树碱衍生物ADC以增加治疗效果是我们研究的方向。
表皮生长因子受体(HER)家族包含4个结构相似的受体分子，ErbB1(EGFR)、ErbB2(HER2)、ErbB3(HER3)和ErbB4(HER4)，同属于受体酪氨酸激酶。
表皮生长因子受体(Epidermal Growth Factor Receptor，EGFR，也称为HER1，ErbB1)是由1186个氨基酸残基组成，分子量为170kDa的跨膜糖蛋白，具有酪氨酸激酶活性。EGFR表达于许多上皮组织，包括皮肤和毛囊。正常状态下EGFR是单体，当其与相关配体如表皮生长因子(EGF)、转化生长因子(TGF-α)等结合后，形成同源/异源二聚体，二聚体发生磷酸化进而激活多种下游信号通路，促进细胞增殖、血管生成、转移、侵袭和抑制细胞凋亡。很多实体瘤发现过度表达EGFR，如结直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌和食管癌等。使EGFR成为良好的肿瘤治疗靶点。
目前抗EGFR药物主要包括酪氨酸激酶抑制剂(TKI)、抗EGFR的单克隆抗体(mAb)和抗体-药物偶联物等。TKI与腺苷5'三磷酸(ATP)竞争结合EGFR的细胞内催化结构域，从而抑制EGFR自身磷酸化和下游信号传导。由于EGFR易突变的特点，EGFR-TKIs竞争结合的方式也由第一代的非选择性可逆结合(吉非替尼、厄洛替尼、埃克替尼为代表)发展到第二代非选择性不可逆结合(阿法替尼和达克替尼)，一直到第三代选择性不可逆结合(奥希替尼和阿美替尼为代表)，EGFR TKI 结合能力越来越强，选择性越来越高，但EGFR其他罕见突变(例如20号染色体外显子插入突变)仍然是治疗盲区，所以需要其他的治疗方法。
抗EGFR单克隆抗体或者双特异性抗体通过与配体竞争性结合EGFR细胞外结构域结合，来阻断配体诱导的EGFR酪氨酸激酶活化，国内已获批上市的EGFR类单抗药物包括西妥昔单抗和尼妥珠单抗，临床数据显示EGFR单抗仅对KRAS野生型(KRAS wild type)有疗效，对KRAS突变体没有展现出肿瘤抑制活性。
抗体-药物偶联物通过结合肿瘤细胞表面EGFR而内吞，释放小分子毒素而杀伤肿瘤细胞。目前乐普生物旗下的美雅珂公司MRG003是国内首款进入临床阶段的EGFR ADC药物，由人源化抗EGFR单抗JMT101与毒素微管蛋白抑制剂MMAE通过可降解的mc-vc-PAB(简称Vc)连接子偶联而成。Ib期临床结果显示非小细胞肺癌患者的客观缓解率(ORR,Objective response rate)为40％(4/10)，鼻咽癌患者的ORR为44％(4/9)，展现良好的药效。但是MRG003 Ib期入组患者为EGFR阳性，且经多种治疗手段依然进展的晚期或转移性结直肠癌、头颈部鳞状细胞癌、鼻咽癌，临床试验数据并没有阐述MRG003对EGFR-TKI耐药患者是否具有药效(phase I dose escalation and dose expansion study of anti-EGFR ADC MRG003 in patients with advanced solid tumors.doi.org/10.1016/j.annonc.2021.08.1315)。为了解决EGFR-TKI耐药问题，开发安全、有效的ADC药物是我们研究的方向。
ADC药物靶向的靶抗原若在正常组织表达广泛，很可能造成靶点介导的on-target毒性，从而限制ADC药物的临床开发。为了尽可能降低ADC药物的on-target毒性，条件激活型ADC以降低靶点毒性应运而生。肿瘤微环境在理化性质方面与人体正常内环境存在着许多不同点，比较显著的是其低氧、低PH以及高压。正是因为这些特点，使得肿瘤微环境中存在大量的生长因子、细胞趋化因子和各种蛋白水解酶所产生的免疫炎性反应，这种特性十分利于肿瘤的增殖、侵袭、粘附、血管生成以及抗放射化疗。其中肿瘤在缺氧条件下进行葡萄糖分解，糖酵解产生大量乳酸而引起了pH的下降。目前，pH依赖性ADC药物的开发已经应用于临床，BioAtla公司目前有2个ADC项目在临床阶段，分别是AXL靶向性的BA3011和ROR2靶向性的BA3021，从该公司公布的早期数据显示，没有出现明显的靶点相关毒性，可见pH依赖性抗体技术能够降低ADC药物的靶向毒性。
pH依赖性抗体改造的目的是使抗体改造后，构建的ADC分子在健康组织 或循环系统中不能结合或较低结合抗原，而在肿瘤微环境中保持与抗原的结合能力，从而降低ADC药物的靶向毒性，扩展治疗窗。进行抗体改造，开发高效、低毒的ADC药物是我们的研究方向。
发明内容
本发明提供式I所示的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物：
Figure PCTCN2022132627-appb-000002
其中Ab为靶向HER2、HER3或EGFR的抗体或其抗原结合片段，R选自C 1-6烷基，n选自1-8的整数或1-8的小数。
进一步，n选自4-8的整数或4-8的小数。
进一步，当n为选自1-8的整数时，其可以为1、2、3、4、5、6、7、8；当n为选自4-8的整数时，其可以为4、5、6、7、8。
进一步，当n为小数时，其指每个抗体单元缀合的接头-药物分子个数的平均数。
在本发明的一个优选实施例中，R选自C 1-3烷基。
在本发明的一个优选实施例中，R选自甲基、乙基、丙基、异丙基。
在本发明的一个优选实施例中，R选自甲基。
进一步本发明提供式Ia或式Ib所示的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物：
Figure PCTCN2022132627-appb-000003
其中，式Ia和式Ib中，Ab、R和n的定义如式I。
进一步本发明提供式I-1、Ia-1和式Ib-1所示的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物：
Figure PCTCN2022132627-appb-000004
Figure PCTCN2022132627-appb-000005
其中，式I-1、式Ia-1和式Ib-1中，Ab和n的定义如式I。
本发明的另一方面，提供式II所示的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物：
Figure PCTCN2022132627-appb-000006
其中，Ab为靶向HER2，HER3或EGFR的抗体，n选自1-8的整数或1-8的小数。
进一步，n选自4-8的整数或4-8的小数。
进一步，当n为选自1-8的整数时，其可以为1、2、3、4、5、6、7、8；当n 为选自4-8的整数时，其可以为4、5、6、7、8。
进一步，当n为小数时，其指每个抗体单元缀合的接头-药物分子个数的平均数。
在本发明的一个优选实施例中，上述抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物中，所述Ab为靶向HER2的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中，重链可变区序列包括重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，轻链可变区包括轻链互补决定区1(LCDR1)、轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，其中HCDR1的氨基酸序列如SEQ ID NO:1所示，HCDR2的氨基酸序列如SEQ ID NO:2所示，HCDR3的氨基酸序列如SEQ ID NO:3所示，和/或LCDR1的氨基酸序列如SEQ ID NO:4所示，LCDR2的氨基酸序列如SEQ ID NO:5所示，LCDR3的氨基酸序列如SEQ ID NO:6所示。
进一步优选地，所述Ab为靶向HER2的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中HV的氨基酸序列如SEQ ID NO:7所示，LV的氨基酸序列如SEQ ID NO:8所示。
更优选地，所述Ab为靶向HER2的抗体或其抗原结合片段时，其包括重链(HC)和轻链(LC)，其中HC的氨基酸序列如SEQ ID NO:9所示，LC的氨基酸序列如SEQ ID NO:10所示。
在本发明的一个优选实施例中，所述Ab为靶向HER3的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中，重链可变区序列包括重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，轻链可变区包括轻链互补决定区1(LCDR1)、轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，其中HCDR1的氨基酸序列如SEQ ID NO:11所示，HCDR2的氨基酸序列如SEQ ID NO:12所示，HCDR3的氨基酸序列如SEQ ID NO:13所示，和/或LCDR1的氨基酸序列如SEQ ID NO:14所示，LCDR2的氨基酸序列如SEQ ID NO:15所示，LCDR3的氨基酸序列如SEQ ID NO:16所示。
进一步优选地，所述Ab为靶向HER3的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中HV的氨基酸序列如SEQ ID NO:17所示，LV的氨基酸序列如SEQ ID NO:18所示。
更优选地，所述Ab为靶向HER3的抗体或其抗原结合片段时，其包括重链(HC)和轻链(LC)，其中HC的氨基酸序列如SEQ ID NO:19所示，LC的氨基酸序列如SEQ ID NO:20所示。
在本发明的一个优选实施例中，所述Ab为靶向EGFR的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中，重链可变区序列包括重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，轻链可变区包括轻链互补决定区1(LCDR1)、轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，其中HCDR1的氨基酸序列如SEQ ID NO:21所示，HCDR2的氨基酸序列如SEQ ID NO:22所示，HCDR3的氨基酸序列如SEQ ID NO:23所示，和/或LCDR1的氨基酸序列如SEQ ID NO:24所示，LCDR2的氨基酸序列如SEQ ID NO:25所示，LCDR3的氨基酸序列如SEQ ID NO:26所示。
在本发明的一个优选实施例中，所述Ab为靶向EGFR的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中HCDR1的氨基酸序列如SEQ ID NO:31或SEQ ID NO:35或SEQ ID NO:38所示，HCDR2的氨基酸序列如SEQ ID NO:22所示，HCDR3的氨基酸序列如SEQ ID NO:23或SEQ ID NO:32所示，和/或LCDR1的氨基酸序列如SEQ ID NO:24所示，LCDR2的氨基酸序列如SEQ ID NO:25所示，LCDR3的氨基酸序列如SEQ ID NO:26所示。
在本发明的一个优选实施例中，所述Ab为靶向EGFR的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中HCDR1的氨基酸序列如SEQ ID NO:31或SEQ ID NO:35或SEQ ID NO:38所示，HCDR2的氨基酸序列如SEQ ID NO:22所示，HCDR3的氨基酸序列如SEQ ID NO:32所示，和LCDR1的氨基酸序列如SEQ ID NO:24所示，LCDR2的氨基酸序列如SEQ ID NO:25所示，LCDR3的氨基酸序列如SEQ ID NO:26所示。
进一步优选地，所述Ab为靶向EGFR的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中HV的氨基酸序列如SEQ ID NO:27所示，LV的氨基酸序列如SEQ ID NO:28所示。
进一步优选地，所述Ab为靶向EGFR的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中HV的氨基酸序列如SEQ ID NO:33或SEQ ID NO:36或SEQ ID NO:39所示，LV的氨基酸序列如SEQ ID NO:28所示。
更优选地，所述Ab为靶向EGFR的抗体或其抗原结合片段时，其包括重链(HC)和轻链(LC)，其中HC的氨基酸序列如SEQ ID NO:29所示，LC的氨基酸序列如SEQ ID NO:30所示。
更优选地，所述Ab为靶向EGFR的抗体或其抗原结合片段时，其包括重链(HC)和轻链(LC)，其中HC的氨基酸序列如SEQ ID NO:34或SEQ ID NO:37或SEQ ID NO:40所示，LC的氨基酸序列如SEQ ID NO:30所示。
当Ab为靶向HER2、HER3或EGFR的抗体时，所述抗体优选为单克隆抗体，所述单克隆抗体优选选自人源抗体、人源化抗体、嵌合抗体。
当Ab为靶向HER2、HER3或EGFR的抗原结合片段时，所述抗原结合片段优选选自Fab’、(Fab’) 2、Fab、Fv、scFv、dAb。
本发明中，抗体氨基酸序列的编号规则采用Kabat编号规则。
本发明还提供未经偶联的上述任意抗体(Ab)或其抗原结合片段。
进一步本发明提供以下的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物：
Figure PCTCN2022132627-appb-000007
Figure PCTCN2022132627-appb-000008
Figure PCTCN2022132627-appb-000009
Figure PCTCN2022132627-appb-000010
Figure PCTCN2022132627-appb-000011
Figure PCTCN2022132627-appb-000012
Figure PCTCN2022132627-appb-000013
Figure PCTCN2022132627-appb-000014
上述抗体-药物偶联物中：
DP001为具有两条如SEQ ID NO:9所示氨基酸序列的重链和两条如SEQ ID NO:10所示氨基酸序列的轻链的抗体；
Patritumab为具有两条如SEQ ID NO:19所示氨基酸序列的重链和两条如SEQ ID NO:20所示氨基酸序列的轻链的抗体；
SWY2110为具有两条如SEQ ID NO:29所示氨基酸序列的重链和两条如SEQ ID NO:30所示氨基酸序列的轻链的抗体；
SWY2111为具有两条如SEQ ID NO:34所示氨基酸序列的重链和两条如SEQ ID NO:30所示氨基酸序列的轻链的抗体；
SWY2112为具有两条如SEQ ID NO:37所示氨基酸序列的重链和两条如SEQ ID NO:30所示氨基酸序列的轻链的抗体；
SWY2113为具有两条如SEQ ID NO:40所示氨基酸序列的重链和两条如SEQ ID NO:30所示氨基酸序列的轻链的抗体；
n选自1-8的整数或1-8的小数。
进一步，n选自4-8的整数或4-8的小数。
进一步，当n为选自1-8的整数时，其可以为1、2、3、4、5、6、7、8；当n为选自4-8的整数时，其可以为4、5、6、7、8。
本发明的另一方面，提供式III所示的接头-药物化合物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物：
Figure PCTCN2022132627-appb-000015
其中，R的定义同式I。
进一步本发明提供式IIIa和式IIIb所示的接头-药物化合物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物：
Figure PCTCN2022132627-appb-000016
其中，式IIIa和式IIIb中，R的定义同式III。
进一步本发明提供下式的接头-药物化合物，及其药用盐、水合物、溶剂合 物、立体异构体或同位素标记物：
Figure PCTCN2022132627-appb-000017
进一步本发明提供一种药物组合物，其包含本发明所述的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物。所述药物组合物 进一步包括药学上可接受的辅料和载体。
本发明提供本发明所述的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物在制备用于治疗增殖性疾病的药物中的用途。所述增殖性疾病优选为与HER2、HER3或EGFR异常表达相关的疾病，包括癌症，所述癌症选自乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、肺癌(例如小细胞肺癌和非小细胞肺癌)、结肠癌、直肠癌、结直肠癌、白血病(例如急性淋巴细胞白血病、急性髓细胞白血病、急性早幼粒细胞白血病、慢性髓细胞白血病、慢性淋巴细胞白血病)骨癌、皮肤癌、甲状腺癌、胰腺癌或淋巴瘤(例如霍奇金淋巴瘤、非霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤)。
本发明提供一种用于治疗或预防增殖性疾病的方法，该方法包括向需要其患者施用治疗有效剂量的本发明的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物或包含其的药物组合物；所述增殖性疾病优选为与HER2、HER3或EGFR异常表达相关的疾病，包括癌症。所述癌症选自乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、肺癌(例如小细胞肺癌和非小细胞肺癌)、结肠癌、直肠癌、结直肠癌、白血病(例如急性淋巴细胞白血病、急性髓细胞白血病、急性早幼粒细胞白血病、慢性髓细胞白血病、慢性淋巴细胞白血病)骨癌、皮肤癌、甲状腺癌、胰腺癌或淋巴瘤(例如霍奇金淋巴瘤、非霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤)。
本发明提供本发明所述的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物在制备用于治疗产生耐药性的增殖性疾病的药物中的用途。所述产生耐药性的增殖性疾病为产生耐药性的与HER2、HER3或EGFR异常表达相关的疾病，包括癌症。所述产生耐药性的癌症优选为HER2、HER3或EGFR基因突变导致的耐药癌症，所述癌症优选乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、肺癌(例如小细胞肺癌和非小细胞肺癌)、结肠癌、直肠癌、结直肠癌、白血病(例如急性淋巴细胞白血 病、急性髓细胞白血病、急性早幼粒细胞白血病、慢性髓细胞白血病、慢性淋巴细胞白血病)骨癌、皮肤癌、甲状腺癌、胰腺癌或淋巴瘤(例如霍奇金淋巴瘤、非霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤)，进一步优选为结肠癌、直肠癌、肺癌或胰腺癌。其中所述耐药性优选对受体酪氨酸激酶抑制剂耐药，进一步优选为对第一代、第二代或第三代EGFR抑制剂耐药，更进一步优选对吉非替尼、厄洛替尼、埃克替尼、阿法替尼、达克替尼、奥希替尼或阿美替尼耐药，特别是对吉非替尼、阿法替尼、奥希替尼或阿美替尼耐药，更优选为对吉非替尼或奥希替尼耐药。
本发明提供一种用于治疗或预防产生耐药性的增殖性疾病的方法，该方法包括向有需要的患者施用治疗有效剂量的本发明的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物或包含其的药物组合物；所述产生耐药性的增殖性疾病为产生耐药性的与HER2、HER3或EGFR异常表达相关的疾病，包括癌症。所述产生耐药性的癌症优选为HER2、HER3或EGFR基因突变导致的耐药癌症。所述癌症优选乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、肺癌(例如小细胞肺癌和非小细胞肺癌)、结肠癌、直肠癌、结直肠癌、白血病(例如急性淋巴细胞白血病、急性髓细胞白血病、急性早幼粒细胞白血病、慢性髓细胞白血病、慢性淋巴细胞白血病)骨癌、皮肤癌、甲状腺癌、胰腺癌或淋巴瘤(例如霍奇金淋巴瘤、非霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤)，进一步优选为结肠癌、直肠癌、肺癌或胰腺癌。其中所述耐药性优选对受体酪氨酸激酶抑制剂耐药，进一步优选为对第一代、第二代或第三代EGFR抑制剂耐药，更进一步优选对吉非替尼、厄洛替尼、埃克替尼、阿法替尼、达克替尼、奥希替尼或阿美替尼耐药，特别是对吉非替尼、阿法替尼、奥希替尼或阿美替尼耐药，更优选为对吉非替尼或奥希替尼耐药。
本发明提供本发明所述的接头-药物化合物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物在制备抗体-药物偶联物中的用途。
本发明的抗体-药物偶联物可以单独使用，也可以与其它抗肿瘤剂组合使用。
本发明进一步提供一种药物组合物，包含本发明的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物和其它抗肿瘤剂。
本发明提供本发明所述的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物的制备方法，其包括如下步骤
S1：抗体的还原；
具体为：将抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。按照还原量加入适量10mM TCEP水溶液及1M磷酸氢二钾水溶液，确认了该溶液的pH为正确后，于37℃孵育数小时，由此将抗体内的二硫键还原。
S2：抗体与接头-药物化合物的偶联；
具体为：在室温下向上述溶液中添加适量二甲基亚砜溶液溶解的10mM化合物，混匀，在室温下反应0.5-4小时，将接头-药物化合物连接于抗体。接下来，添加100mM NAC水溶液，进而在室温下搅拌，终止反应。
S3：抗体-药物偶联物的纯化；
具体为：使用超滤离心管对反应液进行超滤纯化。向反应液中加入作为纯化缓冲液的25mM 2-(N-吗啉)乙磺酸(MES)，pH＝6.5，将溶液浓缩至1-2mL，再次加入纯化缓冲液，置换倍数大于1000倍，由此，将未偶联的接头-药物化合物及其他低分子量试剂除去，得到纯化的抗体-药物偶联物。
本发明的抗体-药物偶联物相比现有技术已知的抗体-药物偶联物如DS-8201或目前在不同靶点效果较好的实验样品作为对照，均展现出具有更好的抗细胞增殖效果，具有显著的抑瘤效果，对产生耐药性的肿瘤如对HER2、HER3或EGFR基因突变导致的耐药肿瘤，特别是对受体酪氨酸激酶抑制剂类抗肿瘤剂耐药的肿瘤具有优于DS-8201的抑制活性，具有更好的稳定性，包括血浆稳定性和缓冲液稳定性，具有更低的毒性，具有更好的旁观者效应，具有更宽的治疗窗口。本发明对抗体进行pH依赖性改造，将改造后的抗体与接头-药物化合物偶联后获得的ADC，在药效方面与原ADC具有相当的肿瘤抑制活性，在安全性方面显示对正常组织的on target毒性减轻，抗体经优化后获得的ADC进一步扩大了治疗窗。
定义
除另有规定外，术语“抗体-药物偶联物”是指将抗体(如单克隆抗体)或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒素药物相连。
除另有规定外，术语“接头-药物化合物”是指“抗体-药物偶联物”中由接头化合物和药物化合物组成的部分结构。
本发明中所述接头-药物化合物与所述抗体通过本领域常规的偶联方式进行连接，包括：赖氨酸偶联、轻重链间还原性二硫键偶联和定向偶联(Beck A,Reichert JM.Antibody-drug conjugates:Present and future；MAbs,2014,6:15-17；McCombs J R,Owen S C.Antibody drug conjugates:design and selection of linker,payload and conjugation chemistry.The AAPS journal,2015,17:339-351)。本发明优选通过轻重链间还原性二硫键偶联，即轻重链间二硫键位点(重链之间的两个位点，重点与轻链之间的两个位点)中的一个或多个被还原后形成的硫醇基(半胱氨酸残基的硫原子)反应进行连接。
除另有规定外，术语“烷基”指一价饱和脂肪族烃基团，包含1-20个碳原子的直链或支链基团，优选包含1-10个碳原子(即C 1-10烷基)，进一步优选包含1-8个碳原子(C 1-8烷基)，更优选包含1-6个碳原子(即C 1-6烷基)。例如“C 1-6烷基”指的是该基团为烷基，且碳链上的碳原子数量在1-6之间(具体地为1个、2个、3个、4个、5个或6个)，实例包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、新戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基等。
除另有规定外，术语“药物上可接受的盐”或“可药用盐”是指在合理医学判断范围内适用于与哺乳动物特别是人的组织接触而无过度毒性、刺激、过敏反应等并与合理的效益/风险比相称的盐，比如胺、羧酸和其他类型化合物的医学上可接受的盐在所属领域中是被熟知的。可以在本发明化合物的最终分离和纯化期间原位制备所述盐，或单独通过将游离碱或游离酸与合适的试剂反应制备所述盐。
除另有规定外，术语“同位素标记物”是指本发明的化合物可以以同位素示踪的或富集形式存在，含有一个或多个原子，这些原子的原子量或质量数不同于自然界中发现的最大量的原子的原子量或质量数。同位素可以是放射性或非放射性的同位素。通常用作同位素标记的同位素是：氢同位素， 2H和 3H；碳同位素： 13C和 14C；氯同位素： 35Cl和 37Cl；氟同位素： 18F；碘同位素： 123I和 125I；氮同位素： 13N和 15N；氧同位素： 15O, 17O和 18O和硫同位素 35S。这些同位素标记化合物可以用来研究药用分子在组织中的分布情况。尤其是 2H和 13C，由于它们容易标记且方便检测，运用更为广泛。某些重同位素，比如重氢(2H)，的取代能增强代谢的稳定性，延长半衰期从而达到减少剂量的目的而提供疗效优势的。同位素标记 的化合物一般从已被标记的起始物开始，用已知的合成技术象合成非同位素标记的化合物一样来完成其合成。
除另有规定外，术语“溶剂合物”、“溶剂化物”意指本发明化合物与一个或多个溶剂分子(无论有机的还是无机的)的物理缔合。该物理缔合包括氢键。在某些情形中，例如当一个或多个溶剂分子纳入结晶固体的晶格中时，溶剂化物将能够被分离。溶剂化物中的溶剂分子可按规则排列和/或无序排列存在。溶剂合物可包含化学计量或非化学计量的溶剂分子。“溶剂合物”涵盖溶液相和可分离的溶剂合物。示例性溶剂合物包括但不限于水合物、乙醇合物、甲醇合物和异丙醇合物。溶剂化方法是本领域公知的。
除另有规定外，术语“立体异构体”是指具有相同化学构造，但原子或基团在空间上排列方式不同的化合物。立体异构体包括对映异构体、非对映异构体、构象异构体(旋转异构体)、几何异构体(顺/反)异构体、阻转异构体等。所得的任何立体异构体的混合物可以依据组分物理化学性质上的差异被分离成纯的或基本纯的几何异构体，对映异构体，非对映异构体，例如，通过色谱法和/或分步结晶法。
附图说明
图1为DP001-JSSW-001DAR值检测结果
图2为DP001-ZW-002DAR值检测结果
图3为DP001-JSSW-001DAR值检测结果
图4为partitumab-JSSW-001DAR值检测结果
图5为partitumab-ZW-002DAR值检测结果
图6为SWY2110-JSSW-001DAR值检测结果
图7为SWY2111-JSSW-001DAR值检测结果
图8为SWY2112-JSSW-001DAR值检测结果
图9为SWY2113-JSSW-001DAR值检测结果
图10为partitumab-Dxd DAR值检测结果
图11为SWY2110-Dxd DAR值检测结果
图12为SWY2110-Vc MMAE DAR值检测结果
图13为DP001-ADC对NU/NU小鼠HER2-表达的人乳腺癌JIMT-1细胞移植瘤肿瘤体积的影响
图14为patritumab-ADC对NU/NU小鼠人肺腺癌PC9-GR(Gefitinib耐药细胞)细胞移植瘤肿瘤体积的影响
图15为SWY2110-ADC对NU/NU小鼠人结直肠癌DiFi细胞移植瘤肿瘤体积的影响
图16为SWY2110-ADC对NU/NU小鼠人肺腺癌PC9-GR(Gefitinib耐药细胞)细胞移植瘤肿瘤体积的影响
图17为SWY2110-ADC对DiFi细胞抑制活性检测结果
图18为SWY2110-ADC对PC9-GR细胞抑制活性检测结果
图19为DP001-ADC旁观者效应检测结果
图20为SWY2110-ADC对NU/NU小鼠人肺腺癌PC9-AR(PC9-Del19/T790M/C797S、Osimertinib耐药细胞)细胞移植瘤肿瘤体积的影响
图21为SWY2110-ADC、SWY2111-ADC、SWY2112-ADC、SWY2113-ADC对NU/NU小鼠人肺腺癌NCI-H1975细胞移植瘤肿瘤体积的影响
图22为SWY2110-ADC、SWY2111-ADC、SWY2112-ADC、SWY2113-ADC对NOD-SCID小鼠人乳腺癌MDA-MB-468细胞移植瘤肿瘤体积的影响
图23为SWY2110-ADC、SWY2111-ADC、SWY2112-ADC、SWY2113-ADC对NU/NU小鼠人结直肠癌DiFi细胞移植瘤肿瘤体积的影响
图24为SWY2110-ADC、SWY2111-ADC、SWY2112-ADC、SWY2113-ADC对NU/NU小鼠人肺腺癌PC9-GR(Gefitinib耐药细胞)细胞移植瘤肿瘤体积的影响
图25为patritumab-ADC对NOD-SCID小鼠人肺腺癌PC9-DTC(Del19/T790M/C797S，Osimertinib耐药细胞)细胞移植瘤体积的影响
图26为patritumab-ADC对NU/NU小鼠人结肠癌SW620细胞移植瘤体积的影响
图27为patritumab-ADC对DiFi细胞抑制活性检测结果
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或者按照制造厂商所建议的条件。除非另行定义，文中所使用的所有专业与科学用语与本领域专业人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法之中。文中所示的较佳实施方法与材料仅做示范之用。
本发明的化合物结构是通过核磁共振(NMR)或/和液质联用色谱(LC-MS)或/和液相色谱(HPLC)来确定的。NMR的测定使用的仪器是Bruker AvanceⅢ400MHz核磁共振仪；LC-MS使用的仪器是SHIMADZU LC-20AD-PDA-LCMS-2020；HPLC使用的仪器是SHIMADZU LC-20AD-PDA高效液相色谱仪。
本发明实施例中的起始原料是已知的并且可以在市场上买到，或者可以采用或按照本领域已知的方法来合成。
本发明中抗体的制备可用由Kohler等，自然(1975)首次描述的杂交瘤技术制备，或用重组DNA方法制备(美国专利4,816,567等)。
制备实施例
将具有两条如SEQ ID NO:9所示氨基酸序列的重链和两条如SEQ ID NO:10所示氨基酸序列的轻链的抗体定义为DP001；
将具有两条如SEQ ID NO:19所示氨基酸序列的重链和两条如SEQ ID NO:20所示氨基酸序列的轻链的抗体定义为patritumab；
将具有两条如SEQ ID NO:29所示氨基酸序列的重链和两条如SEQ ID NO:30所示氨基酸序列的轻链的抗体定义为SWY2110；
将具有两条如SEQ ID NO:34所示氨基酸序列的重链和两条如SEQ ID NO:30所示氨基酸序列的轻链的抗体定义为SWY2111；
将具有两条如SEQ ID NO:37所示氨基酸序列的重链和两条如SEQ ID NO:30所示氨基酸序列的轻链的抗体定义为SWY2112；
将具有两条如SEQ ID NO:40所示氨基酸序列的重链和两条如SEQ ID NO:30所示氨基酸序列的轻链的抗体定义为SWY2113。
抗体序列说明
1.HER2抗体DP001(trastuzumab)
轻链(SEQ ID NO:10)：
Figure PCTCN2022132627-appb-000018
重链(SEQ ID NO:9)：
Figure PCTCN2022132627-appb-000019
DP001 HCDR1：GFNIKDTYIH(SEQ ID NO:1)
DP001 HCDR2：RIYPTNGYTRYAD(SEQ ID NO:2)
DP001 HCDR3：WGGDGFYAMDY(SEQ ID NO:3)
DP001 LCDR1：RASQDVNTAVA(SEQ ID NO:4)
DP001 LCDR2：SASFLYS(SEQ ID NO:5)
DP001 LCDR3：QQHYTTPPT(SEQ ID NO:6)
DP001 HV：SEQ ID NO:7
Figure PCTCN2022132627-appb-000020
DP001 LV：SEQ ID NO:8
Figure PCTCN2022132627-appb-000021
2.HER3抗体patritumab
轻链(SEQ ID NO:20)：
Figure PCTCN2022132627-appb-000022
重链(SEQ ID NO:19)：
Figure PCTCN2022132627-appb-000023
Patritumab HCDR1：GGSFSGYYWS SEQ ID NO:11
Patritumab HCDR2：EINHSGSTNYNPSLKS SEQ ID NO:12
Patritumab HCDR3：DKWTWYFDL SEQ ID NO:13
Patritumab LCDR1：RSSQSVLYSSSNRNYLA SEQ ID NO:14
Patritumab LCDR2：WASTRES SEQ ID NO:15
Patritumab LCDR3：QQYYSTPRT SEQ ID NO:16
Patritumab HV：SEQ ID NO:17
Figure PCTCN2022132627-appb-000024
Patritumab LV：SEQ ID NO:18
Figure PCTCN2022132627-appb-000025
Figure PCTCN2022132627-appb-000026
3.EGFR抗体SWY2110
轻链(SEQ ID NO:30)：
Figure PCTCN2022132627-appb-000027
重链(SEQ ID NO:29)：
Figure PCTCN2022132627-appb-000028
SWY2110 HCDR1：NYDVH SEQ ID NO:21
SWY2110 HCDR2：VIWSGGNTDYNTPFTS SEQ ID NO:22
SWY2110 HCDR3：ALDYYDYEFAY SEQ ID NO:23
SWY2110 LCDR1：RASQSIGTNIH SEQ ID NO:24
SWY2110 LCDR2：YASESIS SEQ ID NO:25
SWY2110 LCDR3：QQNNEWPTS SEQ ID NO:26
SWY2110 HV：SEQ ID NO:27
Figure PCTCN2022132627-appb-000029
SWY2110 LV：SEQ ID NO:28
Figure PCTCN2022132627-appb-000030
4.EGFR抗体SWY2111
轻链(SEQ ID NO:30)：
Figure PCTCN2022132627-appb-000031
重链(SEQ ID NO:34)：
Figure PCTCN2022132627-appb-000032
SWY2111 HCDR1：DYDVH SEQ ID NO:31
SWY2111 HCDR2：VIWSGGNTDYNTPFTS SEQ ID NO:22
SWY2111 HCDR3：ALDDYDYEFAY SEQ ID NO:32
SWY2111 LCDR1：RASQSIGTNIH SEQ ID NO:24
SWY2111 LCDR2：YASESIS SEQ ID NO:25
SWY2111 LCDR3：QQNNEWPTS SEQ ID NO:26
SWY2111 HV：SEQ ID NO:33
Figure PCTCN2022132627-appb-000033
Figure PCTCN2022132627-appb-000034
SWY2111 LV：SEQ ID NO:28
Figure PCTCN2022132627-appb-000035
5.EGFR抗体SWY2112
轻链(SEQ ID NO:30)：
Figure PCTCN2022132627-appb-000036
重链(SEQ ID NO:37)：
Figure PCTCN2022132627-appb-000037
SWY2112 HCDR1：EYDVH SEQ ID NO:35
SWY2112 HCDR2：VIWSGGNTDYNTPFTS SEQ ID NO:22
SWY2112 HCDR3：ALDDYDYEFAY SEQ ID NO:32
SWY2112 LCDR1：RASQSIGTNIH SEQ ID NO:24
SWY2112 LCDR2：YASESIS SEQ ID NO:25
SWY2112 LCDR3：QQNNEWPTS SEQ ID NO:26
SWY2112 HV：SEQ ID NO:36
Figure PCTCN2022132627-appb-000038
Figure PCTCN2022132627-appb-000039
SWY2112 LV：SEQ ID NO:28
Figure PCTCN2022132627-appb-000040
6.EGFR抗体SWY2113
轻链(SEQ ID NO:30)：
Figure PCTCN2022132627-appb-000041
重链(SEQ ID NO:40)：
Figure PCTCN2022132627-appb-000042
SWY2113 HCDR1：HYDVH SEQ ID NO:38
SWY2113 HCDR2：VIWSGGNTDYNTPFTS SEQ ID NO:22
SWY2113 HCDR3：ALDDYDYEFAY SEQ ID NO:32
SWY2113 LCDR1：RASQSIGTNIH SEQ ID NO:24
SWY2113 LCDR2：YASESIS SEQ ID NO:25
SWY2113 LCDR3：QQNNEWPTS SEQ ID NO:26
SWY2113 HV：SEQ ID NO:39
Figure PCTCN2022132627-appb-000043
SWY2113 LV：SEQ ID NO:28
Figure PCTCN2022132627-appb-000044
实施例1
N-((2R，10S)-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[3'，4'：6,7]中氮茚并[1,2-b]喹啉-1-基)氨基)-2-甲基-1,6,9,12,15-五氧-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-1H-吡咯1-基)己酰胺(JSSW-001)
Figure PCTCN2022132627-appb-000045
方案一：
Figure PCTCN2022132627-appb-000046
第一步：化合物B2-1的制备：在100ml三口瓶中加入B1-1(1.0g,2.28mmol)、无水四氢呋喃(30mL)、甲苯(10ml)、吡啶(0.5ml)，四乙酸铅(1.83g，4.12mmol)，溶液变成橙色，加热回流，一小时后溶液变为无色，有白色固体生成，反应三小时后，溶液使用硅藻土过滤，固体使用乙酸乙酯淋洗，滤液使用旋转蒸发仪干燥。得到的粗品使用柱层析提纯，得到固体(640mg)。LC-MS[M+Na]+:m/z 391.1。
第二步：化合物A2-1的制备：在100ml三口瓶中加入A1-1(2.7g,30.0mmol)和N,N-二甲基甲酰胺(30mL)，然后添加碳酸钾(4.17g,30.0mmol)和溴苄(2.4mL,20.0mmol)。在室温下反应14小时后把反应液滴加水中，使用乙酸乙酯进行萃取，合并有机层后有机层使用饱和食盐水水洗，后有机层使用无水硫酸镁干燥后过滤浓缩，粗品使用柱层析提纯(PE/EA＝3:1)，得到目标化合物2.61g无色液体。
1H NMR(400MHz,CDCl3):δ7.33-7.41(m,5H),5.21(s,2H),4.29-4.35(m,1H),2.77(d,J＝5.6Hz,1H),1.44(d,J＝6.8Hz,3H)。
第三步：化合物A3-1的制备：在100ml三口瓶中加入A2-1(720mg,4.0mmol)、B2-1(368mg，1.0mmol)和二氯甲烷(5mL)，添加4-甲基苯磺酸吡啶(75mg,0.30mmol)，反应液加热回流过夜，反应结束后，反应液中加入乙酸乙酯(20mL)，水洗三次后使用无水硫酸镁干燥，过滤除去无水硫酸镁，溶液进行浓缩，得到的粗品使用柱层析提纯(PE/EA＝2:1)，得到白色固体A3-1(290mg)。
LC-MS[M+Na] +:m/z 511.1.1H NMR(400MHz,CDCl3):δ7.77(d,J＝7.2Hz, 2H),7.58(d,J＝7.6Hz,2H),7.40(t,J＝7.6Hz,2H),7.26-7.37(m,6H),6.70(brs,1H),5.12-5.30(m,3H),4.76-4.89(m,2H),4.45(d,J＝6.8Hz,1H),4.19-4.27(m,2H),3.69-3.85(m,2H),1.41(d,J＝6.8Hz,3H)。
第四步：化合物A4-1的制备：在100ml三口瓶中加入A3-1(291mg,0.60mmol)溶解在四氢呋喃：乙酸乙酯(4mL/2mL)中，加入Pd/C(10％w/w,60mg)，使用氢气进行置换3次后套上氢气球搅拌过夜，反应结束后，反应液过硅藻土，使用甲醇进行淋洗，有机层进行浓缩，得到产品A4-1。LC-MS[M+Na]+:m/z 399.1。
第五步：化合物A6-1的制备：在25ml单口瓶中加入A4-1(50mg,0.13mmol，1.3eq)、依喜替康甲磺酸盐(A5-1)(50mg,0.049mmol，1.0eq)，2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(48mg，0.13mmol，1.3eq)，N,N-二异丙基乙胺(36mg，0.28mmol，3.0eq)，在N,N-二甲基甲酰胺(2ml)中溶解，在室温下搅拌过夜，使用乙酸乙酯进行稀释后用半饱和柠檬酸进行水洗，随后使用食盐水水洗，碳酸氢钠溶液水洗，食盐水水洗，有机层使用无水硫酸钠干燥后过滤，去除无水硫酸钠，浓缩，使用prep-TLC展开剂体系为(DCM/MeOH＝15:1)进行提纯，得到产物A6-1(56mg)，LC-MS[M+H]+:m/z 816.3.
第六步：化合物A7-1的制备：在25ml单口瓶中加入A6-1(56mg,0.069mmol)、N,N-二甲基甲酰胺(2ml)、哌啶(12mg,0.14mmol)，搅拌2小时，溶液浓缩得到白色固体。LC-MS[M+H]+:m/z 594.3。
第七步：化合物JSSW-001的制备：在25ml单口瓶中加入A7-1(0.069mmol)、C7(66mg,0.14mmol)、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(53mg，0.14mmol)，N,N-二异丙基乙胺(36mg，0.28mmol)在N,N-二甲基甲酰胺(2ml)中溶解，室温下搅拌2小时，使用乙酸乙酯(30ml)进行稀释后用半饱和柠檬酸进行水洗，随后使用食盐水水洗，碳酸氢钠溶液水洗，食盐水水洗，有机层使用无水硫酸钠干燥后过滤，去除无水硫酸钠，浓缩，使用prep-TLC展开剂体系为(DCM/MeOH＝15:1)进行提纯，得到产物JSSW-001(21mg)。
LC-MS[M+H]+:m/z 1048.1H NMR(400MHz,DMSO-d6):δ8.63(t,J＝6.4Hz,1H),8.53(d,J＝8.8Hz,1H),8.28(t,J＝6.0Hz,1H),8.03-8.10(m,2H),7.98(t,J＝6.0Hz,1H),7.77(d,J＝11.2Hz,1H),7.30(s,1H),7.14-7.25(m,5H),6.99(s,2H),6.51(s,1H),5.56-5.62(m,1H),5.40(s,2H),5.08-5.23(m,2H),4.66-4.71(m,1H),4.42-4.56(m,2H),4.09-4.15(m,1H),3.54-3.76(m,7H),3.09-3.27(m,1H), 2.97-3.02(m,1H),2.67-2.77(m,1H),2.38(s,3H),2.16-2.21(m,2H),2.05-2.10(m,2H),1.83-1.89(m,2H),1.37-1.46(m,7H),1.14-1.33(m,4H),0.87(t,J＝7.2Hz,3H)。
方案二：
Figure PCTCN2022132627-appb-000047
第一步：将化合物C1-1(8g,88.86mmol)和C2-1(22.66g,133.33mmol)加入到250ml反应瓶中，加入DMAc(40mL,5v/w)，搅拌溶清，控温至0-10℃，然后滴加DIEA(34.45g,266.6mmol)，滴加完后让其自然升温至25℃左右，在该温度下搅拌16小时。TLC监测原料反应完全后。将反应液倒入120mL冰水中，加入MTBE(40mL)搅拌，静置分液，水相用MTBE(40mL*4)萃取4次，合并有机相，有机相依次用0.5M的盐酸溶液(40mL)洗涤一次，水(40mL)洗涤一次，饱和食盐水(40mL*2)洗涤两次。用无水硫酸钠干燥，过滤，浓缩，湿法上样过柱纯化，石油醚：乙酸乙酯＝30:1→20:1→10:1的体系过柱，得到浅黄色液体C3-1，LC-MS[M+Na]+:m/z 203。
第二步：氮气保护下，将C4-1(6g,16.29mmol,1.0eq)和C3-1(3.23g，17.92mmol，1.1eq)溶解于THF(60mL,10V/w)中，降温到10～15℃，加入TsOH(281mg,1.63mmol,0.1eq)，加完，在14～18℃下反应4h，TLC监控，反应结束后。将反应液加入到冰水(60mL)中，再用EA(60mL*3)萃取，合并有机相，用饱和NaHCO 3水溶液(60mL)洗涤，水(60mL*2)洗涤，饱和食盐水(60mL)洗涤，用无水硫酸钠干燥，过滤，浓缩蒸干，拌样过柱纯化，正己烷：乙酸乙酯＝10:1→5:1→3:1→2:1→1:1的体系过柱，拿到无色油状物C5-1，LC-MS[M+Na]+: m/z 511。
第三步：氮气保护下，将C5-1(2.8g,5.74mmol,1.0eq)溶解于DMAc(28mL,10V/w)中，降温到14～18℃，滴加DBU(436.6mg,2.87mmol,0.5eq)，并在该温度下搅拌反应1.5h，TLC监控原料反应完全，降温到0～10℃，加入PPTS(721.23mg，2.87mmol,0.5eq)，EDCI(1.1g，5.74mmol,1.0eq)，HOBT(775mg,5.74mmol,1.0eq)和C7-1(2.45g，4.88mmol,0.85eq)，加完，在0～10℃反应3～4h，TLC监控C7-1反应完全后。将反应液加入到冰水(100mL)中，再用2-甲基THF(100mL*3)萃取，合并有机相，用0.5M盐酸(150mL*2)洗涤，饱和NaHCO 3水溶液(100mL*3)洗涤，水(100mL)洗涤，饱和食盐水(100mL)洗涤，用无水硫酸钠干燥，过滤，浓缩蒸干，拌样过柱纯化，DCM→DCM：MeOH＝80:1→60:1→50:1→40:1→30:1→20:1的体系过柱，拿到白色泡状固体C8-1，3.1g，收率72％，LC-MS[M+Na]+:m/z 772。
第四步：氮气保护下，将C8-1(3.1g,4.14mmol,1.0eq)溶解于DCM(46.5mL,15V/w)中，室温25～30℃下，滴加DBU(314.6mg,2.07mmol,0.5eq)，并在该温度下搅拌反应16h，TLC监控原料反应完全。将反应液直接湿法过柱，DCM→DCM：MeOH＝50:1→30:1→20:1→10:1的体系过柱，拿到白色泡状固体C9-1。LC-MS[M+H]+:m/z 528。
第五步：氮气保护下，将C9-1(1.7g,3.2mmol,1.0eq)溶解于水(25.5mL,15V/w)和叔丁醇(8.5mL,5V/w)中，搅拌溶清，氮气置换一次，加入Pd/C(0.34g，20％w/w)，氢气置换三次，室温下25～30℃下氢化反应12h，LCMS监控原料反应完全。将反应液过滤，滤饼用水(25mL*2)洗涤，滤液再次过滤一次，浓缩除去叔丁醇，水相直接冻干，拿到白色粉末状固体C10-1共1.5g，收率100％。LC-MS[M+H]+:m/z438。
第六步：氮气保护下，将C10-1(1.4g,3.2mmol,1.0eq)溶解于乙腈(14mL,10V/w)和水(28mL,20V/w)中，加入C11-1(1.86g,6.097mmol,1.1eq)，搅拌溶清，降温至0～10℃下，滴加DIEA(330.24mg,2.56mmol)，滴加完在该温度下搅拌反应16h，LC-MS监控原料反应完全。将Na 2HPO 4(260mg)，NaH 2PO 4(5.6g)加入20mL水中，搅拌溶清配成缓冲液，将缓冲液降温至0-5℃。将反应液倒入冻好的缓冲液中，并用DCM/异丙醇＝4/1(50mL*4)萃取，合并有机相，干燥，过滤，浓缩蒸干，过柱纯化。DCM→DCM：MeOH＝50:1→30:1→20:1→15:1的体系过柱，拿到黄色 固体C12-1。
第七步：氮气保护下，将A5-1(547.4mg,1.03mmol,1.0eq)溶解于5％的无水硫酸钠的水(6.5mL)和THF(7.8mL)中，搅拌溶解不清，降温到0～10℃下，加入NMM(104mg,1.03mmol,1.0eq)，加完在此温度下反应1h。依次加入C12-1(650mg，1.03mmol，1.1eq)，EDCI(296.2mg，1.545mmol，1.5eq)和2-肟氰基乙酸乙酯(73.9mg，0.52mmol，0.5eq)，加完，在0～10℃下反应4～5h，TLC监控原料反应完全。将反应液加入到冰浴下的0.1M盐酸(13mL，20V/w)中，用2-甲基THF(13mL*3)萃取，合并有机相，再用0.05M盐酸(13mL，20V/w)洗一次，水(13mL*2)洗涤，干燥，过滤，浓缩蒸干，过柱纯化。DCM→DCM：MeOH＝50:1→30:1→20:1→15:1→12:1的体系过柱，拿到黄色泡状固体JSSW-001。LC-MS[M+H]+:m/z 1048。
实施例2
N-((S)-((2R，10S)-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[3'，4'：6,7]中氮茚并[1,2-b]喹啉-1-基)氨基)-2-甲基-1,9,12,1,15,18-五氧-3,6二恶沙,8,11,14,17-四氮杂十六烷-19-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯1-基)己酰胺(ZW-002)
Figure PCTCN2022132627-appb-000048
方案一：
Figure PCTCN2022132627-appb-000049
第一步：化合物A2-2的制备：在100ml三口瓶中加入A1-2(4.98g,30mmol)和N,N-二甲基甲酰胺(40ml)，放入0℃搅拌，加入氢化钠(1.26g,31.5mmol)，反应液继续搅拌30分钟后，加入2-(2-溴乙氧基)四氢-2H-吡喃(6.59g,31.5mmol)，之后反应过夜，反应结束后在反应中加入水(100mL)把反应进行淬灭，之后反应液使用乙酸乙酯(150mL*3)进行萃取，合并有机层并用饱和食盐水进行水洗，有机层使用无水硫酸钠进行干燥，过滤除去固体，液体进行浓缩，使用柱层析(PE/EA＝10/1)进行提纯，得到无色液体A2-2(6.39g)。LC-MS[M+Na]+:m/z 317.2。
第二步：化合物A3-2的制备：在100ml三口瓶中加入A2-2(760mg,2.58mmol)在二氯甲烷(50ml)搅拌，加入盐酸甲醇溶液(0.01mL)，反应在室温下搅拌1小时，反应结束后使用反向(H 2O/ACN＝1:1)进行提纯，得到目标化合物A3-2(660mg)无色液体。
1H NMR(400MHz,CDCl3):δ7.37-7.36(m,5H),5.20(s,2H),4.18(s,2H),3.75(t,J＝1.6Hz,2H),3.70(t,J＝1.6Hz,2H)。
第三步：化合物A5-2的制备：在100ml三口瓶中加入A3-2(105mg,0.5mmol)和二氯甲烷(10ml)，搅拌，加入A4-2(61mg,0.17mmol)和4-甲基苯磺酸吡啶(13mg,0.05mmol)，之后反应回流过夜，反应结束后在反应液中加入乙酸乙酯(50mL)，水洗三次(20mL*3)，有机层使用无水硫酸镁干燥，过滤去除固体，有机层浓缩干燥，得到的粗品使用柱层析(PE/EA＝1:1)提纯，得到目标化合物A5-2(80mg) 淡黄色液体。LC-MS[M+H]+:m/z 519.1.
第四步：化合物A6-2的制备：在100ml三口瓶中加入A5-2(80mg,0.15mmol)、四氢呋喃：乙酸乙酯(10mL/5mL)和钯碳(10％w/w,80mg)，反应使用氢气置换三次后用氢气进行反应过夜，反应结束后过硅藻土，使用二氯甲烷：甲醇＝10：1进行淋洗，反应液进行浓缩得到目标化合物A6-2(73mg)。LC-MS[M+Na]+:m/z451.3。
第五步：化合物A8-2的制备：在25ml单口瓶中加入A6-2((73mg,0.17mmol)、依喜替康甲磺酸盐(45mg,0.085mmol)，2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(48mg，0.13mmol)，N,N-二异丙基乙胺(110mg，0.85mmol)在N,N-二甲基甲酰胺(2ml)中溶解，在室温下搅拌过夜，使用乙酸乙酯进行稀释后用氯化铵水洗，随后使用食盐水水洗，有机层使用无水硫酸镁干燥后过滤，浓缩，使用prep-TLC展开剂体系为(DCM/MeOH＝15:1)进行提纯，得到产物A8-2(30mg)。
LC-MS[M+H]+:m/z 846.4.1H NMR(400MHz,DMSO-d6):δ8.62(t,J＝9.2Hz,1H),8.41(d,J＝12Hz,1H),7.87(d,J＝10.8Hz,2H),7.78(d,J＝13.6Hz,1H),7.68(d,J＝8.4Hz,2H),7.52(t,J＝8Hz,1H),7.39(t,J＝6.8Hz,2H),7.32-7.28(m,3H),6.50(s,1H),5.62-5,54(m,1H),5.41(s,2H),5.19(s,2H),4.50-4.44(m,2H),4.27-4.23(m,2H),4.20-4.14(m,1H),4.01(s,2H),3.64-3.57(m,4H),3.53-3.48(m,2H),3.21-3.12(m,2H),2.38(s,3H),2.22-2.13(m,2H),1.88-1.80(m,2H),0.85(t,J＝7.6Hz,3H)。
第六步：化合物A9-2的制备：在25ml单口瓶中加入A8-2(30mg,0.036mmol)、N,N-二甲基甲酰胺(2ml)、哌啶(6mg,0.07mmol)，搅拌2小时，溶液浓缩得到黄色固体A9-2(30mg)。LC-MS[M+H]+:m/z 624.3。
第七步：化合物ZW-002的制备：在25ml单口瓶中加入A9-2(30mg,0.048mmol)、C7(23mg,0.048mmol)、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(28mg,0.072mmol)，N,N-二异丙基乙胺(31mg,0.24mmol)，在N,N-二甲基甲酰胺(5ml)中溶解，室温下搅拌1小时，使用乙酸乙酯(30ml)进行稀释后用氯化铵水洗，随后使用食盐水水洗，有机层使用无水硫酸镁干燥后过滤，去除固体，浓缩，使用制备液相进行提纯，得到产物ZW-002(4.2mg)。LC-MS[M+H]+:m/z 1078.2.
1H NMR(400MHz,DMSO-d6):δ8.46-8.39(m,2H),8.28(t,J＝5.6Hz,1H), 8.11-7.96(m,3H),7.78(d,J＝10.8Hz,1H),7.29(s,1H),7.20-7.16(m,5H),6.97(s,2H),6.5(s,1H),5.63-5.55(m,1H),5.41(s,2H),5.19(s,2H),4.49-4.42(m,3H),4.01(s,2H),3.74-3.64(m,3H),3.64-3.63(m,2H),3.63-3.40(m,2H),3.34-3.32(m,1H),3.20-3.14(m,1H),3.03-2.98(dd,J＝5.2Hz,J＝14.4Hz,1H),2.78-2.73(m,1H),2.38(s,3H),2.24-2.13(m,2H),2.10(t,J＝7.2Hz,2H),1.91-1.77(m,2H),1.50-1.39(m,5H),1.31-1.12(m,6H),0.85(t,J＝7.2Hz,3H).
方案二：
Figure PCTCN2022132627-appb-000050
第一步：将B1-2(24g,169mmol,1.0eq)溶于DMSO(1200mL,50v/v)，搅拌下滴加B2-2(43.35g,253mmol,5eq)，加完，室温20-30℃反应16h。反应结束后，反应液倒入冰水(250mL,10v/v)中，控温0-10度，加EA(300mL,12-15v/v)*3次萃取，有机相用水(200mL,8-10v/v)*2，饱和氯化钠水溶液(150mL，6-10v/v)*1分别洗涤，有机相无水硫酸钠(50g)干燥，过滤浓缩，过柱得黄色油状物B3-2。
第二步：B3-2(14g，66.6mmol，1.0eq)溶于THF(280mL，20v/v)中，加入B4-2(29.47g，79.92mmol，1.2eq)，TsOH(1.15g，6.66mmol，0.1eq)，加完，室温20-30℃搅拌反应3h。反应结束后，反应液倒入冰水(500mL，35-40v/v)，EA(500mL，35-40v/v)的混合液中，水相用EA(500mL，35-40v/v)*3萃取，有机层用饱和NaHCO 3水溶液(250mL，15-20v/v)，水(250mL，15-20v/v)，各洗一次，有机层加入无水硫酸钠(80g)干燥，浓缩成黄色油，柱层析得白色固体B5-2。
第三步：B5-2(12g，23.16mmol，1.0eq)溶于DMAc(240mL，20v/v)，滴加DBU(1.76g，11.8mmol，0.5eq)，加完，10-15℃反应60min，加PPTS(2.91g，11.8mmol，0.5eq)，HOBt(3.12g，23.16mmmol，1.0eq)，B7-2(11.6g，23.16mmmol，1.0eq),EDCI(4.4g，23.16mmmol，1.0eq)，10-15℃，反应3h。反应结束后，反应液倒入冰水(300mL，25v/v)，甲基四氢呋喃(500mL，40-45v/v)的混合液中(控温0-10度)，水相用甲基四氢呋喃(300mL，25v/v)*3次萃取，有机相合并，饱和NaHCO 3水溶液(200mL，15-20v/v)，水(200mL，15-20v/v)各洗一次，有机相加入无水硫酸钠(100g)干燥，过滤去干燥剂，滤液水泵20-30度真空浓缩成黄色油，柱层析得白色固体B8-2。
第四步：B8-2(13g，16.68mmol，1.0eq)溶于ACN(130mL，10v/v)，滴加DBU(3.0g，20.01mmol，1.2eq)，加完室温20-30℃反应60min。反应结束后，反应液加入冰水(130mL，10v/v)，1M HCl调PH＝3-4，EA(150mL，10-15v/v)*4萃取(有机相废弃)，水相饱和NaHCO 3水溶液调PH＝8-9，加DCM:异丙醇4:1(150mL，10-15v/v)*4萃取，合并有机相，无水硫酸钠(80g)干燥，浓缩得白色固体B9-2。
第五步：B9-2(5.5g，9.86mmol，1.0eq)溶于THF(82.5mL，15v/v)和水(27.5mL，5v/v)，加入10％钯碳(0.55g，重量比0.1)，加完氢气保护，20-30℃反应16h。
反应后处理1:反应液过滤去钯碳，补加水(100mL，18-20v/v)，EA(100mL，18-20v/v)*2萃取(有机相废弃)，水相记为A溶液(B10-2)。
操作2：反应瓶加A溶液(B10-2)(130mL)，B11-2(3.6g，11.83mmol，1.2eq)溶于ACN(49.5mL，9v/v)，滴加DIEA(1.27g，9.86mmol，1.0eq)，加完10-15度反应5.0h。
反应后处理2:反应液倒入冰水(100mL，18-20v/v)，水相EA(150mL，10-15v/v)*3萃取，水相控温0-10度，0.5MHCl水溶液调PH＝2-3，加DCM:异丙醇4:1(150mL，10-15v/v)*4萃取，合并有机相，无水硫酸钠(50g)干燥，滤液浓缩，油泵拉干,得白色固体B12-2。
第六步：无水硫酸钠(0.8g，0.5v/v)，水(16mL，10v/v)，THF(19.2mL，12v/v)，搅拌下加A5-1(1.6g，3.0mmol，1.0eq)，降温5-10度，滴加N-甲基吗啡啉(0.304g，3.0mmol，1.0eq)，5-10度反应40min，依次加B12-2(2.4g，3.6mmol，1.2eq)，EDCI (0.863g，4.5mmol，1.5eq)，2-肟氰基乙酸乙酯(0.214g，1.5mmol，0.5eq)，加完5-10℃反应3.0h。反应结束后，反应液倒入冰水150mL中，EA(150mL*3)萃取，0.1MHCl(50mL)，水(150mL)各洗一次，有机相无水硫酸钠30g干燥，浓缩干后柱层析，得淡黄色固体ZW-002。
实施例3抗体-药物偶联物(DP001-JSSW-001 ADC,DAR＝4.0)
Figure PCTCN2022132627-appb-000051
第一步 抗体的还原：
通过公知技术将编码轻、重链基因的质粒瞬时转染HEK293细胞表达后纯化后得到的DP001抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加10mM TCEP(百灵威科技有限公司)水溶液(17μL；相对于一分子抗体为2.5当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司，50μL)。确认了该溶液的pH为7.4±0.1内后，于37℃孵育1小时，由此将抗体内的二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加实施例1得到的JSSW-001化合物10mM的二甲基亚砜溶液(31.28μL；相对于一分子抗体为4.6当量)，混匀，在室温下反应30分钟，将接头-药物化合物连接于抗体。接下来，添加100mM NAC(百灵威科 技有限公司)水溶液(10.2μL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体-药物偶联物的纯化:
使用超滤离心管(Merck，
Figure PCTCN2022132627-appb-000052
Regenerated CellμLose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为纯化缓冲液的25mM 2-(N-吗啉)乙磺酸(MES)，pH＝6.5，将溶液浓缩至1-2mL，再次加入纯化缓冲液，置换倍数大于1000倍，由此，将未偶联的接头-药物化合物及其他低分子量试剂除去，得到纯化的抗体-药物偶联物(DP001-JSSW-001 ADC,DAR＝4.0)。
第四步 抗体-药物偶联物的药物抗体偶联比(DAR)的测定：
向2mg/kg抗体—药物偶联物中加入终浓度为20mM的二硫苏糖醇，于37℃水浴30分钟，由此将切断了抗体-药物偶联物链间二硫键的样品用于HPLC分析。HPLC系统选择Agilent Technologies 1260 Infinity HPLC，色谱柱选择PLRP-S(5μm粒径；2.1mm×50mm；Agilent Technologies)，柱温80℃，流动相A为0.1％三氟乙酸(TFA)水溶液，流动相B为0.1％三氟乙酸(TFA)乙腈溶液。上样量为10μL，梯度程序为0-3分钟27％-27％,3-8分钟27％-35％,8-25分钟35％-43％，25-26分钟43％-95％,26-31分钟95％-95％,31-32分钟95％-27％,32-40分钟27％-27％。相对于未连接的轻链(L0)及重链(H0)，连接了药物的轻链(连接一个药物的轻链，L1)及重链(连接了一个药物的重链，H1；连接了两个药物的重链，H2；连接三个药物的重链，H3)而言，疏水性随着连接药物的数目的增加而增加，因此按照L0、L1、H0、H1、H2、H3顺序洗脱。根据280nm下的峰面积计算DAR值。DAR值结果如图1所示。
Figure PCTCN2022132627-appb-000053
实施例4抗体-药物偶联物(DP001-ZW-002 ADC,DAR＝5.78)
Figure PCTCN2022132627-appb-000054
第一步 抗体的还原：
通过公知技术将编码轻、重链基因的质粒瞬时转染HEK293细胞表达后纯化后得到的DP001抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加100mM TCEP(百灵威科技有限公司)水溶液(6.66μL；相对于一分子抗体为10当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司，18μL)。确认了该溶液的pH为7.0±0.1内后，于37℃孵育3小时，由此将抗体内的二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加实施例2得到的化合物ZW-002 10mM的二甲基亚砜溶液(87μL；相对于一分子抗体为13当量)，混匀，在室温下反应30分钟，将药物接头连接于抗体。接下来，添加100mM NAC(百灵威科技有限公司)水溶液(10.2μL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体-药物偶联物的纯化:
使用超滤离心管(Merck，
Figure PCTCN2022132627-appb-000055
Regenerated CellμLose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为纯化缓冲液的25mM 2-(N-吗啉)乙磺酸(MES)，pH＝6.5，将溶液浓缩至1-2mL，再次加入纯化缓冲液，置换倍数大于1000倍，由此，将未偶联的接头-药物化合物及其他 低分子量试剂除去，得到纯化的抗体-药物偶联物(DP001-ZW-002 ADC,DAR＝5.78)。DAR值测定方法同实施例3第四步，DAR值结果如图2所示。
实施例5抗体-药物偶联物(DP001-JSSW-001 ADC,DAR＝7.32)
Figure PCTCN2022132627-appb-000056
第一步 抗体的还原：
通过公知技术将编码轻、重链基因的质粒瞬时转染HEK293细胞表达后纯化后得到的DP001抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加100mM TCEP(百灵威科技有限公司)水溶液(6.66μL；相对于一分子抗体为10当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司，18μL)。确认了该溶液的pH为7.0±0.1内后，于37℃孵育3小时，由此将抗体内的二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加实施例1得到的化合物JSSW-001 10mM的二甲基亚砜溶液(87μL；相对于一分子抗体为13当量)，混匀，在室温下反应30分钟，将药物接头连接于抗体。接下来，添加100mM NAC(百灵威科技有限公司)水 溶液(10.2μL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体-药物偶联物的纯化：
使用超滤离心管(Merck，
Figure PCTCN2022132627-appb-000057
Regenerated CellμLose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为纯化缓冲液的25mM 2-(N-吗啉)乙磺酸(MES)，pH＝6.5，将溶液浓缩至1-2mL，再次加入纯化缓冲液，置换倍数大于1000倍，由此，将未偶联的接头-药物化合物及其他低分子量试剂除去，得到纯化的抗体-药物偶联物(DP001-JSSW-001 ADC,DAR＝7.32)。DAR值测定方法同实施例3第四步，DAR值结果如图3所示。
实施例6抗体-药物偶联物(patritumab-JSSW-001 ADC,DAR＝7.36)
Figure PCTCN2022132627-appb-000058
第一步 抗体的还原：
通过公知技术将编码轻、重链基因的质粒瞬时转染HEK293细胞表达后纯化后得到的patritumab抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加100mM TCEP(百灵威科技有限公司)水溶液(6.66μL；相对于一分子抗体为10当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司，18μL)。确认了该溶液的pH 为7.0±0.1内后，于37℃孵育3小时，由此将抗体内的二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加实施例1得到的化合物JSSW-001 10mM的二甲基亚砜溶液(87μL；相对于一分子抗体为13当量)，混匀，在室温下反应30分钟，将药物接头连接于抗体。接下来，添加100mM NAC(百灵威科技有限公司)水溶液(10.2μL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体-药物偶联物的纯化：
使用超滤离心管(Merck，
Figure PCTCN2022132627-appb-000059
Regenerated CellμLose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为纯化缓冲液的25mM 2-(N-吗啉)乙磺酸(MES)，pH＝6.5，将溶液浓缩至1-2mL，再次加入纯化缓冲液，置换倍数大于1000倍，由此，将未偶联的接头-药物化合物及其他低分子量试剂除去，得到纯化的抗体-药物偶联物(patritumab-JSSW-001 ADC,DAR＝7.36)。DAR值测定方法同实施例3第四步，DAR值结果如图4所示。
实施例7抗体-药物偶联物(patritumab-ZW-002 ADC,DAR＝7.45)
Figure PCTCN2022132627-appb-000060
第一步 抗体的还原：
通过公知技术将编码轻、重链基因的质粒瞬时转染HEK293细胞表达后纯化 后得到的patritumb抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加100mM TCEP(百灵威科技有限公司)水溶液(6.66μL；相对于一分子抗体为10当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司，18μL)。确认了该溶液的pH为7.0±0.1内后，于37℃孵育3小时，由此将抗体内二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加实施例2得到的化合物ZW-002 10mM的二甲基亚砜溶液(87μL；相对于一分子抗体为13当量)，混匀，在室温下反应30分钟，将药物接头连接于抗体。接下来，添加100mM NAC(百灵威科技有限公司)水溶液(10.2μL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体-药物偶联物的纯化:
使用超滤离心管(Merck，
Figure PCTCN2022132627-appb-000061
Regenerated CellμLose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为纯化缓冲液的25mM 2-(N-吗啉)乙磺酸(MES)，pH＝6.5，将溶液浓缩至1-2mL，再次加入纯化缓冲液，置换倍数大于1000倍，由此，将未偶联的接头-药物化合物及其他低分子量试剂除去，得到纯化的抗体-药物偶联物(patritumab-ZW-002 ADC,DAR＝7.45)。DAR值测定方法同实施例3第四步，DAR值结果如图5所示。
实施例8抗体-药物偶联物(SWY2110-JSSW-001 ADC,DAR＝7.12)
Figure PCTCN2022132627-appb-000062
第一步 抗体的还原：
通过公知技术将编码轻、重链基因的质粒瞬时转染HEK293细胞表达后纯化后得到的SWY2110抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加10mM TCEP(百灵威科技有限公司)水溶液(66.6uL；相对于一分子抗体为10当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司)。确认了该溶液的pH为7.0±0.1内后，于37℃孵育3小时，由此将抗体内二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加实施例1得到的化合物JSSW-001 10mM的二甲基亚砜溶液(87uL；相对于一分子抗体为13当量)，混匀，在室温下反应30分钟，将药物接头连接于抗体。接下来，添加100mM NAC(百灵威科技有限公司)水溶液(10.2uL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体－药物偶联物的纯化:
超滤离心管(Merck，
Figure PCTCN2022132627-appb-000063
Regenerated Cellulose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为缓冲液L-组氨酸0.89mg/ml，L-组氨酸盐酸盐4.04mg/ml，Tween80 0.03％，蔗糖90mg/ml，pH＝5.5，将未偶联的药物接头及其他低分子量试剂除去，得到纯化的抗体-药物偶联物 (SWY2110-JSSW-001 ADC,DAR＝7.12)。DAR值测定方法同实施例3第四步，DAR值结果如图6所示。
实施例9 pH依赖抗体的制备
通过计算机模拟辅助技术，将SWY2110与EGFR蛋白进行建模和对接，获得抗原-抗体复合体，将复合体
Figure PCTCN2022132627-appb-000064
以内的氨基酸进行D、E、H扫描，获得数条在pH 7.4的亲和力降低，在pH 6.0条件下亲和力基本保持不变或亲和力下降程度较低的pH依赖性抗体。将获得的数条抗体序列基因克隆到哺乳动物表达载体，并通过HEK-293细胞转染的方式进行抗体的表达，表达完成后收集表达上清，使用AKTA系统搭载Protein A预装柱进行抗体纯化，获得目的抗体。
实施例10抗体亲和力测定
使用0.25％胰酶-EDTA将MDA-MB-468细胞消化成单细胞，并平均分成多份，每份细胞数大于10 6个。向细胞中加入300μL连续5倍梯度稀释的SWY2110(对照)及实施例9得到的抗体(抗体分别用pH7.4和pH6.0的PBS缓冲溶液稀释)，4℃孵育1h，1000rpm离心4min去掉上清。使用PBS溶液洗两遍，之后加入200μL 1：200稀释的FITC标记的鼠抗人Fc的二抗，4℃孵育1h，孵育结束后1000rpm离心4min去掉上清。使用流式细胞仪检测荧光强度，并保存数据，通过相关软件进行数据分析。筛选后的抗体亲和力测定结果见表1。经pH依赖性优化后抗体在与靶抗原进行亲和力测定结果显示，在pH7.4的条件下，pH优化后抗体SWY2111、SWY2112、SWY2113相对于SWY2110亲和力在较大程度上降低(约3.0～9.7倍)，在pH6.0条件下，SWY2111和SWY2112相对于SWY2110亲和力基本保持不变(1.2～1.3倍)，SWY2113抗体亲和力下降程度为6.6倍。
表1、抗体亲和力测定结果
FACS(EC50) SWY2110 SWY2111 SWY2112 SWY2113
pH7.4(nM) 0.49 1.45 2.35 4.76
pH6.0(nM) 0.51 0.60 0.67 3.39
实施例11抗体-药物偶联物(SWY2111-JSSW-001 ADC,DAR＝7.48)
Figure PCTCN2022132627-appb-000065
第一步 抗体的还原：
将得到的SWY2111抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加10mM TCEP(百灵威科技有限公司)水溶液(66.6uL；相对于一分子抗体为10当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司)。确认了该溶液的pH为7.0±0.1内后，于37℃孵育3小时，由此将抗体内二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加实施例1得到的化合物JSSW-001 10mM的二甲基亚砜溶液(87uL；相对于一分子抗体为13当量)，混匀，在室温下反应30分钟，将药物接头连接于抗体。接下来，添加100mM NAC(百灵威科技有限公司)水溶液(10.2uL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体－药物偶联物的纯化:
超滤离心管(Merck，
Figure PCTCN2022132627-appb-000066
Regenerated Cellulose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为缓冲液L-组氨酸0.89mg/ml，L-组氨酸盐酸盐4.04mg/ml，Tween80 0.03％，蔗糖90mg/ml，pH＝5.5，将未偶联的药物接头及其他低分子量试剂除去，得到纯化的抗体-药物偶联物(SWY2111-JSSW-001 ADC,DAR＝7.48)。DAR值测定方法同实施例3第四步， DAR值结果如图7所示。
实施例12抗体-药物偶联物(SWY2112-JSSW-001 ADC,DAR＝7.51)
Figure PCTCN2022132627-appb-000067
第一步 抗体的还原：
将得到的SWY2112抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加10mM TCEP(百灵威科技有限公司)水溶液(66.6uL；相对于一分子抗体为10当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司)。确认了该溶液的pH为7.0±0.1内后，于37℃孵育3小时，由此将抗体内二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加实施例1得到的化合物JSSW-001 10mM的二甲基亚砜溶液(87uL；相对于一分子抗体为13当量)，混匀，在室温下反应30分钟，将药物接头连接于抗体。接下来，添加100mM NAC(百灵威科技有限公司)水溶液(10.2uL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体－药物偶联物的纯化:
超滤离心管(Merck，
Figure PCTCN2022132627-appb-000068
Regenerated Cellulose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为缓冲液L-组氨酸 0.89mg/ml，L-组氨酸盐酸盐4.04mg/ml，Tween80 0.03％，蔗糖90mg/ml，pH＝5.5，将未偶联的药物接头及其他低分子量试剂除去，得到纯化的抗体-药物偶联物(SWY2112-JSSW-001 ADC,DAR＝7.51)。DAR值测定方法同实施例3第四步，DAR值结果如图8所示。
实施例13抗体-药物偶联物(SWY2113-JSSW-001 ADC,DAR＝7.50)
Figure PCTCN2022132627-appb-000069
第一步 抗体的还原：
将得到的SWY2113抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加10mM TCEP(百灵威科技有限公司)水溶液(66.6uL；相对于一分子抗体为10当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司)。确认了该溶液的pH为7.0±0.1内后，于37℃孵育3小时，由此将抗体内二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加实施例1得到的化合物JSSW-001 10mM的二甲基亚砜溶液(87uL；相对于一分子抗体为13当量)，混匀，在室温下反应30分钟，将药物接头连接于抗体。接下来，添加100mM NAC(百灵威科技有限公司)水溶液(10.2uL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体－药物偶联物的纯化:
超滤离心管(Merck，
Figure PCTCN2022132627-appb-000070
Regenerated Cellulose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为缓冲液L-组氨酸0.89mg/ml，L-组氨酸盐酸盐4.04mg/ml，Tween80 0.03％，蔗糖90mg/ml，pH＝5.5，将未偶联的药物接头及其他低分子量试剂除去，得到纯化的抗体-药物偶联物(SWY2113-JSSW-001 ADC,DAR＝7.50)。DAR值测定方法同实施例3第四步，DAR值结果如图9所示。
参考例1抗体-药物偶联物(DP001-DXD ADC,DAR＝4.3)
Figure PCTCN2022132627-appb-000071
第一步 抗体的还原：
通过公知技术将编码轻、重链基因的质粒瞬时转染HEK293细胞表达后纯化后得到的DP001抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加10mM TCEP(百灵威科技有限公司)水溶液(17μL；相对于一分子抗体为2.5当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司，50μL)。确认了该溶液的pH为7.4±0.1内后，于37℃孵育1小时，由此将抗体内二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加购买的Deruxtecan(CAS No.:1599440-13-7)10mM的二甲基亚砜溶液(31.28μL；相对于一分子抗体为4.6当量)，混匀，在室温下反应30分钟，将接头-药物化合物连接于抗体。接下来，添加100mM NAC(百灵威科技有限公司)水溶液(10.2μL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体-药物偶联物的纯化：
使用超滤离心管(Merck，
Figure PCTCN2022132627-appb-000072
Regenerated CellμLose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为纯化缓冲液的25mM 2-(N-吗啉)乙磺酸(MES)，pH＝6.5，将溶液浓缩至1-2mL，再次加入纯化缓冲液，置换倍数大于1000倍，由此，将未偶联的接头-药物化合物及其他低分子量试剂除去，得到纯化的抗体-药物偶联物(DP001-DXD ADC,DAR＝4.3)。抗体-药物偶联物的药物抗体偶联比(DAR)的测定方法同实施例3第四步。
参考例2抗体-药物偶联物(DP001-DXD ADC,DAR＝7.0)
Figure PCTCN2022132627-appb-000073
第一步 抗体的还原：
通过公知技术将编码轻、重链基因的质粒瞬时转染HEK293细胞表达后纯化后得到的DP001抗体将介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加100mM TCEP(百灵威科技有限公司)水溶液(6.66μL；相对于一分子抗体为10当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司，18μL)。确认了该溶液的pH为7.0±0.1内后，于37℃孵育3小时，由此将抗体内的二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加购买的Deruxtecan(CAS No.:1599440-13-7)10mM的二甲基亚砜溶液(87μL；相对于一分子抗体为13当量)，混匀，在室温下反应30分钟，将接头-药物化合物连接于抗体。接下来，添加100mM NAC(百灵威科技有限公司)水溶液(10.2μL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体-药物偶联物的纯化：
使用超滤离心管(Merck，
Figure PCTCN2022132627-appb-000074
Regenerated CellμLose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为纯化缓冲液的25mM 2-(N-吗啉)乙磺酸(MES)，pH＝6.5，将溶液浓缩至1-2mL，再次加入纯化缓冲液，置换倍数大于1000倍，由此，将未偶联的接头-药物化合物及其他低分子量试剂除去，得到纯化的抗体-药物偶联物(DP001-DXD ADC,DAR＝7.0)。抗体-药物偶联物的药物抗体偶联比(DAR)的测定方法同实施例3第四步。
参考例3抗体-药物偶联物(patritumab-DXD ADC,DAR＝7.3)
Figure PCTCN2022132627-appb-000075
第一步 抗体的还原：
通过公知技术将编码轻、重链基因的质粒瞬时转染HEK293细胞表达后纯化后得到的patritumab抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加100mM TCEP(百灵威科技有限公司)水溶液(6.66μL；相对于一分子抗体为10当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司，18μL)。确认了该溶液的pH为7.0±0.1内后，于37℃孵育3小时，由此将抗体内的二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加购买的Deruxtecan(CAS No.:1599440-13-7)10mM的二甲基亚砜溶液(87μL；相对于一分子抗体为13当量)，混匀，在室温下反应30分钟，将接头-药物化合物连接于抗体。接下来，添加100mM NAC(百灵威科技有限公司)水溶液(10.2μL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体-药物偶联物的纯化：
使用超滤离心管(Merck，
Figure PCTCN2022132627-appb-000076
Regenerated CellμLose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为纯化缓冲液的25mM 2-(N-吗啉)乙磺酸(MES)，pH＝6.5，将溶液浓缩至1-2mL，再次加入 纯化缓冲液，置换倍数大于1000倍，由此，将未偶联的接头-药物化合物及其他低分子量试剂除去，得到纯化的抗体-药物偶联物(patritumab-DXD ADC,DAR＝7.3)。DAR值测定方法同实施例3第四步，DAR值结果如图10所示。
参考例4抗体-药物偶联物(SWY2110-DXD ADC,DAR＝6.93)
Figure PCTCN2022132627-appb-000077
第一步 抗体的还原：
通过公知技术将编码轻、重链基因的质粒瞬时转染HEK293细胞表达后纯化后得到的SWY2110抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加10mM TCEP(百灵威科技有限公司)水溶液(66.6uL；相对于一分子抗体为10当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司)。确认了该溶液的pH为7.0±0.1内后，于37℃孵育3小时，由此将抗体二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加购买的Deruxtecan(CAS No.:1599440-13-7)化合物10mM的二甲基亚砜溶液(87uL；相对于一分子抗体为13当量)，混匀，在室温下反应30分钟，将接头-药物化合物连接于抗体。接下来，添加100mM  NAC(百灵威科技有限公司)水溶液(10.2uL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体－药物偶联物的纯化:
使用超滤离心管(Merck，
Figure PCTCN2022132627-appb-000078
Regenerated Cellulose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为纯化缓冲液的25mM 2-(N-吗啉)乙磺酸(MES)，pH＝6.5，将溶液浓缩至1-2mL，再次加入纯化缓冲液，置换倍数大于1000倍，由此，将未偶联的接头-药物化合物及其他低分子量试剂除去，得到纯化的抗体-药物偶联物(SWY2110-DXD ADC,DAR＝6.93)。DAR值测定方法同实施例3第四步，DAR值结果如图11所示。
参考例5抗体-药物偶联物(SWY2110-VC MMAE ADC,DAR＝4.53)
Figure PCTCN2022132627-appb-000079
第一步 抗体的还原：
通过公知技术将编码轻、重链基因的质粒瞬时转染HEK293细胞表达后纯化后得到的SWY2110抗体介质替换为PBS6.0/EDTA，制备成10mg/mL的抗体浓度。将该溶液(1.0mL)放入到1.5mL聚丙烯制管(tube)中，向其中添加10mM TCEP(百灵威科技有限公司)水溶液(17uL；相对于一分子抗体为2.5当量)及1M磷酸氢二钾水溶液(天津市光复科技发展有限公司)。确认了该溶液的pH为 7.4±0.1内后，于37℃孵育1小时，由此将抗体二硫键还原。
第二步 抗体与接头-药物化合物的偶联：
在室温下向上述溶液中添加购买的mc-vc-PAB-MMAE,(CAS No.:646502-53-6，简称Vc MMAE)得到的化合物10mM的二甲基亚砜溶液(31.28uL；相对于一分子抗体为4.6当量)，混匀，在室温下反应30分钟，将接头-药物化合物连接于抗体。接下来，添加100mM NAC(百灵威科技有限公司)水溶液(10.2uL)，进而在室温下搅拌20分钟，终止反应。
第三步 抗体-药物偶联物的纯化:
使用超滤离心管(Merck，
Figure PCTCN2022132627-appb-000080
Regenerated Cellulose(30kDa MWCO),15mL sample volume)对反应液进行超滤纯化。向反应液中加入作为纯化缓冲液的25mM 2-(N-吗啉)乙磺酸(MES)，pH＝6.5，将溶液浓缩至1-2mL，再次加入纯化缓冲液，置换倍数大于1000倍，由此，将未偶联的接头-药物化合物及其他低分子量试剂除去，得到纯化的抗体-药物偶联物抗体-药物偶联物(SWY2110-VC MMAE ADC,DAR＝4.53)。
第四步 抗体-药物偶联物的药物抗体偶联比(DAR)的测定：
取10μg SWY2110-Vc MMAE ADC样品上色谱柱(2.5μm粒径；4.6mm×10cm；TSKgel Butyl-NPR)，洗脱流动相A液为20mM PB，pH7.0，1.5M(NH4) 2SO 4水溶液，流动相B液为20mM PB，pH7.0，25％异丙醇溶液，控制流速为0.8mL/min，柱温为30℃，检测波长为280nm，梯度程序为0-20分钟，流动相B由0-100％，20-25分钟，100％流动相B，25-30分钟，100％流动相A。未连接的为DAR0，连接2个小分子为DAR2，连接4个小分子为DAR4，连接6个小分子为DAR6，连接8个小分子为DAR8。因此按照DAR0、DAR2、DAR4、DAR6、DAR8的顺序洗脱。根据280nm下的峰面积计算DAR值，其中A代表各峰面积的百分数。DAR值结果如图12所示。
DAR＝2×ADAR 2+4×ADAR 4+6×ADAR 6+8×ADAR 8
生物实施例
试验一 抗体-药物体外细胞活性
1、试验目的：
本试验的目的是为了检测本发明抗体-药物偶联物对SK-BR-3(ATCC/HTB-30)肿瘤细胞、人结直肠癌细胞DiFi、人肺癌吉非替尼耐药细胞PC9-GR、人肺癌突变细胞PC-9(Del19-T790M-C797S)肿瘤细胞体外增值的抑制活性，以不同浓度的化合物体外处理细胞，经培养后，然后加入Resazurin(刃天青)读ex550nm/em610nm的荧光值，四参数拟合处理数据得出IC50值，从而计算出化合物的生物活性。
2、试验材料与设备：
材料
产品名称 厂家/货号
SK-BR-3细胞系 ATCC/HTB-30
DiFi细胞系 上海津曼特生物科技有限公司
PC9-GR细胞系 南京科佰生物科技有限公司
MTT Assay Kit Abcam
DMEM，高糖 Hyclone/SH30022.01
0.25％Trypsin-EDTA Gibco/25200-072
胎牛血清(FBS) Gibco/16000-044
100×双抗 Gibco/15240-062
黑壁，透明底的组织培养板 Corning/3603
刃天青钠 Sigma Aldrich/199303-25G
设备
名称 型号 厂家
生物安全柜 1300系列A2型 Thermo Fisher Scientific
CO 2培养箱 3111型 Thermo Fisher Scientific
倒置显微镜 CKX31 奥林巴斯
酶标仪 Infinite M200 Tecan公司
微量振荡器 MM-I型 上海亚荣生化仪器厂
3、试验操作方法1：
3.1培养基：DMEM，10％FBS，1×双抗
3.2细胞培养：从液氮中取出一支冷冻的SK-BR-3/MDA-MB-468细胞，复苏至75cm 2培养瓶中培养。生长到细胞的汇合度达到＞75％，并且已经进行至少3次传代。
3.2.1如果不进行细胞传代，每3～4天更换一次培养基。
3.2.2如果需要扩增细胞，将细胞传代接种到更大的培养瓶中，并且在测定使用之前使细胞的汇合度达到＞75％。
3.3收集细胞：培养瓶接近长满时收集SK-BR-3/MDA-MB-468细胞。
3.3.1弃除培养基，并用PBS清洗去除死细胞和残留培养基。
3.3.2加入2-3mL0.25％Trypsin-EDTA，轻轻摇晃培养瓶然后37℃孵育2-3分钟消化细胞。
3.3.3立即向培养瓶中加入5mL培养基，并用移液管轻轻上下吸打使细胞 分散开。
3.3.4将细胞悬液转移到无菌离心管中200×g离心3分钟。
3.3.5弃除管中培养基，加入5～10mL新鲜培养基重悬细胞。
3.4细胞密度测定：显微镜下细胞计数板计数。
3.5分析板接种
3.5.1用培养基将细胞稀释至1×10 5个细胞/mL，100μL/孔接种至分析板中(A和H行除外)。
3.5.2向A和H行中每孔加入120μL培养基作为空白对照。
3.5.3 37℃，5％CO 2孵育4-6h使细胞贴壁。
3.6准备稀释化合物样品
将化合物稀释至起始浓度为18μg/ml，再进行3倍稀释，共获得11个梯度溶液，第12列为空白对照。
3.7加药处理
3.7.1将稀释板中1～11列中的药物稀释液各取20μL分别加入到分析板中的1～11列中。
3.7.2第12列和第A、H行中各加入20μL新鲜培养基。
3.7.4在平板震荡器上轻轻震荡10-15秒钟，然后将培养板37℃孵育3天。
3.8分析
3.8.1孵育结束后，每孔加入0.03％刃天青(1×PBS稀释)20μL并轻轻震荡10～15秒。
3.8.2 37℃孵育3～4小时后酶标仪读数，参数设置如下：
激发光：550nm
发射光：610nm
积分时间：50
震荡：15秒，涡旋
读数：顶读
读次数/孔：1
增益：设置优化(应在35～42之间)
如果要读多块板，确保所有板子所用的增益设置一致。
3.8.3利用EXCEL数据作图拟合出参考标准品和样品的IC 50。
3.8.3.1使用模型201进行数据点绘图。
3.8.3.2对于Fit参数，请使用以下命令：
a.A：预配
b.B：预配
c.C：预配
d.D：预配
e.没有为所有约束
3.8.3.3输出参数C为IC 50，单位为ng/mL。
结果见表1。
试验操作方法2：
本试验验证SWY2110单克隆抗体(SWY2110mAb)、SWY2110-DXD ADC(DAR＝6.93)和SWY2110-JSSW-001 ADC(DAR＝7.12)对DiFi(人结直肠癌细胞)、PC9-GR(人肺癌吉非替尼耐药细胞)2株细胞增殖抑制试验，具体浓度设置如下：
DiFi：SWY2110mAb、SWY2110-DXD ADC(DAR＝6.93)和SWY2110-JSSW-001 ADC(DAR＝7.12)以3000ng/ml为起始浓度，药物3×递减稀释共设10个浓度，分别为3000、1000、333.33、111.11、37.04、12.35、4.12、1.37、0.46、0.15ng/ml，药物作用144h。
PC9-GR：SWY2110mAb、SWY2110-DXD ADC(DAR＝6.93)、SWY2110-JSSW-001 ADC(DAR＝7.12)以1000ng/ml为起始浓度，5×递减稀释共设8个浓度，分别为1000、200、40、8、1.6、0.32、0.64、0.013ng/ml，药物作用144h。
将处于对数生长期的细胞以一定数量接种于96孔板(100μL/孔)，贴壁细胞贴壁24h后，当天每孔加入100μL含不同浓度梯度SWY2110mAb、SWY2110-DXD ADC(DAR＝6.93)或SWY2110-JSSW-001 ADC(DAR＝7.12)的培养液，每个药物浓度设3个复孔，并设相应的空白孔(只有培养基)及正常孔(药物浓度为0)。药物作用144小时后，加入MTT工作液(5mg/mL)，每孔20μL；37℃作用4小时，甩板去除上清液，加入DMSO(分析纯)150μL；微孔振荡器震荡混匀，将板擦拭干净，酶标仪550nm处检测光密度值(OD)。
采用下列公式计算细胞生长的抑制率：
抑制率(％)＝(OD值 正常孔－OD值 给药孔)/(OD值 正常孔－OD值 空白孔)×100％
根据各浓度抑制率，用SPSS19.0计算药物半数抑制浓度IC 50。结果见表2、表3和图17、图18
试验数据：
表2：DP001-ADC对SK-BR-3细胞的IC 50值
化合物编号 IC 50值(ng/mL)
DP001-DXD ADC(DAR＝7.0) 7.2481
DP001-JSSW-001 ADC(DAR＝7.32) 5.1824
从表2记载的数据可以看出，本发明抗体-药物DP001-JSSW-001 ADC(DAR＝7.12)对SK-BR-3细胞，具有比DP001-DXD ADC(DAR＝6.93)更低的IC50值，表现出更好的抗细胞增殖效果。
表3：SWY2110-ADC对2株细胞的IC 50值(ng/ml)
Figure PCTCN2022132627-appb-000081
其中“--”表示未展现抗细胞增殖效果。
从表3数据可以看出，本发明的抗体-药物偶联物SWY2110-JSSW-001ADC(DAR＝7.12)与SWY2110-DXD ADC(DAR＝6.93)对EGFR高表达细胞株DiFi以及吉非替尼耐药株PC9-GR具有相似的IC 50值，相对于SWY2110mAb表现出更好的抗细胞增殖效果。
试验二 抗体-药物偶联物对NU/NU小鼠HER2-表达的人乳腺癌JIMT-1细胞移植瘤药效试验
1.试验动物
NU/NU小鼠，5～6周龄，24只
2.试验目的
采用HER2-表达的人乳腺癌JIMT-1细胞NU/NU小鼠移植瘤模型，考察抗体-药物偶联物体内抗肿瘤作用。
3.药物剂量和分组
表4动物分组及剂量表
组别 动物数量(只) 剂量(mg/kg) 给药途径/频次
溶剂组(Vehicle) 6 0 i.v./1次
DP001-DXD ADC(DAR＝4.30) 6 3 i.v./1次
DP001-JSSW-001 ADC(DAR＝4.0) 6 3 i.v./1次
DP001-ZW-002 ADC(DAR＝5.78) 6 3 i.v./1次
注：i.v.：静脉注射。
4.试验方法
本试验选用人乳腺癌细胞JIMT-1构建裸小鼠肿瘤移植瘤模型，待肿瘤体积至约127mm 3时，按表4将动物按肿瘤体积均衡分为4组(d0)，每组6只动物，单次静脉给药，给药体积是10mL/kg，溶剂对照组(Vehicle)给予0.9％氯化钠注射液，给药后观察13天，比较表中3个抗体-药物偶联物对HER2表达细胞系人乳腺癌JIMT-1裸小鼠移植瘤的抑制作用。
5.观测指标
5.1评价指标
(1)肿瘤体积：V＝1/2×A×B 2
(2)相对肿瘤体积：
Figure PCTCN2022132627-appb-000082
(3)相对肿瘤体积增殖率：
Figure PCTCN2022132627-appb-000083
(4)肿瘤抑制率：
Figure PCTCN2022132627-appb-000084
注：V：肿瘤体积
A：肿瘤长
B：肿瘤宽
RTV：相对肿瘤体积
TVnd：第n天肿瘤体积
TV0d：第0天肿瘤体积
RTVxnd：第n天平均相对肿瘤体积
TV Xn：给药组第n天平均肿瘤体积
TV X0：给药组第0天平均肿瘤体积
TV Mn：溶剂组第n天平均肿瘤体积
TV M0：溶剂组第0天平均肿瘤体积
6结果
6.1肿瘤体积
试验终点，与溶剂组相比，DP001-DXD ADC(DAR＝4.3)(P＜0.05)、DP001-JSSW-001 ADC(DAR＝4.0)(P＜0.01)和DP001-ZW-002 ADC(DAR＝5.78) (P＜0.05)均能显著抑制移植瘤的体积。详见图13和表5。
表5抗体-药物偶联物给药13天各组肿瘤参数表
Figure PCTCN2022132627-appb-000085
*：与溶剂组相比P＜0.05，**：与溶剂组相比P＜0.01
试验三 抗体-药物偶联物对NU/NU小鼠人肺腺癌PC9-GR(Gefitinib耐药细胞)细胞移植瘤药效试验
1.试验动物
NU/NU小鼠，5～6周龄，16只
2.试验目的
采用人肺腺癌PC-9/GR(Gefitinib耐药细胞)细胞NU/NU小鼠移植瘤模型，考察2个patritumab-ADC药物体内抗肿瘤作用。
3.药物剂量和分组
表6动物分组及剂量表
Figure PCTCN2022132627-appb-000086
注：i.v.：静脉注射，qw*4w：每周给药一次，给药四周。
4.试验方法
本试验选用人肺腺癌PC9-GR(Gefitinib耐药细胞)细胞构建裸小鼠肿瘤移植瘤模型，待肿瘤体积至约110mm 3时，按表6将动物按肿瘤体积分为3组(d0)，溶媒组6只动物，patritumab-DXD ADC(DAR＝7.3)组与patritumab-JSSW-001 ADC(DAR＝7.36)组各5只动物，静脉给药，每周给药一次，连续给药4周，给药体积是10mL/kg，溶媒对照组(Vehicle)给予0.9％氯化钠注射液，给药后28天结束试验，比较patritumab-DXD ADC(DAR＝7.3)及patritumab-JSSW-001  ADC(DAR＝7.36)对人肺腺癌PC9-GR(Gefitinib耐药细胞)裸小鼠移植瘤的抑制作用。
5.观测指标
5.1评价指标
参见试验二5.1评价指标部分
6结果
6.1肿瘤体积
试验终点，patritumab-JSSW-001 ADC(DAR＝7.36)组(P＜0.001)相对于patritumab-DXD ADC(DAR＝7.3)(P＜0.01)组具有更优的抑制移植瘤的体积的效果。详见图14和表7。
表7 patritumab-ADC给药28天各组肿瘤参数表
Figure PCTCN2022132627-appb-000087
**：与溶媒组相比P＜0.01，***：与溶剂组相比P＜0.001
试验四：SWY2110-ADC对NU/NU小鼠人结直肠癌DiFi细胞移植瘤药效试验
1.试验动物
NU/NU小鼠，5～6周龄，14只
2.试验目的
采用人结直肠癌DiFi细胞NU/NU小鼠移植瘤模型，考察2个SWY2110-ADC药物体内抗肿瘤作用。
3.药物剂量和分组
表8动物分组及剂量表
Figure PCTCN2022132627-appb-000088
注：i.v.：静脉注射。
4.试验方法
本试验选用人结直肠癌DiFi细胞构建裸小鼠肿瘤移植瘤模型，待肿瘤体积至约110mm 3时，按表8将动物按肿瘤体积均衡分为3组(d0)，溶媒组和SWY2110-JSSW-001 ADC(DAR＝7.12)各5只，SWY2110-Vc MMAE ADC(DAR＝4.53)组4只，单次静脉给药，给药体积是10mL/kg，溶媒对照组(Vehicle)给予0.9％氯化钠注射液，给药后观察27天，比较表中2个SWY2110-ADC对人结直肠癌DiFi裸小鼠移植瘤的抑制作用。
5.观测指标
5.1评价指标
参见试验二5.1评价指标部分
6结果
6.1肿瘤体积
试验终点，与溶媒组相比，SWY2110-Vc MMAE ADC(DAR＝4.53)(P＜0.05)和SWY2110-JSSW-001 ADC(DAR＝7.12)(P＜0.05)均能显著抑制移植瘤的体积，SWY2110-JSSW-001 ADC(DAR＝7.12)组27天肿瘤抑制率高达74.6％，SWY2110-Vc MMAE ADC(DAR＝4.53)肿瘤抑制率次之为69.7％，由于JSSW-001小分子的毒性低于Vc MMAE，预示SWY2110-JSSW-001 ADC(DAR＝7.12)能够实现更为高效低毒的作用效果。详见图15和表9。
表9 SWY2110-ADC给药27天各组肿瘤参数表
Figure PCTCN2022132627-appb-000089
*：与溶媒组相比P＜0.05
试验五 SWY2110-ADC对NU/NU小鼠人肺腺癌PC9-GR(Gefitinib耐药细胞)细胞移植瘤药效试验
1.试验动物
NU/NU小鼠，5～6周龄，30只
2.试验目的
采用人肺腺癌PC-9-GR(Gefitinib耐药细胞)细胞NU/NU小鼠移植瘤模型， 考察SWY2110单抗和3个SWY2110-ADC药物体内抗肿瘤作用。
3.药物剂量和分组
表10动物分组及剂量表
Figure PCTCN2022132627-appb-000090
注：i.v.：静脉注射，qw*4w：每周给药一次，给药四周。
4.试验方法
本试验选用人肺腺癌PC9-GR(Gefitinib耐药细胞)细胞构建裸小鼠肿瘤移植瘤模型，待肿瘤体积至约110mm 3时，按表10将动物按肿瘤体积均衡分为5组(d0)，每组6只动物，静脉给药，每周给药一次，连续给药4周，给药体积是10mL/kg，溶媒对照组(Vehicle)给予0.9％氯化钠注射液，给药后28天结束试验，比较SWY2110单抗和3个SWY2110-ADC药物对人肺腺癌PC-9-GR(Gefitinib耐药细胞)裸小鼠移植瘤的抑制作用。
5.观测指标
5.1评价指标
参见试验二5.1评价指标部分
6结果
6.1肿瘤体积
试验终点，与溶媒组相比，SWY2110-Vc MMAE ADC(DAR＝4.53)(P＜0.05)、SWY2110-DXD ADC(DAR＝6.93)(P＜0.05)和SWY2110-JSSW-001 ADC(DAR＝7.12)(P＜0.001)均能抑制移植瘤的体积。抗体SWY2110对肿瘤的抑制效果不佳，肿瘤抑制率仅为20.3％，SWY2110-JSSW-001 ADC(DAR＝7.12)组对PC9-GR的移植瘤体积抑制效果最显著，肿瘤抑制率高达77.7％，SWY2110-Vc MMAE ADC(DAR＝4.53)与SWY2110-DXD ADC(DAR＝6.93)的肿瘤抑制率分别为40.4％、50.2％，SWY2110-JSSW-001 ADC(DAR＝7.12)是SWY2110-DXD ADC(DAR＝6.93)对PCR-GR的肿瘤抑制效果的154％，SWY2110-JSSW-001  ADC(DAR＝7.12)是SWY2110-Vc MMAE ADC(DAR＝4.53)对PCR-GR的肿瘤抑制效果的192％，预示SWY2110-JSSW-001 ADC(DAR＝7.12)对吉非替尼耐药的肿瘤细胞依然有显著抑制作用。详见图16和表11。
表11 SWY2110-ADC给药28天各组肿瘤参数表
Figure PCTCN2022132627-appb-000091
*：与溶媒组相比P＜0.05，***：与溶媒组相比P＜0.001
试验六 抗体-药物体外血浆稳定性
DP001-DXD ADC(DAR＝7.0)、DP001-JSSW-001 ADC(DAR＝7.32)以及DP001-ZW-002 ADC(DAR＝5.78)分别与0.5％BSA-PBS和ICR小鼠血浆进行孵育，孵育浓度分别为DP001-DXD ADC(DAR＝7.0)(91.7μg/mL)、DP001-JSSW-001 ADC(DAR＝7.32)(80μg/mL)以及DP001-ZW-002 ADC(DAR＝5.78)(80μg/mL)，分别在37℃无菌条件下温孵0h、24h、48h、72h、96h、120h、144h、168h、336h和504h后收集样品，采用LC-MS/MS法检测血浆样品中脱落分子的浓度，结果如下：
表12体外血浆稳定性
Figure PCTCN2022132627-appb-000092
注：1.脱落率的计算：根据DAR值，计算DXD/ADC的质量比，得到DP001-DXD ADC(DAR＝7.0)(91.7μg/mL)、DP001-JSSW-001 ADC(DAR＝7.32)(80μg/mL)以及DP001-ZW-002 ADC(DAR＝5.78)(80μg/mL)。脱落率(％)＝C t/C total*100％，其中Ctotal为总的小分子浓度，C t为每个时间点的游离的小分子浓度。2.“-”表示未取样。
结果表明，ADC在37℃温孵0h、24h、48h、72h、96h、120h、144h、168h、336h和504h，0.5％在BSA-PBS和ICR小鼠血浆中均有游离小分子生成。温孵504h后，在0.5％BSA-PBS中脱落率分别为DP001-DXD ADC(DAR＝7.0)(3.90％)、DP001-JSSW-001ADC(DAR＝7.32)(1.09％)和DP001-ZW-002 ADC(DAR＝5.78)(2.91％)，在ICR小鼠血浆中脱落率分别为DP001-DXD ADC(DAR＝7.0)(4.07％)、DP001-JSSW-001 ADC(DAR＝7.32)(2.75％)。在0.5％BSA-PBS中，小分子从抗体上脱落率均不足4.0％：DP001-DXD ADC(DAR＝7.0)＞DP001-ZW-002 ADC(DAR＝5.78)＞DP001-JSSW-001ADC(DAR＝7.32)，表明DP001-JSSW-001ADC(DAR＝7.32)和DP001-ZW-002 ADC(DAR＝5.78)在0.5％BSA-PBS中更稳定。在ICR小鼠血浆中，小分子从抗体上脱落率均不足5.0％：DP001-DXD ADC(DAR＝7.0)＞DP001-JSSW-001 ADC(DAR＝7.32)，表明DP001-JSSW-001 ADC(DAR＝7.32)比DP001-DXD ADC(DAR＝7.0)在ICR小鼠血浆中更较稳定。
试验七 抗体-药物体外旁观效应研究
对于旁观效应试验首先采用阳性细胞系SK-BR-3和阴性对照细胞MDA-MB-468，分别与DP001-DXD ADC(DAR＝7.0)和DP001-JSSW-001ADC(DAR＝7.32)共同孵育，释放的小分子毒素能够引起周围共培养的阴性细胞的凋亡，即旁观效应，本试验主要通过报告基因细胞HEK293-Luc分别与阳性细胞系SK-BR-3和阴性对照细胞MDA-MB-468共培养四天后，通过加入化学发光底物来判断释放的小分子毒素对HEK293-Luc的增长影响。研究结果显示：DP001-DXD ADC(DAR＝7.0)和DP001-JSSW-001 ADC(DAR＝7.32)能够与肿瘤细胞表面的HER2受体结合后，通过内吞作用进入到细胞内释放小分子毒素；同时DP001-JSSW-001 ADC(DAR＝7.32)表现出对旁观细胞的增殖更强抑制作用。
2.试验准备
2.1试验设备
仪器设备 生产厂家 型号
二氧化碳培养箱 Thermo Fisher 3111型
生物安全柜 ThermoFisher 1300Series A2 6英寸
台式高速冷冻离心机 Thermo Fisher SORVALL Stratos
倒置显微镜 奥林巴斯 CKX31倒置显微镜
移液器 Eppendorf Research Plus
酶标仪 Tecan M200
2.2试验耗材
名称 生产厂家 货号
96孔透明平底培养板 Corning 3599
细胞培养板 Corning 3917
50mL离心管 Corning 430828
15mL离心管 Corning 430790
T75细胞培养瓶 Corning 430641
T25细胞培养瓶 Corning 430639
2.3试验试剂
Figure PCTCN2022132627-appb-000093
2.4细胞信息
Figure PCTCN2022132627-appb-000094
3.试验方法
3.1试剂配制
3.1.1细胞复苏培养基的制备
DMEM完全培养基：DMEM基础培养基中加入终体系为10％的Fetal Bovine Serum、1％的Penicilin/Streptomycin，充分混匀后于2-8℃保存。
3.1.2细胞培养基(含筛选剂)的制备
DMEM基础培养基中加入终体系为10％的Fetal Bovine Serum、1％的Penicilin/Streptomycin，并添加200μg/ml的G418，充分混匀后于2-8℃保存。
3.2细胞准备
3.2.1细胞复苏
将细胞冻存管置于温度为37℃的水浴锅中快速晃动至完全融化，转移至超净工作台或生物安全柜中。将细胞悬液用细胞复苏培养基稀释后离心，弃去离心上清，并使用细胞复苏培养基重悬细胞，测定细胞密度和活率。用细胞培养基调整细胞密度，混合均匀后，将细胞悬液转移至细胞培养瓶中，显微镜镜检无误后，转移至培养箱中，37℃，5％CO 2培养。
3.2.2细胞传代
将细胞培养瓶从CO 2培养箱中取出并转移至超净工作台或生物安全柜中，细胞经过PBS洗涤，胰酶消化，细胞生长培养基终止消化后，收集细胞悬液，测定细胞密度和活率。取适量细胞悬液加入到适量新鲜培养基(含或者不含筛选剂)中，根据细胞生长特性和试验需求进行传代扩增。传代后，将细胞置于培养箱中继续培养。
3.3旁观效应试验方法
3.3.1待细胞融汇度达到90％时，分别胰酶消化收集SK-BR-3、MDA-MB-468，以及HEK293-Luc细胞，并使用相对应的培养基重悬细胞；
3.3.2轻柔吹打重悬靶细胞数次至单细胞悬液，使用台盼蓝染色法鉴定细胞活力和细胞计数；调整细胞密度为2.0×10 5cells/ml；
3.3.3调整好密度的SK-BR-3和MDA-MB-468细胞分别与HEK293-Luc细胞悬液进行1:1混匀；每孔50μl加至96孔透明平底培养板；阳性细胞组即SK-BR-3和HEK293-Luc的混合细胞；阴性细胞组即MDA-MB-468和HEK293-Luc的混合细胞；
3.3.4 DP001-DXD ADC(DAR＝7.0)和DP001-JSSW-001 ADC(DAR＝7.32)给药组共设3个浓度，终浓度分别为：100ng/ml、500ng/ml，以及1000ng/ml；每孔50μl加至已接种细胞的96孔透明平底培养板；放置于37℃二氧化碳培养箱中培养4天；
3.3.5孵育结束后，从培养箱中取出培养板，平衡温度至室温；以100μL/孔将Bright-Glo TM Luciferase Assay System加入培养板中，避光5-15分钟读取化学发光。
4.试验结果：DP001-DXD(DAR＝7.0)和DP001-JSSW-001 ADC(DAR＝7.32)能够与肿瘤细胞表面的HER2受体结合后，通过内吞作用进入到细胞内释放小分子毒素；同时DP001-JSSW-001 ADC(DAR＝7.32)表现出比DP001-DXD ADC(DAR＝7.0)对旁观细胞的更强抑制作用。试验结果见表13和图19
表13：本发明抗体-药物对荧光细胞的抑制率。
Figure PCTCN2022132627-appb-000095
试验八 接头-药物化合物稳定性研究
不同的接头-药物化合物如：Deruxtecan、JSSW-001和ZW-002化合物分别在高温(40℃、60℃)、高湿(25℃/75％RH和25℃/92.5％RH)、光照(照度4500Lux，近紫外能量90μw/cm 2)或0.5％BSA-PBS进行研究，分别在不同条件下放置后收集不同时间样品，采用LC-MS/MS法检测样品中接头-药物化合物的浓度变化情况，试验结果表明JSSW-001、ZW-002化合物的稳定性更好。
试验九 SWY2110-ADC对人肺腺癌PC9-AR(PC9-Del19/T790M/C797S、Osimertinib耐药细胞)细胞移植瘤药效试验
1.试验动物
Balb/c nude小鼠，5～6周龄，24只
2.试验目的
采用人肺腺癌PC9-Del19/T790M/C797S(Osimertinib耐药细胞,以下简称PC9-AR)细胞NU/NU小鼠移植瘤模型，考察3个SWY2110-ADC药物体内抗肿瘤作用。
3.药物剂量和分组
表14动物分组及剂量表
Figure PCTCN2022132627-appb-000096
注：i.v.：静脉注射，i.v./1次：给药一次。
4.试验方法
本试验选用人肺腺癌PC9-Del19/T790M/C797S(Osimertinib耐药细胞)细胞构建裸小鼠肿瘤移植瘤模型，待肿瘤体积至约110mm 3时，按表14将动物按肿瘤体积均衡分为4组(d0)，每组6只动物，静脉给药，给药一次，给药体积是10mL/kg，溶媒对照组(Vehicle)给予0.9％氯化钠注射液，给药后24天结束试验，比较3个SWY2110-ADC药物对人肺腺癌PC9-Del19/T790M/C797S(Osimertinib耐药细胞)裸小鼠移植瘤的抑制作用。
5.观测指标
5.1评价指标
参见试验二5.1评价指标部分
6结果
6.1肿瘤体积
试验终点，与溶媒组相比，SWY2110-Vc MMAE ADC(DAR＝4.53)(P＜0.001)、SWY2110-Dxd ADC(DAR＝6.93)(P＜0.001)和SWY2110-JSSW-001 ADC(DAR＝7.12)(P＜0.001)均能抑制移植瘤的体积。SWY2110-JSSW-001 ADC(DAR＝7.12)组对PC9-AR的移植瘤体积抑制效果最显著，肿瘤抑制率高达97.5％，SWY2110-Vc MMAE ADC(DAR＝4.53)与SWY2110-DXD ADC(DAR＝6.93)的肿瘤抑制率分别为89.7％、76.2％，SWY2110-JSSW-001 ADC(DAR＝7.12)是SWY2110-DXD ADC(DAR＝6.93)肿瘤抑制效果的128％，SWY2110-JSSW-001 ADC(DAR＝7.12)是SWY2110-Vc MMAE ADC(DAR＝4.53)肿瘤抑制效果的109％，预示SWY2110-JSSW-001 ADC(DAR＝7.12)对Osimertinib耐药的肿瘤细胞依然有显著抑制作用。详见图20和表15。
表15 SWY2110-ADC给药24天各组肿瘤参数表
Figure PCTCN2022132627-appb-000097
***：相对于溶媒组的统计学显著性p＜0.001
试验十 SWY2110-ADC、SWY2111-ADC、SWY2112-ADC、SWY2113-ADC对人肺腺癌NCI-H1975(人肺腺癌细胞)细胞移植瘤药效试验
1.试验动物
NU/NU小鼠，5～6周龄，30只
2.试验目的
采用人肺腺癌NCI-H1975(人肺腺癌细胞)细胞NU/NU小鼠移植瘤模型，考察SWY2110-JSSW-001、SWY2111-JSSW-001、SWY2112-JSSW-001、SWY2113-JSSW-001药物体内抗肿瘤作用。
3.药物剂量和分组
表16动物分组及剂量表
Figure PCTCN2022132627-appb-000098
注：i.v.：静脉注射，i.v./1次：给药一次；
4.试验方法
本试验选用人肺腺癌NCI-H1975(人肺腺癌细胞)细胞构建裸小鼠肿瘤移植瘤 模型，待肿瘤体积至约110mm 3时，按表16将动物按肿瘤体积均衡分为5组(d0)，每组6只动物，每剂量组为单次给药1mg/kg。
静脉给药，给药一次，给药体积是10mL/kg，溶媒对照组(Vehicle)给予0.9％氯化钠注射液，给药后21天结束试验，比较SWY2110-JSSW-001、SWY2111-JSSW-001、SWY2112-JSSW-001、SWY2113-JSSW-001对人肺腺癌NCI-H1975裸小鼠移植瘤的抑制作用。
5.观测指标
5.1评价指标
参见试验二5.1评价指标部分
6结果
6.1肿瘤体积
试验终点，与溶媒组相比，SWY2110-JSSW-001 ADC(DAR＝7.12)、SWY2112-JSSW-001 ADC(DAR＝7.51)、SWY2111-JSSW-001 ADC(DAR＝7.48)、SWY2113-JSSW-001 ADC(DAR＝7.50)(P＜0.001)均具有很好的抑瘤效果，肿瘤抑制率达到84.1％，83.0％、80.9％和74.2％(P＜0.001)，显示4种ADC均对人肺腺癌NCI-H1975肿瘤细胞有显著抑制作用。详见图21和表17。
表17 SWY2110～SWY2113-ADC给药21天各组肿瘤参数表
Figure PCTCN2022132627-appb-000099
***：与溶媒组相比P＜0.001
试验十一 SWY2110-ADC、SWY2111-ADC、SWY2112-ADC、SWY2113-ADC对人乳腺癌MDA-MB-468(人乳腺癌细胞)细胞移植瘤药效试验
1.试验动物
NOD-SCID小鼠，5～6周龄，28只
2.试验目的
采用人乳腺癌MDA-MB-468(人乳腺癌细胞)细胞NOD-SCID小鼠移植瘤模型，考察SWY2110-JSSW-001、SWY2111-JSSW-001、SWY2112-JSSW-001、SWY2113-JSSW-001药物体内抗肿瘤作用。
3.药物剂量和分组
表18动物分组及剂量表
Figure PCTCN2022132627-appb-000100
注：i.v.：静脉注射，i.v./1次：给药一次；
4.试验方法
本试验选用人乳腺癌MDA-MB-468(人乳腺癌细胞)细胞构建小鼠肿瘤移植瘤模型，待肿瘤体积至约150mm 3时，按表18将动物按肿瘤体积分为5组(d0)，对照组8只动物，实验组5只动物，静脉给药，给药一次，给药体积是10mL/kg，溶媒对照组(Vehicle)给予0.9％氯化钠注射液，给药后28天结束试验，考察4个ADC药物体内抗肿瘤作用。
5.观测指标
5.1评价指标
参见试验二5.1评价指标部分
6结果
6.1肿瘤体积
试验终点，与溶媒组相比，SWY2110-JSSW-001(DAR＝7.12)、SWY2111-JSSW-001 ADC(DAR＝7.48)、SWY2112-JSSW-001 ADC(DAR＝7.51)和SWY2113-JSSW-001 ADC(DAR＝7.50)给药肿瘤抑制率分别达65.8％、65.1％、58.3％、47.1％(P＜0.001)，均显示良好的肿瘤抑制活性。预示4种ADC对人乳腺 癌MDA-MB-468肿瘤细胞有显著抑制作用。详见图22和表19。
表19 SWY2110～SWY2113-ADC给药28天各组肿瘤参数表
Figure PCTCN2022132627-appb-000101
***：与溶媒组相比P＜0.001
试验十二 SWY2110-ADC、SWY2111-ADC、SWY2112-ADC、SWY2113-ADC对人结直肠癌DiFi(人结直肠癌细胞)细胞移植瘤药效试验
1.试验动物
NU/NU小鼠，5～6周龄，35只
2.试验目的
采用人结直肠癌DiFi(人结直肠癌细胞)NU/NU小鼠移植瘤模型，考察SWY2110-JSSW-001、SWY2111-JSSW-001、SWY2112-JSSW-001、SWY2113-JSSW-001药物体内抗肿瘤作用。
3.药物剂量和分组
表20动物分组及剂量表
Figure PCTCN2022132627-appb-000102
注：i.v.：静脉注射，i.v./qw*2次：每周给药，给药两次；
4.试验方法
本试验选用人结直肠癌DiFi(人结直肠癌细胞)细胞构建小鼠肿瘤移植瘤模型，待肿瘤体积至约100mm 3时，按表20将动物按肿瘤体积分为5组(d0)，各组7只动物，静脉给药，给药两次，给药体积是10mL/kg，溶媒对照组(Vehicle)给予0.9％氯化钠注射液，给药后21天结束试验，考察4个ADC药物体内抗肿瘤作用。
5.观测指标
5.1评价指标
参见试验二5.1评价指标部分
6结果
6.1肿瘤体积
试验终点，与溶媒组相比，SWY2110-JSSW-001(DAR＝7.12)、SWY2111-JSSW-001 ADC(DAR＝7.48)、SWY2112-JSSW-001 ADC(DAR＝7.51)、SWY2113-JSSW-001 ADC(DAR＝7.50)给药肿瘤抑制率分别达86.5％、82.3％、83.5％、76.8％(P＜0.001)，均显示良好的肿瘤抑制活性。预示4种ADC对人结直肠癌DiFi肿瘤细胞有显著抑制作用。详见图23和表21。
表21 SWY2110～SWY2113-ADC给药21天各组肿瘤参数表
Figure PCTCN2022132627-appb-000103
***：与溶媒组相比P＜0.001
试验十三 SWY2110-ADC、SWY2111-ADC、SWY2112-ADC、SWY2113-ADC对人肺腺癌PC9-GR(人肺腺癌吉非替尼获得性耐药株)细胞移植瘤药效试验
1.试验动物
NU/NU小鼠，5～6周龄，30只
2.试验目的
采用人肺腺癌细胞PC9-GR NU/NU小鼠移植瘤模型，考察SWY2110-JSSW-001、SWY2111-JSSW-001、SWY2112-JSSW-001、SWY2113-JSSW-001药物体内抗肿瘤作用。
3.药物剂量和分组
表22动物分组及剂量表
Figure PCTCN2022132627-appb-000104
注：i.v.：静脉注射，i.v./1次：给药一次；
4.试验方法
本试验选用人肺腺癌细胞PC9-GR细胞构建小鼠肿瘤移植瘤模型，待肿瘤体积至约110mm 3时，按表22将动物按肿瘤体积分为5组(d0)，各组6只动物，静脉给药，给药一次，给药体积是10mL/kg，溶媒对照组(Vehicle)给予0.9％氯化钠注射液，给药后28天结束试验，考察4个ADC药物体内抗肿瘤作用。
5.观测指标
5.1评价指标
参见试验二5.1评价指标部分
6结果
6.1肿瘤体积
试验终点，与溶媒组相比，SWY2110-JSSW-001(DAR＝7.12)、SWY2111-JSSW-001 ADC(DAR＝7.48)、SWY2112-JSSW-001 ADC(DAR＝7.51)、SWY2113-JSSW-001 ADC(DAR＝7.50)给药肿瘤抑制率分别为99.0％、100.1％、92.4％、89.3％(P＜0.001)，均显示良好的肿瘤抑制效果。预示4种ADC对人肺腺癌细胞PC9-GR肿瘤细胞有显著抑制作用。详见图24和表23。
表23 SWY2110～SWY2113-ADC给药28天各组肿瘤参数表
Figure PCTCN2022132627-appb-000105
***：与溶媒组相比P＜0.001
试验十四 抗体-药物偶联物对NOD-SCID小鼠人肺腺癌PC9-DTC(Del19/T790M/C797S，Osimertinib耐药细胞)细胞移植瘤药效试验
1.试验动物
NOD-SCID小鼠，5～6周龄，18只
2.试验目的
采用人肺腺癌PC9-DTC(Del19/T790M/C797S，Osimertinib耐药细胞)细胞NU/NU小鼠移植瘤模型，考察2个patritumab-ADC药物体内抗肿瘤作用。
3.药物剂量和分组
表24动物分组及剂量表
Figure PCTCN2022132627-appb-000106
注：i.v.：静脉注射，qw*3w：每周给药一次，给药三周。
4.试验方法
本试验选用人肺腺癌PC9-DTC(Del19/T790M/C797S，Osimertinib耐药细胞)细胞构建裸小鼠肿瘤移植瘤模型，待肿瘤体积至约115mm 3时，按表24将动物按肿瘤体积分为3组(d0)，溶媒组、patritumab-DXD ADC(DAR＝7.3)组与 patritumab-JSSW-001 ADC(DAR＝7.36)组各6只动物，静脉给药，每周给药一次，连续给药3周，给药体积是10mL/kg，溶媒对照组(Vehicle)给予0.9％氯化钠注射液，给药后26天结束试验，比较patritumab-DXD ADC(DAR＝7.3)及patritumab-JSSW-001 ADC(DAR＝7.36)对人肺腺癌PC9-DTC(Del19/T790M/C797S，Osimertinib耐药细胞)裸小鼠移植瘤的抑制作用。
5.观测指标
5.1评价指标
参见试验二5.1评价指标部分
6结果
6.1肿瘤体积
验终点，patritumab-JSSW-001 ADC(DAR＝7.36)组(P＜0.001)相对于patritumab-DXD ADC(DAR＝7.3)(P＜0.001)组具有更优的抑制移植瘤的体积的效果。详见图25和表25
表25patritumab-ADC给药26天各组肿瘤参数表
Figure PCTCN2022132627-appb-000107
***：与溶媒组相比P＜0.001
试验十五 抗体-药物偶联物对NU/NU小鼠人结肠癌SW620细胞移植瘤药效试验
1.试验动物
NU/NU小鼠，5～6周龄，18只
2.试验目的
采用人结肠癌SW620细胞NU/NU小鼠移植瘤模型，考察2个patritumab-ADC药物体内抗肿瘤作用。
3.药物剂量和分组
表26动物分组及剂量表
Figure PCTCN2022132627-appb-000108
4.试验方法
本试验选用人结肠癌SW620细胞构建裸小鼠肿瘤移植瘤模型，待肿瘤体积至约110mm3时，按表26将动物按肿瘤体积分为3组(d0)，溶媒组、patritumab-DXD ADC(DAR＝7.3)组与patritumab-JSSW-001 ADC(DAR＝7.36)组各6只动物，静脉给药，单次给药，给药体积是10mL/kg，溶媒对照组(Vehicle)给予0.9％氯化钠注射液，给药后26天结束试验，比较patritumab-DXD ADC(DAR＝7.3)及patritumab-JSSW-001 ADC(DAR＝7.36)对人结肠癌SW620裸小鼠移植瘤的抑制作用。
5.观测指标
5.1评价指标
参见试验二5.1评价指标部分
6结果
6.1肿瘤体积
试验终点，patritumab-JSSW-001 ADC(DAR＝7.36)组(P＜0.001)相对于patritumab-DXD ADC(DAR＝7.3)(P＜0.001)组具有更优的抑制移植瘤的体积的效果。详见图26和表27。
表27 patritumab-ADC给药21天各组肿瘤参数表
Figure PCTCN2022132627-appb-000109
***：与溶剂对照组相比P＜0.001
试验十六 抗体-药物偶联物对NU/NU小鼠人结肠癌DiFi细胞移植瘤药效试验
1.试验动物
NU/NU小鼠，5～6周龄，18只
2.试验目的
采用人结直肠癌DiFi细胞NU/NU小鼠移植瘤模型，考察2个patritumab-ADC药物体内抗肿瘤作用。
3.药物剂量和分组
表28动物分组及剂量表
Figure PCTCN2022132627-appb-000110
注：i.v.：静脉注射。
4.试验方法
本试验选用人结直肠癌DiFi细胞构建裸小鼠肿瘤移植瘤模型，待肿瘤体积至约110mm 3时，按表28将动物按肿瘤体积分为3组(d0)，溶媒组6只动物，patritumab-DXD ADC(DAR＝7.3)组与patritumab-JSSW-001 ADC(DAR＝7.36)组各5只动物，静脉给药，单次给药，给药体积是10mL/kg，溶媒对照组(Vehicle)给予0.9％氯化钠注射液，给药后28天结束试验，比较patritumab-DXD ADC(DAR＝7.3)及patritumab-JSSW-001 ADC(DAR＝7.36)对人结直肠癌DiFi裸小鼠移植瘤的抑制作用。
5.观测指标
5.1评价指标
参见试验二5.1评价指标部分
6结果
6.1肿瘤体积
试验终点，patritumab-JSSW-001 ADC(DAR＝7.36)组(P＜0.01)相对于patritumab-DXD ADC(DAR＝7.3)(P＜0.05)组具有更优的抑制移植瘤的体积的效果。详见图27和表29。
表29 patritumab-ADC给药28天各组肿瘤参数表
Figure PCTCN2022132627-appb-000111
*：与溶媒组相比P＜0.05，**：与溶媒组相比P＜0.01
试验十七：安评研究
本试验选用适龄食蟹猴6只，雌雄各半，通过静脉注射SWY2110-JSSW-001、SWY2111-JSSW-001、SWY2113-JSSW-001。剂量设计如下表所示，每三周静脉注射1次，连续注射3次，给药后连续观察7天，每天2次一般观察，每天1次详细观察。在给药前、给药后观察体重变化，并进行血液学检测。实验结果显示，pH依赖性抗体制备的ADC(SWY2111-JSSW-001与SWY2113-JSSW-001)相比SWY2110-JSSW-001在皮肤、胃肠道毒性方面下降，没有出现明显的靶点相关毒性(皮肤破溃、结痂，稀便等)，可见pH改造的ADC对正常组织的毒性降低，但三种ADC小分子造成的血液毒性并没有明显差异。
表30 预毒理实验设计方案
Figure PCTCN2022132627-appb-000112
表31预毒理试验结果
Figure PCTCN2022132627-appb-000113
HGB：血红蛋白；WBC：白细胞计数；LYMP：淋巴细胞绝对数。

Claims (16)

  1. 式I或式II所示的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物：
    Figure PCTCN2022132627-appb-100001
    其中Ab为靶向HER2、HER3或EGFR的抗体或其抗原结合片段，R选自C 1-6烷基，n选自1-8的整数或1-8的小数。
  2. 根据权利要求1所述的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物，式I进一步为式Ia或式Ib：
    Figure PCTCN2022132627-appb-100002
    Figure PCTCN2022132627-appb-100003
    其中，式Ia和式Ib中，Ab、R和n的定义如式I。
  3. 根据权利要求1所述的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物，式I进一步为式I-1、式Ia-1或式Ib-1：
    Figure PCTCN2022132627-appb-100004
    Figure PCTCN2022132627-appb-100005
    其中，式I-1、式Ia-1和式Ib-1中，Ab和n的定义如式I。
  4. 根据权利要求1-3任一项所述的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物，其中，
    所述Ab为靶向HER2的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中，重链可变区序列包括重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，轻链可变区包括轻链互补决定区1(LCDR1)、轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，其中HCDR1的氨基酸序列如SEQ ID NO:1所示，HCDR2的氨基酸序列如SEQ ID NO:2所示，HCDR3的氨基酸序列如SEQ ID NO:3所示，和/或LCDR1的氨基酸序列如SEQ ID NO:4所示，LCDR2的氨基酸序列如SEQ ID NO:5所示，LCDR3的氨基酸序列如SEQ ID NO:6所示；进一步优选地，所述Ab为靶向HER2的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中HV的氨基酸序列如SEQ ID NO:7所示，LV的氨基酸序列如SEQ ID NO:8所示；更优选地，所述Ab为靶向HER2的抗体或其抗原结合片段时，其包括重链(HC)和轻链(LC)，其中HC的氨基酸序列如SEQ ID NO:9所示，LC的氨基酸序列如SEQ ID NO:10所示；
    所述Ab为靶向HER3的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中，重链可变区序列包括重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，轻链可变区包括轻链互补决定区1(LCDR1)、轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，其中HCDR1的氨基酸序列如SEQ ID NO:11所示，HCDR2的氨基酸序列如SEQ ID NO:12所示，HCDR3的氨基酸序列如SEQ ID NO:13所示，和/或LCDR1的 氨基酸序列如SEQ ID NO:14所示，LCDR2的氨基酸序列如SEQ ID NO:15所示，LCDR3的氨基酸序列如SEQ ID NO:16所示。进一步优选地，所述Ab为靶向HER3的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中HV的氨基酸序列如SEQ ID NO:17所示，LV的氨基酸序列如SEQ ID NO:18所示；更优选地，所述Ab为靶向HER3的抗体或其抗原结合片段时，其包括重链(HC)和轻链(LC)，其中HC的氨基酸序列如SEQ ID NO:19所示，LC的氨基酸序列如SEQ ID NO:20所示；
    所述Ab为靶向EGFR的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中，重链可变区序列包括重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)和重链互补决定区3(HCDR3)，轻链可变区包括轻链互补决定区1(LCDR1)、轻链互补决定区2(LCDR2)和轻链互补决定区3(LCDR3)，其中HCDR1的氨基酸序列如SEQ ID NO:21或SEQ ID NO:31或SEQ ID NO:35或SEQ ID NO:38所示，HCDR2的氨基酸序列如SEQ ID NO:22所示，HCDR3的氨基酸序列如SEQ ID NO:23或SEQ ID NO:32所示，和/或LCDR1的氨基酸序列如SEQ ID NO:24所示，LCDR2的氨基酸序列如SEQ ID NO:25所示，LCDR3的氨基酸序列如SEQ ID NO:26所示；进一步优选地，所述Ab为靶向EGFR的抗体或其抗原结合片段时，其包括重链可变区(HV)和轻链可变区(LV)，其中HV的氨基酸序列如SEQ ID NO:27或SEQ ID NO:33或SEQ ID NO:36或SEQ ID NO:39所示，LV的氨基酸序列如SEQ ID NO:28所示；更优选地，所述Ab为靶向EGFR的抗体或其抗原结合片段时，其包括重链(HC)和轻链(LC)，其中HC的氨基酸序列如SEQ ID NO:29或SEQ ID NO:34或SEQ ID NO:37或SEQ ID NO:40所示，LC的氨基酸序列如SEQ ID NO:30所示。
  5. 根据权利要求1-4任一项所述的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物，所述抗体优选为单克隆抗体，所述单克隆抗体优选选自人源抗体、人源化抗体、嵌合抗体。
  6. 根据权利要求1-4任一项所述的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物，所述抗原结合片段优选选自Fab’、(Fab’) 2、Fab、Fv、scFv、dAb。
  7. 式III所示的接头-药物化合物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物：
    Figure PCTCN2022132627-appb-100006
    其中R选自C 1-6烷基，优选C 1-3烷基，进一步优选甲基、乙基、丙基、异丙基。
  8. 根据权利要求7所述的接头-药物化合物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物，式III进一步为式IIIa或式IIIb：
    Figure PCTCN2022132627-appb-100007
  9. 下式所示的接头-药物化合物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物：
    Figure PCTCN2022132627-appb-100008
  10. 一种药物组合物，其包含权利要求1-6任一项所述的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物。
  11. 权利要求1-6任一项所述的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、溶剂合物、立体异构体或同位素标记物在制备用于治疗增殖性疾病的药 物中的用途。
  12. 根据权利要求11所述的用途，其特征在于所述增殖性疾病为HER2、HER3或EGFR异常表达相关疾病，包括癌症，所述癌症优选自乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、肺癌(例如小细胞肺癌和非小细胞肺癌)、结肠癌、直肠癌、结直肠癌、白血病(例如急性淋巴细胞白血病、急性髓细胞白血病、急性早幼粒细胞白血病、慢性髓细胞白血病、慢性淋巴细胞白血病)骨癌、皮肤癌、甲状腺癌、胰腺癌或淋巴瘤(例如霍奇金淋巴瘤、非霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤)。
  13. 权利要求1-6任一项所述的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物在制备用于治疗产生耐药性的增殖性疾病的药物中的用途。
  14. 根据权利要求13所述的用途，所述产生耐药性的增殖性疾病为产生耐药性的与HER2、HER3或EGFR异常表达相关的疾病，包括癌症，所述产生耐药性的癌症优选为HER2、HER3或EGFR基因突变导致的耐药癌症，所述癌症优选乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、肺癌(例如小细胞肺癌和非小细胞肺癌)、结肠癌、直肠癌、结直肠癌、白血病(例如急性淋巴细胞白血病、急性髓细胞白血病、急性早幼粒细胞白血病、慢性髓细胞白血病、慢性淋巴细胞白血病)骨癌、皮肤癌、甲状腺癌、胰腺癌或淋巴瘤(例如霍奇金淋巴瘤、非霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤)，进一步优选为结肠癌、直肠癌、肺癌或胰腺癌。
  15. 根据权利要求13所述的用途，其中所述耐药性对受体酪氨酸激酶抑制剂耐药，进一步优选为对第一代、第二代或第三代EGFR抑制剂耐药，更进一步优选对吉非替尼、厄洛替尼、埃克替尼、阿法替尼、达克替尼、奥希替尼或阿美替尼耐药，特别是对吉非替尼、阿法替尼、奥希替尼或阿美替尼耐药，更优选为对吉非替尼或奥希替尼耐药。
  16. 权利要求1-6任一项所述的抗体-药物偶联物，及其药用盐、水合物、溶剂合物、立体异构体或同位素标记物的制备方法，其包括如下步骤：
    S1：抗体的还原；
    S2：抗体与药物接头的偶联；
    S3：抗体－药物偶联物的纯化。
PCT/CN2022/132627 2021-11-17 2022-11-17 抗体-药物偶联物及其用途 WO2023088382A1 (zh)

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