WO2021148003A1 - 艾日布林衍生物的药物偶联物、其制备方法及其在医药上的应用 - Google Patents

艾日布林衍生物的药物偶联物、其制备方法及其在医药上的应用 Download PDF

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WO2021148003A1
WO2021148003A1 PCT/CN2021/073314 CN2021073314W WO2021148003A1 WO 2021148003 A1 WO2021148003 A1 WO 2021148003A1 CN 2021073314 W CN2021073314 W CN 2021073314W WO 2021148003 A1 WO2021148003 A1 WO 2021148003A1
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antibody
seq
cycloalkyl
hydrogen
alkyl
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PCT/CN2021/073314
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English (en)
French (fr)
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黄建
祝令建
于秀招
朱波
任文明
唐蜜
孙星
杨阳
梁金栋
胡齐悦
Original Assignee
上海森辉医药有限公司
上海盛迪医药有限公司
上海恒瑞医药有限公司
江苏恒瑞医药股份有限公司
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Application filed by 上海森辉医药有限公司, 上海盛迪医药有限公司, 上海恒瑞医药有限公司, 江苏恒瑞医药股份有限公司 filed Critical 上海森辉医药有限公司
Priority to KR1020227027736A priority Critical patent/KR20220130160A/ko
Priority to JP2022544350A priority patent/JP2023511163A/ja
Priority to MX2022009044A priority patent/MX2022009044A/es
Priority to EP21744595.6A priority patent/EP4094779A4/en
Priority to US17/794,732 priority patent/US20230144203A1/en
Priority to AU2021210079A priority patent/AU2021210079A1/en
Priority to BR112022014398A priority patent/BR112022014398A2/pt
Priority to CN202180007579.2A priority patent/CN114845739A/zh
Priority to CA3168654A priority patent/CA3168654A1/en
Publication of WO2021148003A1 publication Critical patent/WO2021148003A1/zh

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    • 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
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61K47/6835—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
    • 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
    • A61K47/6851—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 determinant of a tumour cell
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/04—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D307/18—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/20—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/22—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains four or more hetero rings
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present disclosure relates to drug conjugates of Eribulin derivatives, their preparation methods and their applications in medicine.
  • Antibody drug conjugate connects monoclonal antibodies or antibody fragments with biologically active drugs through stable chemical linkers, making full use of the specificity and binding of antibodies to normal cells and tumor cell surface antigens.
  • the high efficiency of the drug avoids the shortcomings of the former's low curative effect and the latter's excessive side effects. This also means that compared with traditional chemotherapy drugs in the past, antibody-drug conjugates can accurately bind to tumor cells and reduce the impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12:329– 332; DiJoseph JF, Armellino DC, (2004) Blood, 103: 1807-1814).
  • the first antibody-drug conjugate Mylotarg (gemtuzumab ozogamicin (gemtuzumab ozogamicin), Wyeth Pharmaceuticals Co., Ltd.) was approved by the US FDA for the treatment of acute myeloid leukemia (Drugs of the Future ( 2000) 25(7): 686; US4970198; US5079233; US5585089; US5606040; US5693762; US5739116; US5767285; US5773001).
  • Adcetris (brentuximab vedotin, Seattle Genetics) passed the U.S. FDA rapid review channel for the treatment of Hodgkin’s lymphoma and recurrent anaplastic large cell lymphoma (Nat.Biotechnol(2003)21(7) ):778-784; WO2004010957; WO2005001038; US7090843A; US7659241; WO2008025020). It is a new type of targeted ADC drug that can cause the drug to directly act on the target CD30 on lymphoma cells and then undergo endocytosis to induce tumor cell apoptosis.
  • Kadcyla (ado-trastuzumab emtansine, T-DM1) was approved by the U.S. FDA for the treatment of HER2-positive and at the same time resistant to trastuzumab (Tratuzumab, trade name: Herceptin) and paclitaxel in late stage or metastasis Patients with breast cancer (WO2005037992; US8088387).
  • Kadcyla is the first ADC drug approved by the US FDA for the treatment of solid tumors.
  • Microtubules are powerful filamentous cytoskeleton proteins related to various cellular functions including intracellular migration and transport, cell signal transduction, and maintenance of cell shape. Microtubules also play a key role in mitotic cell division by forming the mitotic spindle required for the division of chromosomes into two daughter cells.
  • the biological functions of microtubules in all cells are mostly regulated by their polymerization kinetics, which is carried out by the reversible and non-covalent addition of ⁇ and ⁇ tubulin dimers to both ends of the microtubules. This dynamic behavior and the resulting control of microtubule length are indispensable for the proper function of the mitotic spindle.
  • ADC antibody-drug conjugate
  • Ab is an antibody or an antigen-binding fragment thereof
  • L is a linker covalently linking Ab to D
  • k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20 or any value between any two values)
  • R 1a is selected from hydrogen, alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to Cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, said alkyl, cycloalkyl, aryl and heteroaryl each independently optionally selected from alkyl (such as C 1- 6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropyl) One or more of oxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl,
  • R 1b is selected from hydrogen, alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy, cycloalkyl (such as C 3-8 cycloalkyl, including But not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl.
  • alkyl such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl
  • alkoxy such as C 3-8 cycloalkyl, including But not limited to cyclopropyl, cyclopentyl or cyclohexyl
  • cycloalkyl such as C 3-8 cycloalkyl, including But not limited to cyclopropyl, cyclopentyl or cyclohexyl
  • aryl and heteroaryl.
  • alkyl, cycloalkyl, aryl and heteroaryl are each independently optionally selected from alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy) , Isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl
  • alkyl such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl
  • alkoxy such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy
  • Isopropoxy halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro,
  • R 1a and R 1b together with the atoms to which they are attached form a 5- to 8-membered heterocycloalkyl group.
  • the heterocycloalkyl group is optionally substituted by an alkyl group (such as a C 1-6 alkyl group, including but not limited to methyl, ethyl, etc.).
  • isopropyl isopropyl
  • alkoxy such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy
  • halogen such as fluorine, chlorine, bromine
  • deuterium amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), heterocycloalkane
  • R 1a and R 1b are not hydrogen at the same time.
  • R 1a and R 1b together with the atoms to which they are attached in -D form a 6 to 8-membered heterocycloalkyl group.
  • R 1a in -D in the antibody-drug conjugate is selected from methyl.
  • R 1a and R 1b in -D in the antibody-drug conjugate are each independently selected from methyl groups.
  • the -D in the antibody-drug conjugate is
  • k is selected from 1 to 10, and may be an integer or a decimal.
  • ADC antibody-drug conjugate
  • -D is selected from:
  • the linker is stable outside the cell so that the ADC remains intact when present in the extracellular environment, but is capable of lysis when internalized in cells such as cancer cells.
  • the ADC enters a cell expressing an antigen specific to the antibody portion of the ADC, the eribulin analog drug portion is cleaved from the antibody portion, and the lysis releases the unmodified form of the eribulin analog .
  • the cleavable moiety in the linker is a cleavable peptide moiety.
  • ADCs containing cleavable peptide moieties show lower aggregation levels, improved antibody to drug ratios, increased targeted killing of cancer cells, and reduced non- Off-target killing of cancer cells, and/or higher drug load (p).
  • the addition of a cleavable moiety increases cytotoxicity and/or potency relative to a non-cleavable linker.
  • the increased efficacy and/or cytotoxicity is increased efficacy and/or cytotoxicity in cancers that express moderate levels of antigens targeted by the antibody portion of the ADC (e.g., moderate FRA expression).
  • the cleavable peptide portion can be cleaved by an enzyme, and the linker is a linker that can be cleaved by the enzyme.
  • the enzyme is cathepsin, and the linker is a linker capable of cleavage by cathepsin.
  • linkers that can be cleaved by enzymes exhibit one or more of the aforementioned improved properties compared to other cleavage mechanisms.
  • the linker comprises an amino acid unit
  • the amino acid unit preferably comprises 2 to 7 selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, Peptide residues composed of amino acids of aspartic acid, more preferably valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine- Lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe) Or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).
  • the linker including the amino acid unit of the present disclosure is selected from:
  • the amino acid unit comprises valine-citrulline (Val-Cit).
  • ADCs containing Val-Cit show increased stability, reduced off-target cell killing, increased targeted cell killing, and lower ADCs containing other amino acid units or other cleavable moieties. Aggregation level, and/or higher drug load.
  • some embodiments provide a linker that includes a cleavable sulfonamide moiety that is capable of being cleaved under reducing conditions.
  • the linker comprises a cleavable disulfide moiety, and the linker is capable of being cleaved under reducing conditions.
  • the linker in the antibody conjugate of the present disclosure includes at least one spacer unit that connects the Eribulin derivative D to the cleavable moiety.
  • the linker comprises a spacer unit connected to D.
  • the spacer unit comprises p-aminobenzyloxycarbonyl (PAB),
  • the spacer unit comprises a p-aminobenzoyl group
  • the spacer unit comprises:
  • Z 1 to Z 5 are each independently selected from a carbon atom or a nitrogen atom;
  • R 14 is selected from an alkyl group, a cycloalkyl group, an aryl group and a heteroaryl group, the alkyl group, cycloalkyl group, aryl group and hetero
  • the aryl groups are each independently optionally selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl
  • R 11 and R 12 are each independently selected from hydrogen, deuterium, C 1-6 alkyl, C 3-6 cycloalkyl, preferably hydrogen; or, R 11 and R 12 are The connected carbon atoms together form a C3-6 cycloalkyl group;
  • X is selected from -O- or -NH-;
  • L is selected from an integer between 1-4;
  • Q is VE-
  • VE- provides a glycosidic bond that can be cleaved by glycosidase located in the cell
  • E is selected from -O-, -S- or -NR 13 -
  • R 13 is selected from hydrogen or methyl
  • V is selected from Wherein R 15 is selected from -COOH or CH 2 OH. In some embodiments, V is selected from -COOH.
  • the spacer unit comprises:
  • Z 1 , Z 3 , Z 4 , X, Q, R 11 , R 12 , and R 14 are as described above.
  • the spacer unit comprises a part selected from:
  • R a and R b are the same or different, and are each independently selected from hydrogen, deuterium atom, halogen or alkyl;
  • R 8 is selected from hydrogen, C 3-6 cycloalkylalkyl or C 3-6 cycloalkyl;
  • R 9 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen; or, R 8 and R 9 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl;
  • m1 and m2 are each independently selected from 0, 1, 2, or 3.
  • the spacer unit comprises a part selected from:
  • L-D in the antibody conjugate (ADC) of the present disclosure is a chemical moiety represented by the following formula:
  • Str is a stretching unit covalently connected to Ab
  • Pep is selected from amino acid units, disulfide moieties, sulfonamide moieties or the following non-peptide chemical moieties:
  • W is -NH-heterocycloalkyl- or heterocycloalkyl
  • Y is heteroaryl, aryl, -C(O)C 1-6 alkylene, C 2-6 alkenylene, C 1 -6 alkylene or -C 1-6 alkylene -NH-;
  • Each R 2 is independently selected from C 1-10 alkyl, C 2-10 alkenyl, C 1-6 alkylene -NH 2 , -(C 1-10 alkylene) NHC(NH)NH 2 or- (C 1-10 alkylene)NHC(O)NH 2 ;
  • R 3 and R 4 are each independently H, C 1-10 alkyl, C 2-10 alkenyl, arylalkyl, heteroarylalkyl, or R 3 and R 4 together can form a C 3-7 cycloalkane base;
  • R 5 and R 6 are each independently C 1-10 alkyl, C 2-10 alkenyl, arylalkyl, heteroarylalkyl, (C 1-10 alkylene) OCH 2 -, or R 5 and R 6 together can form a C 3-7 cycloalkyl ring.
  • Y in the antibody-drug conjugate (ADC) is selected from the following parts:
  • Str in the antibody-drug conjugate is selected from the chemical moiety represented by the following formula:
  • R 7 is selected from -W1-C(O)-, -C(O)-W1-C(O)-, -(CH 2 CH 2 O) p1 C(O)-, -(CH 2 CH 2 O ) p1 CH 2 C(O)-, -(CH 2 CH 2 O) p1 CH 2 CH 2 C(O)-, where W1 is selected from C 1-8 alkylene, C 1-8 alkylene-ring
  • the group and the linear heteroalkyl group are each independently optionally further selected from one or more of halogen, deuterium, hydroxy, cyano, amino, alkyl, haloalkyl, deuterated alkyl, alkoxy and cycloalkyl Substituent substituted;
  • L 1 is selected from -NR 10 (CH 2 CH 2 O) p1 CH 2 CH 2 C(O)-, -NR 10 (CH 2 CH 2 O) p1 CH 2 C(O)-, -S(CH 2 ) p1 C(O)-, -(CH 2 ) p1 C(O)- or a chemical bond, preferably a chemical bond; wherein p1 is an integer from 1 to 20, and R 10 is selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, and a deuterated alkane And hydroxyalkyl.
  • C 1-8 alkylene-cycloalkyl is selected from methylene-cyclohexyl Ethylene-cyclohexyl Methylene-cyclopentyl
  • the linker may comprise at least one polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • the PEG moiety may, for example, comprise -(PEG) p1 -, Where p1 is an integer from 1 to 20, for example (PEG) 2 ; (PEG) 4 ; (PEG) 5 .
  • the stretching unit in the linker comprises (PEG) 2 .
  • ADCs containing shorter stretch units e.g. (PEG) 2
  • exhibit lower relative to ADCs that include longer stretch units e.g. (PEG) 8 ).
  • Higher levels of aggregation and/or higher drug load e.g. (PEG) 8 .
  • the Str of the antibody-drug conjugate is Where R 7 is selected from C 1-6 alkylene C(O)-, -(CH 2 -CH 2 O) 2 C(O)-, -(CH 2 -CH 2 O) 2 CH 2 C(O) -, -(CH 2 -CH 2 O) 2 CH 2 CH 2 C(O)-, -(CH 2 -CH 2 O) 3 C(O)- and -(CH 2 -CH 2 O) 4 C( O)-.
  • the Str of the antibody-drug conjugate is where R 7 is selected from -C 1-8 alkylene-cycloalkyl-C(O)-, -(CH 2 -CH 2 O) 4 CH 2 C(O)- and -(CH 2 -CH 2 O ) 6 CH 2 C(O)-.
  • the linker L in the antibody-drug conjugate includes: maleimide-(PEG) 2 -Val-Cit, maleimide-(PEG) 6- Val-Cit, maleimide-(PEG) 8 -Val-Cit, maleimide-(PEG) 4 -CH 2 CH 2 C(O)-Val-lys, maleimide Diimide-(CH 2 ) 5 -Val-Cit, maleimide-(CH 2 ) 5 -Val-lys, maleimide-(CH 2 ) 5 -Gly-Gly -Phe-Gly, maleimide-(PEG) 2 -Ala-Ala-Asn, maleimide-(PEG) 6 -Ala-Ala-Asn, maleimide -(PEG) 8 -Ala-Ala-Asn, maleimide-(PEG) 4 -triazole-(PEG) 3 -sulfonamide, maleimide-(PEG) 2- CH 2
  • the linker L in the antibody-drug conjugate comprises: maleimide-(PEG) 4 -CH 2 C(O)-Gly-Gly-Phe-Gly, Diimide-(PEG) 2 -CH 2 CH 2 C(O)-Gly-Gly-Phe-Gly, maleimide-(PEG) 6 -CH 2 C(O)-Gly- Gly-Phe-Gly-, maleimide-(CH 2 ) 5 C(O)-Gly-Gly-Phe-Gly-, maleimide-C 1-8 alkylene- Cycloalkyl-C(O)-NH(CH 2 CH 2 O) 4 CH 2 C(O)-Gly-Gly-Phe-Gly-, maleimide-(PEG) 2 -CH 2 C (O)-Gly-Gly-Phe-Gly-, maleimide-(PEG) 2 -CH 2 CH 2 C(O)-Val-Cit-, maleimide-(PEG) 4 -
  • Str in the antibody-drug conjugate is selected from the chemical moiety represented by the following formula:
  • R 8 is selected from C 1-10 alkylene, C 2-10 alkenylene, (C 1-10 alkylene) O-, N(R d )-(C 2-6 alkylene)-N (R d ) and N(R d )-(C 2-6 alkylene); and each Rd is independently H or C 1 -C 6 alkyl.
  • L-D in the antibody-drug conjugate is represented by a formula selected from the following:
  • R 2 is C 1 - 6 alkylene, (C 1 - 6 alkyl) NHC (NH) NH 2 or (C 1 - 6 alkylene) NHC (O) NH 2;
  • R 2 is C 1-6 alkyl, (C 1-6 alkylene) NHC(NH)NH 2 or (C 1-6 alkylene) NHC(O)NH 2 ;
  • R 2 is C 1-6 alkyl, C 2-6 alkenylene, (C 1-6 alkylene) NHC(NH)NH 2 or (C 1-6 alkylene) NHC(O)NH 2 ;
  • R 2 is C 1-6 alkyl, C 2-6 alkenylene, (C 1-6 alkylene) NHC(NH)NH 2 or (C 1-6 alkylene) NHC(O)NH 2 ;
  • R 2 is C 1-6 alkyl, (C 1-6 alkylene) NHC(NH)NH 2 or (C 1-6 alkylene) NHC(O)NH 2 , and R 5 and R 6 are together Form a C 3-7 cycloalkyl ring;
  • R 2 is C 1-6 alkyl, (C 1-6 alkylene) NHC(NH)NH 2 or (C 1-6 alkylene) NHC(O)NH 2 , and R 5 and R 6 are together Form a C 3-7 cycloalkyl ring;
  • the antibody-drug conjugate (ADC) of the present disclosure is represented by the following formula:
  • R 2 is selected from C 1-6 alkylene -NH 2, (C 1 - 6 alkylene) NHC (NH) NH 2 or (C 1 - 6 alkylene) NHC (O) NH 2, k is selected from From 1 to 10, it can be an integer or a decimal, and p2 is selected from an integer between 2-6;
  • R 2 is selected from C 1-6 alkylene-NH 2 , (C 1-6 alkylene) NHC(NH)NH 2 or (C 1-6 alkylene) NHC(O)NH 2 , k is selected From 1 to 10, it can be an integer or a decimal, and p2 is selected from an integer between 2-6;
  • R 2 is C 1-6 alkylene-NH 2 , (C 1-6 alkylene) NHC(NH)NH 2 or (C 1-6 alkylene) NHC(O)NH 2 , and R 5 And R 6 form a C 3-7 cycloalkyl ring, k is selected from 1 to 10, which can be an integer or a decimal, and p2 is selected from an integer between 2-6;
  • R 2 is C 1-6 alkylene-NH 2 , (C 1-6 alkylene) NHC(NH)NH 2 or (C 1-6 alkylene) NHC(O)NH 2 , and R 5 And R 6 form a C 3-7 cycloalkyl ring, k is selected from 1 to 10, which can be an integer or a decimal, and p2 is selected from an integer between 2-6;
  • ADC antibody-drug conjugate
  • R 8 is selected from hydrogen, C 3-6 cycloalkylalkyl or C 3-6 cycloalkyl, preferably hydrogen;
  • R 9 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen, or R 8 and R 9 together with the carbon atom to which they are connected form a C 3-6 cycloalkyl group, k is selected from 1 to 10, which may be an integer or a decimal, and p2 is selected from an integer between 2-6;
  • R 8 is selected from hydrogen, C 3-6 cycloalkylalkyl or C 3-6 cycloalkyl, preferably hydrogen;
  • R 9 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen; or R 8 and R 9 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group;
  • k is selected from 1 to 10, which can be an integer or a decimal, p1 is selected from 2, 4, 6 or 8, and p3 is selected from 0, 1 or 2;
  • k is selected from 1 to 10, can be an integer or a decimal, p2 is selected from an integer between 2-6;
  • k is selected from 1 to 10, can be an integer or a decimal, p2 is selected from an integer between 2-6;
  • k is selected from 1 to 10, can be an integer or a decimal, p1 is selected from 2, 4, 6 or 8; p3 is selected from 0, 1 or 2;
  • k is selected from 1 to 10, can be an integer or a decimal, p1 is selected from 2, 4, 6 or 8, and p3 is selected from 0, 1 or 2; k is selected from 1 to 10, can be an integer or a decimal, p1 is selected from 2, 4, 6 or 8;
  • k is selected from 1 to 10, can be an integer or a decimal, p1 is selected from 2, 4, 6 or 8; p3 is selected from 0, 1 or 2;
  • k is selected from 1 to 10, can be an integer or a decimal, p1 is selected from 2, 4, 6 or 8;
  • k is selected from 1 to 10, can be an integer or a decimal, p1 is selected from 2, 4, 6 or 8, and p3 is selected from 0, 1 or 2;
  • k is selected from 1 to 10, can be an integer or a decimal, p2 is selected from 2, 4, 6 or 8;
  • k is selected from 1 to 10, can be an integer or a decimal, p2 is selected from 2, 4, 6 or 8;
  • k is selected from 1 to 10, can be an integer or a decimal, p2 is selected from 2, 4, 6 or 8;
  • k is selected from 1 to 10, can be an integer or a decimal, p2 is selected from 2, 4, 6 or 8;
  • k is selected from 1 to 10, can be an integer or a decimal, p2 is selected from 2, 4, 6 or 8;
  • k is selected from 1 to 10, can be an integer or a decimal, p1 is selected from 2, 4, 6 or 8, and p3 is selected from 0, 1 or 2;
  • k is selected from 1 to 10, can be an integer or a decimal, p1 is selected from 2, 4, 6 or 8, and p3 is selected from 0, 1 or 2;
  • R 8 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen
  • R 9 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen, or R 8 and R 9 are connected to it
  • k is selected from 1 to 10, which may be an integer or a decimal
  • p2 is selected from an integer between 2-6;
  • R 8 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen
  • R 9 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen, or R 8 and R 9 are connected to it
  • k is selected from 1 to 10, which can be an integer or a decimal
  • p2 is selected from an integer between 2-6;
  • R 8 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen
  • R 9 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen, or R 8 and R 9 are connected to it
  • k is selected from 1 to 10, which may be an integer or a decimal
  • p1 is selected from 2, 4, 6 or 8
  • p3 is selected from 0, 1 or 2;
  • R 8 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen
  • R 9 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen, or R 8 and R 9 are connected to it
  • k is selected from 1 to 10, which can be an integer or a decimal
  • p1 is selected from 2, 4, 6 or 8
  • p3 is selected from 0, 1, or 2.
  • the antibody-drug conjugate (ADC) is represented by the following formula:
  • k is selected from 1 to 10, which can be an integer or a decimal; further, R 1a in D is preferably a methyl group, and R 1b is preferably a hydrogen.
  • the antibody in the antibody-drug conjugate (ADC) of the present disclosure is selected from a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
  • the antibody or antigen-binding fragment thereof in the antibody-drug conjugate is selected from the group consisting of anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, and anti-c-Met antibody , Anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody , Anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C Antibodies, anti-SLC44A4 antibodies, anti-CD79 antibodies, anti
  • the antibody in the antibody-drug conjugate is a known antibody, selected from but not limited to Trastuzumab, Pertuzumab, Pertuzumab Touzumab (Nimotuzumab), Enoblituzumab (Enoblituzumab), Imatuzumab (Emibetuzumab), Ointuzumab (Inotuzumab), Vitin-Pinatuzumab (Pinatuzumab) , Brentuximab (Brentuximab), Gemtuzumab (Gemtuzumab), Bivatuzumab (Bivatuzumab), Molovastuzumab (Lorvotuzumab), cBR96 and Glematumamab or their antigen binding fragments.
  • Trastuzumab Pertuzumab, Pertuzumab Touzumab (Nimotuzumab), Enoblituzuma
  • the antibody in the antibody-drug conjugate is selected from an anti-CD79B antibody or an antigen-binding fragment thereof, which comprises an antibody heavy chain variable region and/or an antibody light chain variable region, wherein :
  • variable region of the antibody heavy chain including:
  • HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 respectively;
  • variable region of the antibody light chain comprising:
  • LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 respectively;
  • the anti-CD79B antibody in the antibody-drug conjugate (ADC) comprises a heavy chain variable region and a light chain variable region, including any one selected from the following (I) to (II) :
  • the heavy chain variable region which comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 respectively;
  • the light chain variable region which includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively;
  • the heavy chain variable region which comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively;
  • the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively.
  • Heavy chain CDR1 GSSFTSY (SEQ ID NO: 7) GYTFTTY(SEQ ID NO:13) Heavy chain CDR2 FPRSGN(SEQ ID NO: 8) YPRSGN(SEQ ID NO:14) Heavy chain CDR3 GDLGDFDY(SEQ ID NO: 9) GSDYDGDFAY (SEQ ID NO: 15) Light chain CDR1 RSSQSIVHSDGNTYFE(SEQ RSSQSIVHHDGNTYLE(SEQ ID NO: 7)
  • ID NO: 16 Light chain CDR2 KVSNRFS(SEQ ID NO:11) KVSNRFS(SEQ ID NO:17) Light chain CDR3 FQGSHVPWT(SEQ ID NO:12) FQGSHVPWT(SEQ ID NO:18)
  • the anti-CD79B antibody in the antibody-drug conjugate (ADC) comprises a heavy chain variable region and a light chain variable region, wherein:
  • variable region of the heavy chain including:
  • And/or light chain variable region including:
  • the heavy chain variable region of the anti-CD79B antibody or antigen-binding fragment is shown in the sequence SEQ ID NO: 3, and the light chain variable region is shown in the sequence SEQ ID NO: 4; or the heavy chain variable region is shown in The sequence is shown in SEQ ID NO: 5, and the light chain variable region is shown in the sequence of SEQ ID NO: 6.
  • the anti-CD79B antibody in the antibody-drug conjugate (ADC) comprises a heavy chain variable region and a light chain variable region, wherein:
  • variable region of the heavy chain including:
  • And/or light chain variable region including:
  • SEQ ID NO: 20 A sequence shown in SEQ ID NO: 20 or at least 90%, 95%, 98% or 99% identical to SEQ ID NO: 20; or
  • the heavy chain variable region of the anti-CD79B antibody or antigen-binding fragment is shown in the sequence SEQ ID NO: 19, and the light chain variable region is shown in the sequence SEQ ID NO: 20; or the heavy chain variable region is shown in The sequence is shown in SEQ ID NO: 21, and the light chain variable region is shown in the sequence of SEQ ID NO: 22.
  • the antibody in the antibody-drug conjugate (ADC) is selected from anti-TROP-2 antibodies.
  • the antibody in the antibody-drug conjugate (ADC) comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence as shown in SEQ ID NO: 23, The HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 24 and SEQ ID NO: 25, and the light chain variable region includes the sequences shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively LCDR1, LCDR2 and LCDR3.
  • the anti-TROP-2 antibody in the antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the same as SEQ ID NO
  • the heavy chain variable region shown in the sequence 29 has the same sequence of HCDR1, HCDR2 and HCDR3, and the light chain variable region includes LCDR1 with the same sequence as the light chain variable region shown in the sequence of SEQ ID NO: 30 , LCDR2 and LCDR3.
  • the anti-TROP-2 antibody in the antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as SEQ ID It is shown in NO: 29 or has at least 90% identity with the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 30 or has at least 90% identity with it.
  • the anti-TROP-2 antibody in the antibody-drug conjugate comprises a heavy chain variable region with a sequence as shown in SEQ ID NO: 29 and a sequence as shown in SEQ ID NO: 30 The variable region of the light chain.
  • the anti-TROP-2 antibody in the antibody-drug conjugate comprises an antibody heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is selected from human IgG1, IgG2 , IgG3 and IgG4 constant regions and conventional variants thereof, the light chain constant region is selected from human antibody ⁇ and ⁇ chain constant regions and conventional variants thereof; more preferably, the antibody comprises a sequence as shown in SEQ ID NO: 31 The heavy chain constant region and sequence shown are the light chain constant region shown in SEQ ID NO:32.
  • the anti-TROP-2 antibody in the antibody-drug conjugate comprises a heavy chain with a sequence as shown in SEQ ID NO: 33 and a light chain with the sequence as shown in SEQ ID NO: 34.
  • TROP-2 (Genbank: NP_002344.2) is as follows:
  • Antibody Anti-TROP-2 antibody PD3 Heavy chain CDR1 NYGMN (SEQ ID NO: 23) Heavy chain CDR2 WINTYTGEPTYTQDFKG (SEQ ID NO: 24) Heavy chain CDR3 GGFGSSYWYFDV(SEQ ID NO:25) Light chain CDR1 KASQDVSIAVA(SEQ ID NO:26) Light chain CDR2 SASYRYT(SEQ ID NO:27) Light chain CDR3 QQHYITPLT(SEQ ID NO:28)
  • the heavy chain constant region of the antibody can be selected from the constant regions of human IgG1, IgG2, IgG4 and variants thereof, and the light chain constant region can be selected from the light chain of human ⁇ , ⁇ chains or their variants Constant region.
  • the antibody heavy chain constant region is selected from the human IgG1 sequence shown in SEQ ID NO: 31, and the light chain constant region is selected from the human kappa chain constant region shown in SEQ ID NO: 32.
  • the heavy chain constant region of human IgG1 is the heavy chain constant region of human IgG1
  • the above-mentioned light chain/heavy chain constant region is combined with the variable region of the above-mentioned PD3 antibody to form a complete antibody, and the light chain/heavy chain sequence is as follows:
  • Anti-TROP-2 antibody (PD3) heavy chain
  • antibody-drug conjugate (ADC) of the present disclosure is selected from:
  • k is selected from 1 to 10, can be an integer or a decimal; further, R 1a in D is selected from methyl, and R 1b is selected from hydrogen.
  • the present disclosure also provides compounds represented by formula D, or tautomers, mesoisomers, racemates, enantiomers, diastereomers, or mixtures thereof, or Medicinal salt,
  • R 1a is selected from hydrogen, alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to ring Propyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, the alkyl, cycloalkyl, aryl and heteroaryl are each independently optionally selected from alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy) Group), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl one or more Substit
  • R 1a and R 1b in the compound represented by formula D are each independently selected from C 1-6 alkyl groups, including but not limited to methyl, ethyl, and isopropyl.
  • R 1a in the compound represented by formula D is selected from C 1-6 alkyl groups, including but not limited to methyl, ethyl, and isopropyl;
  • R 1b is selected from hydrogen.
  • R 1a and R 1b in the compound represented by formula D together with the atoms to which they are attached form a 5- to 8-membered heterocycloalkyl group.
  • the compound represented by formula D is:
  • the compound represented by formula D is:
  • the compound represented by formula D is:
  • the present disclosure also provides a compound represented by formula DZ, or a tautomer, meso, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable compound thereof Used salt,
  • R 1a is selected from hydrogen, alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to) Limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, the alkyl, cycloalkyl, aryl and heteroaryl are each independently optionally selected from alkyl (such as C 1 -6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, iso Propoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl or
  • R 1b is selected from hydrogen, alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy, cycloalkyl (such as C 3-8 cycloalkyl, including But not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl.
  • alkyl such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl
  • alkoxy such as C 3-8 cycloalkyl, including But not limited to cyclopropyl, cyclopentyl or cyclohexyl
  • cycloalkyl such as C 3-8 cycloalkyl, including But not limited to cyclopropyl, cyclopentyl or cyclohexyl
  • aryl and heteroaryl.
  • alkyl, cycloalkyl, aryl and heteroaryl are each independently optionally selected from alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy) , Isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl
  • alkyl such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl
  • alkoxy such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy
  • Isopropoxy halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro,
  • R 1a and R 1b together with the atoms to which they are attached form a 5-8 membered heterocycloalkyl group, which is optionally substituted by an alkyl group (such as a C 1-6 alkyl group, including but not limited to methyl, ethyl Group, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine ), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), heterocycloalkane Substituted by one or more substituents in the group, aryl group and heteroaryl group; and R 1a and R 1b are not hydrogen at the same time;
  • an alkyl group
  • Y is selected from -O(CR a R b ) m2 -CR 8 R 9 -C(O)-, -NH-(CR a R b ) m2 -CR 8 R 9 -C(O)-, -O-CR 8 R 9 (CR a R b ) m2 -, -OCR 8 R 9 -C(O)-, -O(CR a R b ) m2 C(O)- or -S-(CR a R b ) m2- CR 8 R 9 -C (O) -, wherein R a and R b are identical or different and are each independently selected from hydrogen, a deuterium atom, halogen or alkyl;
  • R 8 is selected from hydrogen, C 3-6 cycloalkylalkyl or C 3-6 cycloalkyl;
  • R 9 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen;
  • R 8 and R 9 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group
  • n2 is selected from 0, 1, 2 or 3.
  • R 1a and R 1b in the compound represented by formula DZ are each independently selected from C 1-6 alkyl groups, including but not limited to methyl, ethyl, and isopropyl.
  • R 1a in the compound represented by formula DZ is selected from C 1-6 alkyl groups, including but not limited to methyl, ethyl, and isopropyl; R 1b is selected from hydrogen.
  • R 1a and R 1b in the compound represented by formula DZ together with the carbon atom to which they are attached form a 5- to 8-membered heterocycloalkyl group.
  • the compound represented by formula DZ, or its tautomer, meso, racemate, enantiomer, diastereomer, or mixture thereof, or Its pharmaceutically acceptable salt is a compound represented by formula DZ-1, or its tautomer, meso, racemate, enantiomer, diastereomer, or Its mixture form, or its pharmaceutically acceptable salt:
  • R 8 is selected from hydrogen, C 3-6 cycloalkylalkyl or C 3-6 cycloalkyl
  • R 9 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen; or, R 8 Together with R 9 and the carbon atom to which it is attached, form a C 3-6 cycloalkyl group
  • m2 is selected from 0, 1, 2 or 3.
  • the compound represented by DZ is selected from:
  • the compound represented by DZ provided by some embodiments may contain one or more asymmetric centers, such as Can be
  • the present disclosure also provides a pharmaceutical composition, which contains a therapeutically effective amount of the aforementioned antibody-drug conjugate, and a pharmaceutically acceptable pharmaceutical carrier, diluent or excipient.
  • the present disclosure also provides a use of the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition in the preparation of a medicine for treating or preventing tumors.
  • the tumor is a cancer associated with the expression of HER2, HER3, B7H3 or EGFR.
  • the present disclosure also provides a use of the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition in the preparation of a medicine for treating and/or preventing cancer.
  • 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, melanin Tumor, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.
  • the present disclosure also provides a method for treating or preventing tumors, which comprises administering a therapeutically effective amount of the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition to a patient in need; wherein the tumor is preferably associated with HER2, HER3, B7H3 Or cancers related to EGFR expression.
  • the present disclosure also provides a method for treating and/or preventing cancer, which comprises administering a therapeutically effective amount of the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition to a patient in need;
  • the cancer is preferably breast cancer or ovarian cancer , Cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer , Colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.
  • the present disclosure additionally provides the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition for the treatment or prevention of tumors; wherein the tumors are preferably cancers related to the expression of HER2, HER3, B7H3 or EGFR.
  • the present disclosure additionally provides the aforementioned antibody-drug conjugate or the aforementioned pharmaceutical composition, which is used for the treatment and/or prevention of cancer;
  • the cancer is preferably breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, and kidney cancer.
  • the active compound can be formulated into a form suitable for administration by any appropriate route, and the active compound is preferably in a unit dose form, or in a form in which the patient can self-administer in a single dose.
  • the unit dosage form of the compound or composition of the present disclosure can be tablet, capsule, cachet, bottled syrup, powder, granule, lozenge, suppository, rejuvenated powder or liquid preparation.
  • the dosage of the compound or composition used in the treatment methods of the present disclosure will generally vary with the severity of the disease, the weight of the patient, and the relative efficacy of the compound.
  • a suitable unit dose may be 0.1-1000 mg.
  • the pharmaceutical composition of the present disclosure may contain one or more excipients selected from the following ingredients: fillers (diluents), binders, wetting agents, disintegrants or excipients Wait.
  • the composition may contain 0.1 to 99% by weight of the active compound.
  • the applicant intends to include the formulation of the trade name product, the generic drug and the active drug portion of the trade name product.
  • drug refers to cytotoxic drugs or immunomodulators. Cytotoxic drugs can have strong chemical molecules in tumor cells that can disrupt their normal growth. In principle, cytotoxic drugs can kill tumor cells at a high enough concentration, but due to lack of specificity, while killing tumor cells, it will also cause normal cell apoptosis, leading to serious side effects.
  • toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (for example, At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and Lu radioisotopes), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes.
  • Immunomodulators are inhibitors of immune checkpoint molecules.
  • the drug is denoted as D, which is an immunomodulator, especially a TLR8 agonist.
  • linker refers to a chemical structure fragment or bond that is connected to a ligand at one end and a drug at the other end. It can also be connected to other linkers. Connect with the drug again.
  • the joint may include one or more joint members.
  • exemplary linker building blocks include 6-maleimidohexanoyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-benzene Alanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate ( SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC, also referred to herein as MCC) and N-succinimidyl (4 -Iodo-acetyl)aminobenzoate (SIAB).
  • MC 6-maleimidohexanoyl
  • MP maleimidopropionyl
  • Vcitrulline Val-Cit or
  • the linker may include a stretching unit, a spacer unit, an amino acid unit, and an extension unit. It can be synthesized by methods known in the art, such as those described in US2005-0238649A1.
  • the linker may be a "cleavable linker" that facilitates the release of the drug in the cell.
  • acid-labile linkers such as hydrazone
  • protease-sensitive such as peptidase-sensitive
  • light-labile linkers dimethyl linkers
  • disulfide-containing linkers Chargei et al., Cancer Research 52:127-131
  • splitting unit refers to a chemical structure fragment with one end covalently connected to the antibody through a carbon atom and the other end to an amino acid unit, a disulfide moiety, a sulfonamide moiety or a non-peptide chemical moiety.
  • spacer unit is a bifunctional compound structural fragment that can be used to couple amino acid units and cytotoxic drugs to form antibody-drug conjugates. This coupling method can selectively connect cytotoxic drugs to amino acid units. superior.
  • amino acid refers to an organic compound that contains amino and carboxyl groups in the molecular structure, and both the amino and carboxyl groups are directly connected to the -CH- structure.
  • the general formula is H 2 NCHRCOOH, R is H, substituted or unsubstituted alkyl, and the like. According to the position of the amino group attached to the carbon atom in the carboxylic acid, it can be divided into ⁇ , ⁇ , ⁇ , ⁇ ...-amino acids.
  • amino acids that constitute natural proteins have their specific structural characteristics, that is, their amino groups are directly connected to ⁇ -carbon atoms, that is, ⁇ -amino acids, including glycine (Glycine), alanine (Alanine), and valine.
  • ⁇ -amino acids including glycine (Glycine), alanine (Alanine), and valine.
  • Valine Leucine, Isoleucine, Phenylalanine, Tryptophan, Tyrosine, Aspartic acid, Histidine, Asparagine, Glutamic acid, Lysine, Glutamine, Methionine, Arginine , Serine, Threonine, Cysteine, Proline, etc.
  • Unnatural amino acids such as citrulline.
  • the spacer unit is PAB
  • the structure is like a p-aminobenzyloxycarbonyl fragment, and its structure is shown in formula (VI), which is connected to D,
  • Joint components include but are not limited to:
  • MC 6-maleimidohexanoyl
  • Val-Cit or "vc” valine-citrulline (an exemplary dipeptide in a protease cleavable linker);
  • Citrulline 2-amino-5-ureidovaleric acid
  • Me-Val-Cit N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to prevent it from being cleaved by cathepsin B);
  • MC(PEG) 6 -OH maleimidohexanoyl-polyethylene glycol (can be attached to antibody cysteine);
  • SPP N-succinimidyl 4-(2-pyridylthio)pentanoate
  • SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
  • SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • PBS phosphate buffered saline solution
  • antibody-drug conjugate refers to that the ligand is connected to a biologically active drug through a stable linking unit.
  • ADC antibody-drug conjugate
  • drug loading can be expressed as the ratio of the amount of drug to the amount of antibody.
  • the drug loading can range from 1-20 per antibody (Ab), preferably 1-10 cytotoxic drugs (D).
  • the drug loading is expressed as k, and exemplary can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the mean value between any two values.
  • the monoclonal antibody molecular size variant determination method (CE-SDS) of the present disclosure can adopt the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method. Under reducing and non-reducing conditions, according to the molecular weight, the electrophoresis method is based on the hair (2015 edition of "Chinese Pharmacopoeia” 0542), quantitatively determine the purity of recombinant monoclonal antibody products.
  • the cytotoxic drug is coupled to the N-terminal amino group of the ligand and/or the ⁇ -amino group of the lysine residue through the linking unit.
  • the cytotoxic drug can be coupled to the antibody in the coupling reaction.
  • the number of drug molecules will be less than the theoretical maximum.
  • the following non-limiting methods can be used to control the loading of antibody-drug conjugates, including:
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain, and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the antibodies described in the present disclosure are preferably specific antibodies against cell surface antigens on target cells.
  • Non-limiting examples are the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met Antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 Antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin- C antibody, anti-SLC44A4 antibody, anti-CD79 antibody, anti-TROP-2 antibody, anti-CD79B antibody, anti-Mesothelin antibody or antigen-bind
  • the antibody is selected from Trastuzumab (Trastuzumab, trade name Herceptin), Pertuzumab (Pertuzumab, also known as 2C4, trade name Perjeta), Nimotuzumab (Nimotuzumab, trade name) Name Tai Xinsheng), Enbotuzumab (Enoblituzumab), Imatuzumab (Emibetuzumab), Ointuzumab (Inotuzumab), Vitin-Pinatuzumab (Pinatuzumab), Vitamin Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96 and Glematumamab.
  • Trastuzumab Trastuzumab, trade name Herceptin
  • Pertuzumab Pertuzumab, also known as 2C4, trade name Perjeta
  • Nimotuzumab Nimotuzumab (N
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.
  • murine-derived antibody in the present disclosure refers to the preparation of antibodies with mice based on knowledge and skills in the art. During preparation, a test subject is injected with a specific antigen, and then hybridomas expressing antibodies with desired sequences or functional properties are isolated.
  • chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma secreting murine-derived specific monoclonal antibodies, then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed, and replace the murine variable region genes. It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the framework of human antibody variable regions, that is, different types of human germline antibodies Antibodies produced in the framework sequence. It can overcome the heterogeneous reaction induced by the chimeric antibody because it carries a large amount of murine protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of the human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase ), as well as in Kabat, EA, etc.
  • human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that have been further subjected to affinity maturation for CDR by phage display. Documents that further describe methods that can be used for mouse antibodies involved in humanization include, for example, Queen et al., Proc., Natl. Acad. Sci.
  • the development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the present disclosure is a fully human monoclonal antibody.
  • the related technologies of fully human antibody preparation mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of antibodies.
  • binding fragments contained in "antigen-binding fragments" include (i) Fab fragments, which are monovalent fragments composed of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, which include A bivalent fragment of two Fab fragments connected by a disulfide bridge; (iii) Fd fragment composed of VH and CH1 domains; (iv) Fv fragment composed of VH and VL domains of one arm of an antibody; (v ) A single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) can optionally be passed A combination of two
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, recombination methods can be used to connect them through a synthetic linker so that it can be produced as a single protein in which the VL and VH regions are paired to form a monovalent molecule.
  • Chain referred to as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • single chain antibodies are also intended to be included in the term "antigen-binding fragments" of antibodies.
  • the antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of the intact immunoglobulin.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibodies.
  • Fab is an antibody fragment that has a molecular weight of about 50,000 and has antigen-binding activity among the fragments obtained by treating IgG antibody molecules with the protease papain (cleaving the amino acid residue at position 224 of the H chain), wherein the N-terminal side of the H chain About half and the entire L chain are joined together by disulfide bonds.
  • F(ab')2 is an antibody with a molecular weight of about 100,000 obtained by digesting the lower part of the two disulfide bonds in the hinge region of IgG with the enzyme pepsin and has antigen-binding activity and contains two Fab regions connected at the hinge position. Fragment.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of F(ab')2 described above.
  • the Fab' can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic organism or eukaryotic organism to express Fab'.
  • single-chain antibody means to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker Of molecules.
  • Such scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of a repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448) .
  • linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
  • CDR refers to one of the six hypervariable regions in the variable domain of an antibody that mainly contribute to antigen binding.
  • One of the most commonly used definitions of the 6 CDRs is provided by Kabat E.A. et al. (1991) Sequences of Proteins of Immunological Interest. NIH Publication 91-3242).
  • the Kabat definition of CDR only applies to the CDR1, CDR2, and CDR3 of the light chain variable domain (CDR L1, CDR L2, CDR L3 or L1, L2, L3), and the heavy chain variable domain.
  • CDR2 and CDR3 CDR H2, CDR H3 or H2, H3.
  • CDR1, HCDR2, HCDR3 there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • Any one of various well-known schemes can be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat” numbering rule (see Kabat et al.
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3);
  • the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).
  • the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residue numbers in VL are 26-32 (LCDR1), 50- 52 (LCDR2) and 91-96 (LCDR3).
  • CDR is defined by amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24-35 in human VL.
  • 34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) constitute.
  • the CDR amino acid residue numbers in VH are roughly 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3)
  • the CDR amino acid residue numbers in VL are roughly 27-32 (CDR1) ), 50-52 (CDR2) and 89-97 (CDR3).
  • the CDR region of an antibody can be determined using the program IMGT/DomainGap Align.
  • antibody framework refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
  • epitopes or "antigenic determinant” refers to the site on an antigen where an immunoglobulin or antibody specifically binds. Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation (see, for example, Epitope Mapping Protocols in Methods in Molecular B iology, Volume 66, GEMorris, Ed. (1996)).
  • antibodies bind with an affinity (KD) of approximately less than 10 -7 M, for example, approximately less than 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • the nucleic acid is "operably linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein can replicate autonomously in the host cell into which they have been introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of the host cell after being introduced into the host cell, so as to follow The host genome replicates together (e.g., a non-episomal mammalian vector).
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions.
  • the human FR germline sequence can be obtained from the ImmunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351 get.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacteria, microorganisms, plant or animal cells.
  • Bacteria that are easily transformed include members of the enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NS0 cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region.
  • Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by conventional techniques. For example, use A or G Sepharose FF column with adjusted buffer for purification.
  • the bound antibody was eluted by the PH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • amino acid sequence identity refers to aligning amino acid sequences and introducing gaps when necessary to achieve the maximum sequence identity percentage, and does not consider any conservative substitutions as part of sequence identity, in the first sequence and in the second sequence
  • the percentage of amino acid residues that are identical can be achieved in a variety of ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • BLAST BLAST-2
  • ALIGN ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN
  • peptide refers to a fragment of a compound between amino acids and proteins. It is composed of two or more amino acid molecules connected to each other by peptide bonds. They are structural and functional fragments of proteins, such as hormones, enzymes, etc. in essence. All are peptides.
  • sucrose refers to a biological macromolecule composed of three elements of C, H, and O, which can be divided into monosaccharides, disaccharides and polysaccharides.
  • fluorescent probe refers to the characteristic fluorescence in the ultraviolet-visible-near-infrared region, and its fluorescent properties (excitation and emission wavelength, intensity, lifetime, polarization, etc.) can vary with the properties of the environment, such as polarity, refractive index A type of fluorescent molecules that change sensitively due to changes in, viscosity, etc., which non-covalently interact with nucleic acids (DNA or RNA), proteins or other macromolecular structures to change one or several fluorescent properties, which can be used for research The nature and behavior of macromolecular substances.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably containing 1 to 10 carbons The most preferred is an alkyl group containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-Dimethylpropyl, 2,2-Dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -Methylhexyl, 3-methylhexyl, 4-methylhe
  • lower alkyl groups containing 1 to 6 carbon atoms More preferred are lower alkyl groups containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and sec-butyl.
  • Alkyl groups may be substituted or unsubstituted.
  • substituents When substituted, substituents may be substituted at any available attachment point.
  • the substituents are preferably one or more of the following groups, which are independently selected from alkanes Group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkane Oxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein the alkyl group is as defined above.
  • a “monovalent group” refers to a compound that "formally” eliminates a monovalent atom or group.
  • “Subunit” refers to a compound “formally” eliminates two monovalent or one divalent atoms or groups of atoms.
  • Exemplary “alkyl” refers to the remaining part after removing 1 hydrogen atom from an alkane molecule, and includes linear and branched monovalent groups of 1 to 20 carbon atoms.
  • An alkyl group containing 1 to 6 carbon atoms non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl , 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl and various branched isomers.
  • alkylene refers to a saturated linear or branched aliphatic hydrocarbon group, which has two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, which is A straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, more preferably an alkylene group containing 1 to 6 carbon atoms.
  • Non-limiting examples of alkylene include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 -) CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -) and 1,5-butylene (-CH 2 CH 2 CH 2 CH 2 CH 2 -) Wait.
  • the alkylene group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituent is preferably independently optionally selected from alkyl, alkenyl, alkynyl , Alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy One or more of group, cycloalkylthio group, heterocycloalkylthio group and oxo group.
  • alkenylene is as defined above.
  • alkoxy refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where the definition of alkyl or cycloalkyl is as described above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • the alkoxy group may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent.
  • the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 Carbon atoms, most preferably 3 to 8 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Groups, cyclooctyl, etc.; polycyclic cycloalkyls include spiro, fused, and bridged cycloalkyls.
  • heterocycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O ) m (where m is an integer of 0 to 2) heteroatoms, but does not include the ring part of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms.
  • Non-limiting examples of monocyclic heterocycloalkyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.
  • Polycyclic heterocycloalkyl groups include spirocyclic, fused ring, and bridged heterocycloalkyl groups.
  • spiroheterocycloalkyl refers to a polycyclic heterocycloalkyl group sharing one atom (called a spiro atom) between 5- to 20-membered monocyclic rings, in which one or more ring atoms are selected from nitrogen, oxygen or S (O) Heteroatoms of m (where m is an integer of 0 to 2), and the remaining ring atoms are carbon. It can contain one or more double bonds, but none of the rings have a fully conjugated ⁇ -electron system. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • the spiroheterocycloalkyl group is divided into a single spiroheterocycloalkyl group, a dispiroheterocycloalkyl group or a polyspiroheterocycloalkyl group, preferably a single spiroheterocycloalkyl group and a double Spiroheterocycloalkyl. More preferably, it is 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered monospiroheterocycloalkyl.
  • spiroheterocycloalkyl groups include:
  • fused heterocycloalkyl refers to a 5- to 20-membered polycyclic heterocycloalkyl group in which each ring in the system shares an adjacent pair of atoms with other rings in the system.
  • One or more rings may contain one or Multiple double bonds, but none of the rings have a fully conjugated ⁇ -electron system, where one or more of the ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (where m is an integer of 0 to 2), The remaining ring atoms are carbon. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • fused heterocycloalkyl groups include:
  • bridge heterocycloalkyl refers to a 5- to 14-membered polycyclic heterocycloalkyl group with any two rings sharing two atoms that are not directly connected. It may contain one or more double bonds, but none of the rings have A fully conjugated ⁇ -electron system in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (where m is an integer of 0 to 2), and the remaining ring atoms are carbon. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • bridged heterocycloalkyl groups preferably bicyclic, tricyclic or tetracyclic, and more preferably bicyclic or tricyclic.
  • bridged heterocycloalkyl groups include:
  • heterocycloalkyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocycloalkyl, non-limiting examples thereof include:
  • the heterocycloalkyl group may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, Alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio Group, heterocycloalkylthio group, oxo group.
  • aryl refers to a 6 to 14-membered all-carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) with a conjugated ⁇ -electron system, preferably 6 to 10 members, such as benzene And naphthyl, preferably phenyl.
  • the aryl ring may be fused to a heteroaryl, heterocycloalkyl or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, non-limiting examples of which include:
  • the aryl group may be substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycle Alkylthio.
  • heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen.
  • Heteroaryl groups are preferably 5 to 10 members, more preferably 5 or 6 members, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrakis Azole and so on.
  • the heteroaryl ring may be fused to an aryl, heterocycloalkyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:
  • Heteroaryl groups may be optionally substituted or unsubstituted.
  • the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio.
  • amino protecting group is to keep the amino group unchanged when other parts of the molecule react, and to protect the amino group with a group that is easy to remove.
  • Non-limiting examples include 9-fluorenylmethyloxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with 1-3 substituents selected from halogen, alkoxy or nitro.
  • the amino protecting group is preferably 9-fluorenylmethyloxycarbonyl.
  • heterocyclic amino group refers to a heterocycloalkyl group substituted with one or more amino groups, preferably a substituted amino group, wherein the heterocyclic group is as defined above, wherein “amino” means -NH 2.
  • amino means -NH 2.
  • heterocycloalkylamino refers to an amino group substituted with one or more heterocycloalkyl groups, preferably with one heterocycloalkyl group, where amino is as defined above and where heterocycloalkyl is as defined above.
  • Representative embodiments of the present disclosure are as follows:
  • cycloalkylamino refers to an amino group substituted with one or more cycloalkyl groups, preferably with a cycloalkyl group, where amino is as defined above and where cycloalkyl is as defined above.
  • Representative embodiments of the present disclosure are as follows:
  • cycloalkylalkyl refers to an alkyl group substituted with one or more cycloalkyl groups, preferably with a cycloalkyl group, where alkyl is as defined above and where cycloalkyl is as defined above.
  • haloalkyl refers to an alkyl group substituted with one or more halogens, where the alkyl group is as defined above.
  • deuterated alkyl refers to an alkyl group substituted with one or more deuterium atoms, where the alkyl group is as defined above.
  • hydroxy refers to the -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH 2 .
  • nitro refers to -NO 2 .
  • the present disclosure also includes compounds of formula (I) in various deuterated forms.
  • Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom.
  • Those skilled in the art can synthesize the compound of formula (I) in the deuterated form with reference to relevant literature.
  • Commercially available deuterated starting materials can be used when preparing the deuterated form of the compound of formula (I), or they can be synthesized using conventional techniques using deuterated reagents.
  • Deuterated reagents include, but are not limited to, deuterated borane and tri-deuterated. Borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide and deuterated methyl iodide, etc.
  • the hydrogen in the functional group of the compound described in the present disclosure is deuterated to obtain the corresponding deuterated compound.
  • the deuterated compound retains the selectivity and potential equivalent to the hydrogen derivative; the deuterium bond is more stable, making "ADME" that is Toxic pharmacokinetics are different, thereby providing clinically beneficial effects.
  • Toxic pharmacokinetics refers to the absorption, distribution, metabolism and excretion of foreign chemicals by the body.
  • Substituted refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, independently of each other, substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without too much effort. For example, an amino group or a hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.
  • pharmaceutical composition means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • pharmaceutically acceptable salt refers to the salt of the antibody-drug conjugate of the present disclosure, or the salt of the compound described in the present disclosure. Such salts have It is safe and effective, and has due biological activity.
  • the antibody-drug conjugate of the present disclosure contains at least one amino group, so it can form a salt with an acid.
  • Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate Salt, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, Maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.
  • solvate refers to the ligand-drug coupling compound of the present disclosure forming a pharmaceutically acceptable solvate with one or more solvent molecules.
  • solvent molecules include water, ethanol, acetonitrile, isopropyl Alcohol, DMSO, ethyl acetate.
  • drug carrier used for the drugs of the present disclosure refers to a system that can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug, and deliver the drug to the targeted organ.
  • the drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure. Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to contain drug molecules and at the same time have good permeability to the membrane, and can be used as excellent drug carriers.
  • excipient is an add-on other than the main drug in a pharmaceutical preparation, and can also be referred to as an adjuvant.
  • adjuvant such as binders, fillers, disintegrants, lubricants in tablets; base parts in semi-solid preparations ointments and creams; preservatives, antioxidants, correctives, fragrances, etc. in liquid preparations
  • Cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be called excipients.
  • diluent is also called a filler, and its main purpose is to increase the weight and volume of the tablet.
  • the addition of diluent not only guarantees a certain volume, but also reduces the deviation of the dosage of the main components, and improves the compression molding of the drug.
  • an absorbent should be added to absorb the oily substance to keep it in a "dry" state to facilitate the manufacture of tablets.
  • the compounds in the present disclosure can contain one or more asymmetric centers, so enantiomers and diastereomers can be produced, and they can be defined as (R)- or (S)- or using absolute stereochemistry. (D)- or (L)- other stereoisomeric forms of amino acids.
  • the present disclosure includes all possible isomers and their racemic and optically pure forms.
  • Optically active (+) and (-), (R)- and (S)- or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or conventional methods can be used For example, chromatography and fractional crystallization preparation.
  • the bond Indicates that the configuration is not specified, that is, if there are chiral isomers in the chemical structure, the bond Can be or Or both and Two configurations.
  • Stereoisomers refer to compounds of the same atomic composition that are bonded by the same bond but have different three-dimensional structures, and they are not interchangeable.
  • Various stereoisomers and mixtures thereof are contemplated in the present disclosure, and include “enantiomers”, which refers to two stereoisomers whose molecules are non-superimposable mirror images of each other.
  • Tautomer refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule.
  • the present disclosure includes tautomers of any of the compounds.
  • Figure 1 The animal weight change trend graph of the blank vehicle group and different test administration groups in Test Example 7 (abscissa: days, ordinate: body weight).
  • Figure 2 The trend graph of animal tumor volume changes in vehicle and different test drug administration groups (abscissa: days, ordinate: tumor volume).
  • the experimental methods without specific conditions in the embodiments of the present disclosure usually follow the conventional conditions or the conditions suggested by the raw material or commodity manufacturers. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory; Contemporary Molecular Biology Methods, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY.
  • the reagents without specific sources are the conventional reagents purchased on the market.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS).
  • NMR shift ( ⁇ ) is given in units of 10 -6 (ppm).
  • NMR was measured with Bruker AVANCE-400 nuclear magnetic instrument, and the solvent was deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), and the internal standard was four Methylsilane (TMS).
  • HPLC High performance liquid chromatography analysis uses Agilent HPLC 1200DAD, Agilent HPLC 1200VWD or Waters HPLC e2695-2489 high pressure liquid chromatograph.
  • HPLC preparation uses Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 preparative chromatographs.
  • CombiFlash rapid preparation instrument uses Combiflash Rf200 (TELEDYNE ISCO).
  • the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the size of the silica gel plate used for thin layer chromatography (TLC) is 0.15mm ⁇ 0.2mm, and the size of the thin layer chromatography separation and purification product is 0.4mm. ⁇ 0.5mm.
  • the silica gel column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.
  • the known starting materials of the present disclosure can be synthesized by or according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Darui Chemicals and other companies.
  • reaction can all be carried out under an argon atmosphere or a nitrogen atmosphere.
  • the argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1L.
  • the hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1L.
  • the pressure hydrogenation reaction uses Parr 3916EKX hydrogenator and Qinglan QL-500 hydrogen generator or HC2-SS hydrogenator.
  • the hydrogenation reaction is usually evacuated and filled with hydrogen, and the operation is repeated 3 times.
  • the microwave reaction uses the CEM Discover-S 908860 microwave reactor.
  • the solution refers to an aqueous solution.
  • reaction temperature is room temperature, which is 20°C to 30°C.
  • the monitoring of the reaction progress in the examples adopts thin layer chromatography (TLC), the developing solvent used in the reaction, the eluent system of column chromatography used to purify the compound, and the developing solvent system of thin layer chromatography, and the volume of the solvent
  • TLC thin layer chromatography
  • the ratio is adjusted according to the polarity of the compound, and it can also be adjusted by adding a small amount of basic or acidic reagents such as triethylamine and acetic acid.
  • Antibodies including light and heavy chains
  • Antigens are constructed by overlap extension PCR methods known in the art, and the DNA fragments obtained by overlap extension PCR are inserted into the expression vector pEE6.4 (Lonza Biologics) with HindIII/BstBI restriction sites. ), expressed in 293F cells (Invitrogen, Cat#R790-07).
  • the obtained recombinant protein is used for immunization or screening.
  • the human CD79B gene sequence is derived from NCBI (NP_000617.1), and its extracellular region (ECD) contains 159 amino acids (Met1-Asp159).
  • Human CD79B extracellular region (ECD) and human Fc region fusion protein (human CD79B ECD-hFc) and human CD79B extracellular region (ECD) and His-tagged fusion protein (human CD79B ECD-His) were used as immunogens, respectively. Intraperitoneal injection was used to immunize Balb/c and SJL mice, and stimulate the mice to produce antibodies against the extracellular domain (ECD) of human CD79B.
  • the fusion protein (cyno CD79B ECD-His) of monkey CD79B extracellular domain (ECD) and His tag (cyno CD79B ECD-His) was used as the immunogen to immunize SJL mice by intraperitoneal injection to stimulate the mice to produce extracellular area targeting monkey CD79B. (ECD) antibody.
  • Intraperitoneal injection immunization method calculate the amount of antigen required for this immunization according to the immunization program.
  • the protein antigen is diluted with PBS to the corresponding concentration as required, and then the antigen is emulsified. Transfer the emulsified mixture of antigen and adjuvant to a 2.0ml sterile syringe, and pay attention to venting air bubbles. Grasp the tail of the mouse with the right hand, gently grasp the skin of the head and neck of the mouse with the thumb and index finger of the left hand, with the abdominal cavity facing upwards, wipe the injection site on the right abdomen of the mouse with a 75% alcohol cotton ball.
  • FACS serum titer determination of immunized mice DoHH2 cells or monkey peripheral blood mononuclear cell suspensions were centrifuged and then resuspended in PBS containing 0.1% BSA and counted. The serum to be tested was added to each group of immunized mice, and incubated at room temperature After 60 minutes, the cells were washed three times and Anti-Mouse IgG (Fc specific)-FITC secondary antibody was added. After incubating at room temperature for 30 minutes in the dark, the cells were washed three times. The cells were gently resuspended in PBS containing 0.1% BSA and tested on the machine.
  • immunized mice produced specific antibodies against CD79B, and the above mice can be used for cell fusion to generate hybridoma cell lines capable of secreting specific antibodies against CD79B.
  • Cell fusion is to promote the fusion of mouse lymphocytes and myeloma cells SP2/0 (ATCC, CCL-121 TM ) into hybridoma cells under spontaneous or artificial induction.
  • Hybridoma cells not only have the function of antibody secretion but also can proliferate indefinitely.
  • the electrofusion method was used to fuse lymphocytes and myeloma cells in the immunized group for subsequent antibody screening.
  • Limiting dilution method for subcloning resuspend the cell lines that need to be subcloned from 24-well culture wells and count them. Dilute the cell concentration of each cell line to 5-10 cells/ml, add the diluted cell suspension to a 15cm disposable culture dish, add 0.2ml to each well of a 96-well culture plate, and each well contains 1- 2 pcs. Place the 96-well plate with the cells in a 37°C, 5% CO 2 incubator for culture. After 7-10 days, detect and screen the subcloning plate according to the growth of the cells, and select positive clones to 24 wells for further positive confirmation.
  • ELISA screening Before the start of the experiment, the 96-well plate should be labeled accordingly, and coated with an antigen concentration of 1ug/ml, 50ul per well, and overnight at 4°C in a refrigerator. The next day, take out the coated antigen plate the day before, and wash it once with a plate washer (washing solution: 1x PBST). After washing, block with 1% BSA blocking solution in 1X PBST at 37°C for 1 hour. After washing the plate 3 times with 1x PBST washing solution, add 50ul of the cell supernatant to be tested in a 37°C incubator and incubate for 1 hour.
  • washing solution 1x PBST
  • FACS screening After centrifugation of the DOHH2 cell suspension, the cells were resuspended in PBS containing 0.1% BSA and counted. The supernatant of the cells to be tested was added. After incubating for 60 minutes at room temperature, the cells were washed three times and then Anti-Mouse IgG (Fc specific) was added. FITC secondary antibody, incubate at room temperature for 30 minutes in the dark, wash the cells three times, gently resuspend the cells in PBS containing 0.1% BSA, and test on the machine.
  • Antibody number Clone number Mean fluorescence value Negative control mIgG 58 mAb015 83B2G2 10036 mAb017 86F11F6 8132
  • SPR detection of anti-human CD79B mouse monoclonal antibody surface plasmon resonance (SPR) technology is used to detect the affinity between anti-human CD79B antibody and its antigen human CD79B-His.
  • the antigen human CD79B-His protein was immobilized to the CM5 chip.
  • the coupling level was set at 100RU.
  • the running buffer is HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05%surfactant P20).
  • the diluted antibody was flowed through the experimental channel and the control channel for 3 minutes at a flow rate of 30 ⁇ l/min, and dissociated for 5 minutes.
  • the regeneration buffer (10 mM Glycine, pH 1.5) was run at a flow rate of 30 ⁇ l/min for 30 seconds.
  • the data is analyzed with Biacore 8K evaluation software.
  • Example 1-3 Determination of the amino acid sequence of the variable region of the mouse monoclonal antibody
  • the high-affinity hybridoma monoclonal cell strain obtained in Example 1-2 was subjected to variable region amino acid sequence determination, and then recombinantly expressed human and mouse chimeric antibody (cAb), and further antibody identification was performed.
  • the heavy and light chain variable regions of the antibody gene were amplified by reverse transcription PCR, connected to the vector and sequenced to obtain the light and heavy chain sequence of the monoclonal antibody.
  • an RNA purification kit (Qiagen company, product number 74134, see this instruction for the steps) was used to extract total cellular RNA from the active single cell line in Example 2.
  • amino acid residues of the VH/VL CDR of the anti-human CD79B antibody are determined and annotated by the Chothia numbering system.
  • mice CDR sequence is shown in Table 3:
  • the mouse antibody light and heavy chain sequence is aligned with the sequence in the model, and the sequence consistent with the mouse antibody sequence in the model is retained, and the structure model of the mouse antibody is obtained, in which inconsistent amino acids are possible back mutation sites.
  • the above-mentioned antibodies were cloned, expressed and purified by gene cloning and recombinant expression methods, and tested by ELISA, FACS and SPR, and finally selected humanized antibodies hAb015-10 and hAb017-10 with the best activity.
  • hAb015-10 humanized antibody heavy chain
  • hAb015-10 humanized antibody light chain
  • hAb017-10 humanized antibody heavy chain
  • hAb017-10 humanized antibody light chain
  • TROP-2 on the surface of CHO-K1 cells infected with lentivirus was detected by FACS, and the CHO-K1/hTROP-2 monoclonal cell line with high TROP-2 expression was selected.
  • TROP-2 (Genbank: NP_002344.2) is as follows:
  • the anti-human TROP-2 monoclonal antibody in the present disclosure is prepared according to the method disclosed in the WO03074566 patent, using the hRS7 antibody variable region gene as a template and using computer software to design point mutations for CDR. It is inserted into the protein expression vector Phr-IgG (with signal peptide and constant region gene (CH1-Fc/CL) fragment) by molecular cloning, and expressed in HEK293 and Expi-CHO-S cells. Purify the antibody according to the conventional method. CHO-K1 cells overexpressing huTROP-2 protein and huTROP-2 protein (His27-Thr274Accession#NP_002344.2) were used for activity verification, and antibodies with better target binding activity were selected.
  • the PD3 variable region sequence is as follows:
  • the underlined part is the CDR region determined according to the Kabat numbering rules.
  • Antibody PD3 Heavy chain CDR1 NYGMN (SEQ ID NO: 23) Heavy chain CDR2 WINTYTGEPTYTQDFKG (SEQ ID NO: 24) Heavy chain CDR3 GGFGSSYWYFDV (SEQ ID NO: 25) Light chain CDR1 KASQDVSIAVA(SEQ ID NO: 26) Light chain CDR2 SASYRYT (SEQ ID NO: 27) Light chain CDR3 QQHYITPLT (SEQ ID NO: 28)
  • the heavy chain constant region of the antibody can be selected from the constant regions of human IgG1, IgG2, IgG4 and variants thereof, and the light chain constant region can be selected from the light chain constant regions of human kappa, lambda chains or their variants.
  • the antibody heavy chain constant region is selected from the human IgG1 sequence shown in SEQ ID NO: 11, and the light chain constant region is selected from the human kappa chain constant region shown in SEQ ID NO: 12.
  • the heavy chain constant region of human IgG1 is the heavy chain constant region of human IgG1
  • the above-mentioned light chain/heavy chain constant region is combined with the variable region of the above-mentioned PD3 antibody to form a complete antibody, and the light chain/heavy chain sequence is as follows:
  • compound 1 (50mg, .08mmol, prepared by referring to the method in WO2017151979) was dissolved in 1.5mL of N,N-dimethylformamide, and then DIPEA (N,N-diisopropylethyl Amine, 18 mg, 0.14 mmol), followed by the addition of bis(p-nitrobenzene) carbonate (49 mg, 0.16 mmol). Then it was stirred at room temperature, 20 mL of methyl tert-butyl ether was added, stirred for 20 minutes, filtered, and dried to obtain 36 mg of solid compound 2.
  • DIPEA N,N-diisopropylethyl Amine, 18 mg, 0.14 mmol
  • bis(p-nitrobenzene) carbonate 49 mg, 0.16 mmol
  • compound D-1a (eribulin, prepared by referring to ZL201010236637.2 method) (72.91mg, 0.1mmol) was dissolved in 10mL of tetrahydrofuran, and Fmoc-OSu (fluorene methoxycarbonyl succinimide Amine, 41mg, 0.12mmol), and then stirred at room temperature until the reaction is complete. Concentrate under reduced pressure to obtain the crude product, which was directly used in the next reaction.
  • Fmoc-OSu fluorene methoxycarbonyl succinimide Amine, 41mg, 0.12mmol
  • the crude compound D-1c obtained in the previous step was dissolved in 10 mL of tetrahydrofuran, 2 mL of diethylamine was added, and then stirred at room temperature until the reaction was complete, and concentrated under reduced pressure to obtain the crude product, which was subjected to silica gel column chromatography (eluent: two (Chloromethane/ethyl acetate/petroleum ether) to obtain 3 mg of the target compound D-1.
  • the compound of formula D-8 was prepared using the starting materials p-hydroxyethyl benzoic acid and E1-30 according to the method of Example 2-2.
  • Test Example 1 In vitro cytotoxic activity screening
  • CTG was used to detect the ATP content, which reflects the survival of tumor cells.
  • the final culture conditions were determined according to IC 50 and maximum inhibition rate. Then test the killing effect of toxin molecules under this condition.
  • SKBR3 tumor cells HER2+, ATCC, article number HTB-30
  • MDA-MB-468 HER2-, ATCC, article number HTB-132
  • A549 human Three strains of non-small cell lung cancer cells, ATCC, catalog number CCL-185
  • Cell culture A549, SK-BR-3 and MDA-MB-468 cells are respectively used in Ham's F-12K (Kaighn's) medium (Gibco, 21127030) and McCoy's 5A containing 10% FBS (Gibco, 10099-141) Culture medium (ThermoFisher, article number 16600108) and Leibovitz's L-15 medium (ThermoFisher, article number 11415-114) for cultivation.
  • Ham's F-12K Ham's F-12K (Kaighn's) medium (Gibco, 21127030) and McCoy's 5A containing 10% FBS (Gibco, 10099-141)
  • Culture medium ThermoFisher, article number 16600108
  • Leibovitz's L-15 medium ThermoFisher, article number 11415-114) for cultivation.
  • CTG test Cell Titer-GloTM, luminescent cell viability test, Promega: Take out the cell plate on the 3rd and 5th day, and equilibrate to room temperature. Each well was added 90ul CTG, in the dark at room temperature the reaction 10min, microplate Luminescence was read and calculate IC 50.
  • Compound D-1 has a good killing effect in the three tumor cell lines, and it is significantly better than the positive drug Eribulin.
  • Test Example 2 In vitro cytotoxic activity screening
  • Cell culture A549, SK-BR-3 and MDA-MB-468 cells are respectively used in Ham's F-12K (Kaighn's) medium (Gibco, 21127030) and McCoy's 5A containing 10% FBS (Gibco, 10099-141) Culture medium (ThermoFisher, article number 16600108) and Leibovitz's L-15 medium (ThermoFisher, article number 11415-114) for cultivation.
  • Ham's F-12K Ham's F-12K (Kaighn's) medium (Gibco, 21127030) and McCoy's 5A containing 10% FBS (Gibco, 10099-141)
  • Culture medium ThermoFisher, article number 16600108
  • Leibovitz's L-15 medium ThermoFisher, article number 11415-114) for cultivation.
  • Dispensing plate 1 Start column 1 to dilute the stock solution 40 times, and column 2 to column 11 are successively diluted by 3 times.
  • Column 12 is DMSO.
  • Dispensing plate 2 Add 196 ul of the corresponding culture solution in column 2 to column 11, and suck 4 ul from column 3 to column 12 of dispensing plate 1 to column 2 to column 11 of dispensing plate 2. Mix well.
  • CTG test Cell Titer-GloTM, luminescent cell viability test: Take out the cell plate and equilibrate to room temperature. Each well was added 75ul CTG, in the dark at room temperature the reaction 10min, microplate Luminescence was read and calculate IC 50.
  • TCEP tris(2-carboxyethyl)phosphine
  • CE-SDS sodium dodecyl sulfate capillary electrophoresis
  • TCEP tris(2-carboxyethyl)phosphine
  • TCEP tris(2-carboxyethyl)phosphine
  • TCEP tris(2-carboxyethyl)phosphine
  • Test Example 3 Evaluation and comparison of the effects of ADC-5 and Polivy on human diffuse large B-cell lymphoma WSU-DLCL2 subcutaneously transplanted tumors in nude mice.
  • ADC-5 colorless and clear liquid, concentration 0.68mg/mL, purity 98.00%, shading, sealed at 2-8°C;
  • WSU-DLCL2 cells Human diffuse large B-cell lymphoma WSU-DLCL2 cells were purchased from DSMZ. WSU-DLCL2 cells are cultured in a 10-cm petri dish. The culture conditions are RPMI 1640 medium (Gibco) with 10% fetal bovine serum and penicillin and streptomycin (GIBCO, catalog number 15070-063) at 37°C, containing 5 Cultivate in an incubator with% CO2 air. Passage 2-3 times a week. When the cells are in the exponential growth phase, the cells are collected, counted, and seeded.
  • RPMI 1640 medium Gibco
  • GIBCO penicillin and streptomycin
  • Each nude mouse was inoculated with 2.0 ⁇ 10 7 WSU-DLCL2 cells subcutaneously. After the tumor grew to ⁇ 100 mm 3 , the animals were divided into groups according to the tumor volume (D0). Mice were administered intravenously (IV) with a volume of 10 mL/kg; see Table 3 for the specific dosage and dosage regimen. The tumor volume was measured twice a week, the mice were weighed, and the data was recorded.
  • IV intravenously
  • the use and welfare of the experimental animals are in accordance with the regulations of the "International Laboratory Animal Evaluation and Accreditation Committee (AAALAC)".
  • the health status and death of the animals are monitored daily. Routine inspections include observing the effects of the test substance and drugs on the daily behavior of the animals, such as behavioral activities, weight changes, physical signs, etc.
  • the experimental index is to investigate the effect of drugs on tumor growth, and the specific index is T/C% or tumor inhibition rate TGI (%).
  • the tumor diameter is measured with a vernier caliper twice a week, and the calculation formula for tumor volume (V) is:
  • V 1/2 ⁇ a ⁇ b 2 where a and b represent length and width respectively.
  • T/C(%) (T-T0)/(C-C0) ⁇ 100 where T and C are the tumor volume at the end of the experiment; T0 and C0 are the tumor volume at the beginning of the experiment.
  • TGI Tumor inhibition rate
  • TGI tumor inhibition rate
  • the tumor is smaller than the initial volume, that is, when T ⁇ T0 or C ⁇ C0, it is defined as partial tumor regression (PR); if the tumor disappears completely, it is defined as complete tumor regression (CR).
  • PR partial tumor regression
  • CR complete tumor regression
  • the animals were killed under CO 2 anesthesia, and then the tumors were dissected and taken and photographed.
  • the two-tailed Student's t test was used to compare the tumor volume between the two groups, and P ⁇ 0.05 was defined as a statistically significant difference.
  • ADC-5 and Polivy (IV; D0, 1 mg/kg) on human diffuse large B-cell lymphoma WSU-DLCL2 nude mice subcutaneously transplanted tumors were 53% and 37%, respectively; tumor-bearing mice were more than The drugs were well tolerated, and no symptoms such as significant weight loss occurred.
  • Anti-tumor inhibition rates of antibody-drug conjugate ADC-5 and Polivy (positive control group) against human diffuse large B-cell lymphoma WSU-DLCL2 nude mice subcutaneously transplanted tumors were 53% and 37%, respectively. They have significant anti-tumor effects. Activity; tumor-bearing mice can tolerate the above drugs well.
  • the purpose of this experiment is to detect the anti-TROP-2 antibody (PD3)-drug conjugate in the present disclosure for different tumor cell lines: Miapaca2 tumor cells (human pancreatic cancer cells, Nanjing Kebai, catalog number CBP60544), Fadu tumors Cells (Human Squamous Cell Carcinoma, ATCC, Item No. HTB-43), SK-OV-3 (Human Ovarian Cancer Cells, ATCC, Item No. HTB-77), K562 (Human Chronic Myeloid Leukemia Cells, ATCC, Item No.
  • Miapaca2 tumor cells human pancreatic cancer cells, Nanjing Kebai, catalog number CBP60544
  • Fadu tumors Cells Human Squamous Cell Carcinoma, ATCC, Item No. HTB-43
  • SK-OV-3 Human Ovarian Cancer Cells, ATCC, Item No. HTB-77
  • K562 Human Chronic Myeloid Leukemia Cells, ATCC, Item No.
  • HCC827 human lung cancer cells, ATCC, article number CRL-2868
  • BXPC3 human pancreatic cancer cells, ATCC, article number CRL-1687 proliferation inhibitory activity.
  • the cells were treated in vitro with different concentrations of antibody drug conjugates. After 6 days of culture, CTG ( Luminescent Cell Viability Assay, Promega, item number: G7573)
  • the reagent detects the proliferation of cells, and evaluates the in vitro activity of the antibody-drug conjugate based on the IC 50 value.
  • Drug preparation Adjust the initial concentration of the ADC mother solution to be tested to 4uM and add it to the first column of the dispensing board (corning, catalog number 3599). In the second column to the 9th column of the dispensing plate, it is diluted 5 times, and the 10th column is PBS. Take 15uL from each well and add it to the cell culture plate.
  • CTG detection After culturing at 37°C for 6 days, take out the cell culture plate and equilibrate to room temperature. Add 75uL CTG to each well and react for 10 minutes at room temperature in the dark, and read the luminescence value with a microplate reader (BMG Labtech, PHERAstar FS).
  • Detection of the antibody-drug conjugates of the present disclosure was co-cultured on TROP-2 positive cells BXPC3 (human pancreatic adenocarcinoma cells, Pronoxil) and TROP-2 negative cells MiaPaCa2 (human pancreatic adenocarcinoma cells, Pronoxil)
  • TROP-2 positive cells BXPC3 human pancreatic adenocarcinoma cells, Pronoxil
  • TROP-2 negative cells MiaPaCa2 human pancreatic adenocarcinoma cells, Pronoxil
  • MiaPaCa2 and BXPC3 cells were cultured with 10% FBS (Gibco, 10099-141) in DMEM/high glucose medium (GE, SH30243.01) and RPIM1640 medium (Gibco, 11875119) respectively.
  • Antibody drug conjugate preparation Dilute the antibody drug conjugates ADC-1, ADC-2, ADC-3 and ADC-4 with RPMI1640 to a concentration of 600nM respectively, and take 50uL of the culture solution and dilute 100uL to 200nM ( 40X, final concentration 5nM). Take 25uL and add it to the cell culture plate. A PBS solvent control group was also set up, and the culture was continued for 6 days.
  • the cells in plate 1 centrifuged at 1000 rpm for 3 min, discarded the supernatant, resuspended in 100 uL of FACS buffer, and added 2 uL of TROP-2 (EGP-1) monoclonal antibody (MR54) (ThermoFisher, catalog number 12-6024-42) , Incubate on ice for 30 min. Then centrifuge at 4°C and 2000rpm for 1 min, add 150uL FACS buffer to resuspend the cells, and repeat the above operation twice. Use flow cytometry (BD, FACSVerse) to detect.
  • the beagle dog was the test animal.
  • the LC/MS/MS method was used to determine the drug concentration in the plasma of the beagle dog at different times after intravenous injection of compound D-1 and Eribulin. To study the pharmacokinetic behavior of the compound of the present disclosure in dogs and evaluate its pharmacokinetic characteristics.
  • a group of dogs were given compound D-1 by intravenous injection at a dosage of 0.5 mg/kg and a volume of 2 ml/kg.
  • the other group of dogs was given intravenous injection of Eribulin at a dose of 0.5 mg/kg and a volume of 2 ml/kg.
  • Compound D-1 was administered to dogs by injection, and 1 ml of blood was collected before and 5 minutes, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, 24.0 hours after the administration, and the collected blood samples were placed in the EDTA-K2 antibody.
  • the collected whole blood is placed on ice, and the plasma is separated by centrifugation within 1 hour (centrifugal force 2200g, centrifugation 10min, 2-8°C).
  • the plasma samples were stored in a refrigerator at -80°C before testing.
  • Dogs were injected with Eribulin compound. 1ml of blood was collected before and 5 minutes, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, 24.0 hours after the administration, and the collected blood samples were placed in EDTA-K2 Place the collected whole blood on ice in the anticoagulated blood collection tube, and centrifuge to separate the plasma within 1 hour (centrifugal force 2200g, centrifugation 10min, 2-8°C). The plasma samples were stored in a refrigerator at -80°C before testing.
  • Test Example 7 Pharmacodynamic effects of ADC-2 and ADC-3 on human pharyngeal squamous cell carcinoma FaDu cell line BALB/c subcutaneously transplanted tumor in nude mice
  • the human pharyngeal squamous cell carcinoma Fadu cell line (ScienCell Laboratory, ml-cs-0374) used in this experiment was in MEM medium (with 10% (v/v) fetal bovine serum (FBS) (GIBCO, catalog number 10099-141) And 0.1% phosphate buffer) in a 37°C incubator containing 5% CO 2.
  • the mice were anesthetized with 3-4% isoflurane before inoculation. Before the cells were continuously cultured for ten generations, 100 ⁇ L of 5 ⁇ 10 6 Fadu cell culture medium was mixed with an equal volume of Matrigel (base gel, solarbio), and then inoculated into the right side of the back of the mouse near the armpit by subcutaneous injection.
  • Matrigel base gel, solarbio
  • mice When the tumor grows to an average of about 100-150 mm 3 , the mice are randomly grouped according to tumor volume and body weight, with 8 mice in each group. The day of group administration is defined as day 0. The grouping situation and dosing schedule are shown in Table 10 below:
  • Dosing volume the animal's dosing volume is adjusted according to 10 ⁇ L/g body weight
  • Tumor volume After grouping, the tumor volume was measured twice a week for 4 consecutive weeks.
  • Animal body weight Measure and record the mouse body weight twice a week after grouping.
  • Body weight The change trend of animal body weight in the vehicle and different test drug administration groups is shown in Figure 1. The body weight of the animals increased normally with the progress of the experiment.
  • the tumor grew slowly; until the 14th day, the tumor volume increased rapidly.
  • the mean tumor volume of the G1 group was 125.05 ⁇ 3.66 mm 3 ; on the 25th day, the mean tumor volume was 1854.48 ⁇ 99.50 mm 3 .
  • the Fadu cell line of human pharyngeal squamous cell carcinoma was successfully established in BALB/c nude mice subcutaneously transplanted tumor model.
  • the mean tumor volume of animals in the ADC-3 high-dose (3mg/kg) group (G2 group) on day 0 was 121.40 ⁇ 3.18 mm 3 ; on day 25, the mean tumor volume was 721.56 ⁇ 169.15 mm 3 .
  • the ADC-3 high-dose group can significantly inhibit tumor growth.
  • T/C relative tumor proliferation rate
  • the tumor volume of the high-dose group was significantly reduced compared with the model group, and the tumor volume of the low-dose group had a decreasing trend compared with the model group, but there was no statistical difference.
  • the drug showed a dose-dependent inhibitory effect on tumor growth.
  • the ADC-3 group had better in vivo efficacy at 25 days than the ADC-2 group, and there was a significant difference between the two.

Abstract

本公开涉及艾日布林衍生物的药物偶联物、其制备方法及其在医药上的应用。具体而言，提供一种抗体‐药物偶联物，所述抗体‐药物偶联物包含艾日布林衍生物药物部分。本公开进一步涉及通过施用本文提供的抗体‐药物偶联物用于治疗癌症的方法。

Description

艾日布林衍生物的药物偶联物、其制备方法及其在医药上的应用 技术领域
本公开涉及艾日布林衍生物的药物偶联物、其制备方法及其在医药上的应用。
背景技术
抗体药物偶联物(antibody drug conjugate，ADC)把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药物相连，充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和药物的高效性，同时又避免了前者疗效偏低和后者毒副作用过大等缺陷。这也就意味着，与以往传统的化疗药物相比，抗体药物偶联物能精准地结合肿瘤细胞并降低将对正常细胞的影响(Mullard A,(2013)Nature Reviews Drug Discovery,12:329–332；DiJoseph JF,Armellino DC,(2004)Blood,103:1807-1814)。
2000年第一个抗体药物偶联物Mylotarg(吉妥珠单抗奥唑米星(gemtuzumab ozogamicin)，惠氏制药有限公司)被美国FDA批准上市，用于治疗急性髓细胞白血病(Drugs of the Future(2000)25(7):686；US4970198；US5079233；US5585089；US5606040；US5693762；US5739116；US5767285；US5773001)。
2011年8月，Adcetris(brentuximab vedotin，西雅图基因遗传公司)通过美国FDA快速审评通道，用于治疗霍奇金淋巴瘤以及复发性间变性大细胞淋巴瘤(Nat.Biotechnol(2003)21(7):778-784；WO2004010957；WO2005001038；US7090843A；US7659241；WO2008025020)。
Figure PCTCN2021073314-appb-000001
是一种新型靶向ADC药物，能使药物直接作用于淋巴瘤细胞上的靶点CD30后发生内吞作用从而诱导肿瘤细胞的凋亡。
Mylotarg和Adcetris都是针对血液肿瘤进行靶向治疗，血液肿瘤和实体肿瘤相比组织结构相对简单。2013年2月，Kadcyla(ado-trastuzumab emtansine，T-DM1)获得美国FDA批准，用于治疗HER2阳性同时对曲妥珠单抗(Tratuzumab，商品名：Herceptin)和紫杉醇有抗药性的晚期或转移性乳腺癌患者(WO2005037992；US8088387)。Kadcyla是美国FDA批准的第一个治疗实体肿瘤的ADC药物。
微管为与包括细胞内迁移和转运、细胞信号传导和维持细胞形状的多种细胞功能相关的有力的细丝状细胞骨架蛋白。微管也在有丝分裂细胞分裂中通过形成染色体分成两个子细胞所需的有丝分裂纺锤体而起到关键作用。所有细胞中微管的生物功能大部分由其聚合动力学调节，这通过α和β微管蛋白二聚物可逆、非共价地加在微管两端进行。这种动力学行为和所产生的对微管长度的控制为有丝分裂纺锤体的适当功能所不可缺少的。甚至微管动力学的微小改变也会牵涉轴检查点，抑制有丝分裂时细胞周期进展，且随后引起细胞死亡(Mukhtar等人(2014)Mol.Cancer Ther.13：275-84)。由于癌细胞的细胞分裂快速，所以与正常细胞相比，其一般对结合于微管蛋白且破坏其正常功能的化合物更加敏感。因此， 微管蛋白抑制剂和其它靶向微管剂有望成为一类治疗癌症的药物(Dumontet和Jordan(2010)Nat.Rev.Drug Discov.9：790-803)。
发明内容
本公开提供了一种抗体-药物偶联物(ADC)，其具有式(I)所示结构或其药学上可接受的盐或溶剂化物：
Ab-(L-D) k
(I)
其中，Ab为抗体或其抗原结合片段，
L为将Ab共价连接于D的连接子，且k为1至20(包括1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或任意两数值之间任意数值)，
-D如下式所示：
Figure PCTCN2021073314-appb-000002
其中R 1a选自氢、烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选甲基；
R 1b选自氢、烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选氢；
或者R 1a与R 1b与其相连接的原子一起形成5至8元杂环烷基，所述杂环烷基任选被烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6 烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，且R 1a和R 1b不同时为氢。
在一些实施方案中，抗体-药物偶联物中-D中R 1a与R 1b与其相连接的原子一起形成6至8元杂环烷基。
在一些实施方案中，抗体-药物偶联物中-D中R 1a选自甲基。
在一些实施方案中，抗体-药物偶联物中-D中R 1a、R 1b各自独立选自甲基。
在一些实施方案中，抗体-药物偶联物中-D为
Figure PCTCN2021073314-appb-000003
在一些实施方案中，抗体-药物偶联物Ab-(L-D) k中，k选自1至10，可以为整数，也可以为小数。
另一方面，本公开还提供了一种抗体-药物偶联物(ADC)，其具有式(I)所示结构或其药学上可接受的盐或溶剂化物：
Ab-(L-D) k
(I)
其中，-D选自：
Figure PCTCN2021073314-appb-000004
在一些实施方案中，连接子在细胞外是稳定的，使得ADC在存在于细胞外环境中时保持完整，但在例如癌细胞的细胞中内化时能够裂解。在一些实施方案中，当ADC进入表达对ADC的抗体部分具有特异性的抗原的细胞时，艾日布林类似物药物部分从抗体部分裂解，且裂解释放艾日布林类似物的未修饰形式。
在一些实施方案中，连接子中的可裂解部分为可裂解肽部分。在一些实施方案中，相对于包含其他可裂解部分的ADC，包含可裂解肽部分的ADC显示较低的聚集水平，改善的抗体与药物比率，增加的癌细胞的靶向杀死，减少的非癌细胞的脱靶杀死，和/或较高的药物负载(p)。在一些实施方案中，相对于不可裂解的 连接子，添加可裂解部分增加细胞毒性和/或效力。在一些实施方案中，增加的效力和/或细胞毒性是在表达中等水平的由ADC的抗体部分所靶向的抗原(例如中等FRA表达)的癌症中具有增加的效力和/或细胞毒性。在一些实施方案中，可裂解肽部分能够由酶裂解，且连接子为酶能够裂解的连接子。在一些实施方案中，酶为组织蛋白酶，且连接子为组织蛋白酶能够裂解的连接子。在某些实施方案中，与其它分裂机制相比，酶能够裂解的连接子(例如组织蛋白酶能够裂解的连接子)显示上述改善特性中的一种或多种。
在一些实施方案中，连接子包含氨基酸单元，所述氨基酸单元优选包含由2至7个选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸的氨基酸构成的肽残基，更优选缬氨酸-瓜氨酸(Val-Cit)、丙氨酸-丙氨酸-天冬酰胺(Ala-Ala-Asn)、甘氨酸-甘氨酸-赖氨酸(Gly-Gly-lys)、缬氨酸-赖氨酸(Val-lys)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-苯丙氨酸(Val-Phe)或甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)。
在一些实施方案中，本公开包括氨基酸单元的连接子选自：
Figure PCTCN2021073314-appb-000005
在一些实施方案中，氨基酸单元包含缬氨酸-瓜氨酸(Val-Cit)。在一些实施方案中，相对于包含其它氨基酸单元或其它可裂解部分的ADC，包含Val-Cit的ADC显示增加的稳定性，减少的脱靶细胞杀死，增加的靶向细胞杀死，较低的聚集水平，和/或较高的药物负载。
另一方面，一些实施方案提供的连接子包含可裂解磺酰胺部分，所述连接子在还原条件下能够裂解。
在一些实施方案中，所述连接子包含可裂解二硫化物部分，所述连接子在还原条件下能够裂解。
另一方面，本公开抗体偶联物中连接子包含至少一种将艾日布林衍生物D连接于可裂解部分的间隔单元。在一些实施方案中，所述连接子包含连接于D的间隔单元。
在一些实施方案中，所述间隔单元包含对氨基苯甲氧基羰基(PAB)，
Figure PCTCN2021073314-appb-000006
在一些实施方案中，所述间隔单元包含对氨基苯甲酰基，
Figure PCTCN2021073314-appb-000007
在一些实施方案中，所述间隔单元包含：
Figure PCTCN2021073314-appb-000008
其中，Z 1～Z 5各自独立地选自碳原子或氮原子；R 14选自烷基、环烷基、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代；R 11和R 12各自独立选自氢、氘、C 1-6烷基、C 3-6环烷基，优选氢；或者，R 11与R 12与其相连接的碳原子一起形成C3-6环烷基；X选自-O-或-NH-；L选自1-4之间整数；
Q为V-E-，V-E-提供了可被位于胞内的糖苷酶切割的糖苷键，E选自-O-、-S-或-NR 13-，R 13选自氢或甲基，进一步地，V选自
Figure PCTCN2021073314-appb-000009
其中R 15选自-COOH或CH 2OH。在一些实施方案中，V选自-COOH。
在一些实施方案中，所述间隔单元包含：
Figure PCTCN2021073314-appb-000010
Figure PCTCN2021073314-appb-000011
Z 1、Z 3、Z 4、X、Q、R 11、R 12、R 14如上所述。
在一些实施方案中，所述间隔单元包含选自以下部分：
-(CR aR b) m1-O(CR aR b) m2-CR 8R 9-C(O)-，
-(CR aR b) m1NH-(CR aR b) m2-CR 8R 9-C(O)-，
-(CR aR b) m1O-CR 8R 9(CR aR b) m2-，
-(CR aR b) m1OCR 8R 9-C(O)-，
-(CR aR b) m1-O-(CR aR b) m2C(O)-或-(CR aR b) m1-S-(CR aR b) m2-CR 8R 9-C(O)-，
其中R a和R b相同或不同，且各自独立地选自氢、氘原子、卤素或烷基；R 8选自氢、C 3-6环烷基烷基或C 3-6环烷基；R 9选自氢、卤代烷基或C 3-6环烷基，优选氢；或者，R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基；m1和m2各自独立选自0、1、2或3。
在一些实施方案中，所述间隔单元包含选自以下部分：
-(CH 2) 3-C(O)-、-CH 2-O-CH 2-C(O)-、-(CH 2) 2-O-CH 2-C(O)-、
Figure PCTCN2021073314-appb-000012
Figure PCTCN2021073314-appb-000013
另一方面，本公开抗体偶联物(ADC)中L-D是由下式表示的化学部分：
-Str-(Pep)-Sp-D
Str是与Ab共价连接的拉伸单元，
Sp为间隔单元，
Pep选自氨基酸单元、二硫化物部分、磺酰胺部分或以下非肽化学部分：
Figure PCTCN2021073314-appb-000014
其中，W是-NH-杂环烷基-或杂环烷基；Y是杂芳基、芳基、-C(O)C 1-6亚烷基、C 2-6亚烯基、C 1-6亚烷基或-C 1-6亚烷基-NH-；
每个R 2独立选自C 1-10烷基、C 2-10烯基、C 1-6亚烷基-NH 2、-(C 1-10亚烷 基)NHC(NH)NH 2或-(C 1-10亚烷基)NHC(O)NH 2；
R 3和R 4各自独立为H、C 1-10烷基、C 2-10烯基、芳基烷基、杂芳基烷基，或R 3和R 4一起可形成C 3-7环烷基；
R 5和R 6各自独立为C 1-10烷基、C 2-10烯基、芳基烷基、杂芳基烷基、(C 1-10亚烷基)OCH 2-，或R 5和R 6一起可形成C 3-7环烷基环。
在一些实施方案中，所述抗体-药物偶联物(ADC)中Y选自以下部分：
Figure PCTCN2021073314-appb-000015
另一方面，所述抗体-药物偶联物(ADC)中Str选自下式表示的化学部分：
Figure PCTCN2021073314-appb-000016
其中R 7选自-W1-C(O)-、-C(O)-W1-C(O)-、-(CH 2CH 2O) p1C(O)-、-(CH 2CH 2O) p1CH 2C(O)-、-(CH 2CH 2O) p1CH 2CH 2C(O)-，其中W1选自C 1-8亚烷基、C 1-8亚烷基-环烷基或1至8个原子的直链杂烷基，所述杂烷基包含1至3个选自N、O或S的杂原子，其中所述的C 1-8亚烷基、环烷基和直链杂烷基各自独立地任选进一步被选自卤素、氘、羟基、氰基、氨基、烷基、卤代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
L 1选自-NR 10(CH 2CH 2O) p1CH 2CH 2C(O)-、-NR 10(CH 2CH 2O) p1CH 2C(O)-、-S(CH 2) p1C(O)-、-(CH 2) p1C(O)-或化学键，优选化学键；其中，p1为1至20的整数，R 10选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基。
在一些实施方案中，C 1-8亚烷基-环烷基选自亚甲基-环已基
Figure PCTCN2021073314-appb-000017
亚乙基‐环已烷基
Figure PCTCN2021073314-appb-000018
亚甲基‐环戊基
Figure PCTCN2021073314-appb-000019
在一些实施方案中，连接子可以包含至少一种聚乙二醇(PEG)部分。PEG部分可以例如包含-(PEG) p1-，
Figure PCTCN2021073314-appb-000020
其中p1为整数1至20，例如
Figure PCTCN2021073314-appb-000021
(PEG) 2；
Figure PCTCN2021073314-appb-000022
(PEG) 4；
Figure PCTCN2021073314-appb-000023
(PEG) 5。
在一些实施方案中，连接子中的拉伸单元包含(PEG) 2。在一些实施方案中，尽管连接子长度较短，但相对于包含较长拉伸单元(例如(PEG) 8)的ADC，包含较短拉伸单元(例如(PEG) 2)的ADC显示较低的聚集水平和/或较高的药物负载。
在一些实施方案中，所述抗体-药物偶联物的Str，
Figure PCTCN2021073314-appb-000024
中R 7选自C 1-6亚烷基C(O)-、-(CH 2-CH 2O) 2C(O)-、-(CH 2-CH 2O) 2CH 2C(O)-、-(CH 2-CH 2O) 2CH 2CH 2C(O)-、-(CH 2-CH 2O) 3C(O)-和-(CH 2-CH 2O) 4C(O)-。
在一些实施方案中，所述抗体-药物偶联物的Str，
Figure PCTCN2021073314-appb-000025
中R 7选自-C 1-8亚烷基-环烷基-C(O)-、-(CH 2-CH 2O) 4CH 2C(O)-和-(CH 2-CH 2O) 6CH 2C(O)-。
在一些实施方案中，所述抗体-药物偶联物中连接子L包含：顺丁烯二酰亚胺-(PEG) 2-Val-Cit、顺丁烯二酰亚胺-(PEG) 6-Val-Cit、顺丁烯二酰亚胺-(PEG) 8-Val-Cit、顺丁烯二酰亚胺-(PEG) 4-CH 2CH 2C(O)-Val-lys、顺丁烯二酰亚胺-(CH 2) 5-Val-Cit、顺丁烯二酰亚胺-(CH 2) 5-Val-lys、顺丁烯二酰亚胺-(CH 2) 5-Gly-Gly-Phe-Gly、顺丁烯二酰亚胺-(PEG) 2-Ala-Ala-Asn、顺丁烯二酰亚胺-(PEG) 6-Ala-Ala-Asn、顺丁烯二酰亚胺-(PEG) 8-Ala-Ala-Asn、顺丁烯二酰亚胺-(PEG) 4-三唑-(PEG) 3-磺酰胺、顺丁烯二酰亚胺-(PEG) 2-CH 2CH 2C(O)-Val-lys、顺丁烯二酰亚胺-(PEG) 4-三唑-(PEG) 3-磺酰胺或Mal-(PEG) 4-三唑-(PEG) 3-二硫化物。
在一些实施方案中，所述抗体-药物偶联物中连接子L包含：顺丁烯二酰亚胺-(PEG) 4-CH 2C(O)-Gly-Gly-Phe-Gly、顺丁烯二酰亚胺-(PEG) 2-CH 2CH 2C(O)-Gly-Gly-Phe-Gly、顺丁烯二酰亚胺-(PEG) 6-CH 2C(O)-Gly-Gly-Phe-Gly-、顺丁烯二酰亚胺-(CH 2) 5C(O)-Gly-Gly-Phe-Gly-、顺丁烯二酰亚胺-C 1-8亚烷基-环烷基-C(O)-NH(CH 2CH 2O) 4CH 2C(O)-Gly-Gly-Phe-Gly-、顺丁烯二酰亚胺-(PEG) 2-CH 2C(O)-Gly-Gly-Phe-Gly-、顺丁烯二酰亚胺-(PEG) 2-CH 2CH 2C(O)-Val-Cit-、顺丁烯二酰亚胺-(PEG) 2-Gly-Gly-Phe-Gly-、顺丁烯二酰亚胺-(PEG) 2-CH 2C(O)-Val-Cit-、顺丁烯二酰亚胺-(PEG) 4-CH 2C(O)-Val-Cit-和顺丁烯二酰亚胺-(PEG) 6-CH 2C(O)-Val-Cit-。
另一方面，一些实施方案提供所述抗体-药物偶联物中Str选自下式表示的化学部分：
Figure PCTCN2021073314-appb-000026
其中R 8选自C 1-10亚烷基、C 2-10亚烯基、(C 1-10亚烷基)O-、N(R d)-(C 2-6亚烷基)-N(R d)和N(R d)-(C 2-6亚烷基)；且每个R d独立为H或C 1-C 6烷基。
在一些实施方案中，所述抗体-药物偶联物中L-D由选自以下的式表示：
Figure PCTCN2021073314-appb-000027
其中R 2是C 1- 6亚烷基、(C 1- 6烷基)NHC(NH)NH 2或(C 1- 6亚烷基)NHC(O)NH 2；
Figure PCTCN2021073314-appb-000028
其中R 2是C 1-6烷基、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2；
Figure PCTCN2021073314-appb-000029
其中R 2是C 1-6烷基、C 2-6亚烯基、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2；
Figure PCTCN2021073314-appb-000030
其中R 2是C 1-6烷基、C 2-6亚烯基、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2；
Figure PCTCN2021073314-appb-000031
其中R 2是C 1-6烷基、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2，且R 5和R 6一起形成C 3-7环烷基环；
Figure PCTCN2021073314-appb-000032
其中R 2是C 1-6烷基、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2，且R 5和R 6一起形成C 3-7环烷基环；
W、Str、D如前所述。
在另一些实施方案中，本公开抗体-药物偶联物(ADC)，其由下式表示：
Figure PCTCN2021073314-appb-000033
其中R 2选自C 1-6亚烷基-NH 2、(C 1- 6 亚烷基)NHC(NH)NH 2或(C 1- 6亚烷基)NHC(O)NH 2，k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
Figure PCTCN2021073314-appb-000034
其中R 2选自C 1-6亚烷基-NH 2、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2，k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
Figure PCTCN2021073314-appb-000035
其中R 2是C 1-6亚烷基-NH 2、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2，且R 5和R 6形成C 3-7环烷基环，k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
Figure PCTCN2021073314-appb-000036
其中R 2是C 1-6亚烷基-NH 2、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2，且R 5和R 6形成C 3-7环烷基环，k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
Y、R 3、R 4、Ab、D如前所述。
另一方面，一些实施方案提供的抗体-药物偶联物(ADC)，其由下式表示：
Figure PCTCN2021073314-appb-000037
其中R 8选自氢、C 3-6环烷基烷基或C 3-6环烷基，优选氢；R 9选自氢、卤代烷基或C 3-6环烷基，优选氢，或者R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基，k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
Figure PCTCN2021073314-appb-000038
其中R 8选自 氢、C 3-6环烷基烷基或C 3-6环烷基，优选氢；R 9选自氢、卤代烷基或C 3-6环烷基，优选氢；或者R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基；k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2；
Figure PCTCN2021073314-appb-000039
k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
Figure PCTCN2021073314-appb-000040
k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
Figure PCTCN2021073314-appb-000041
k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8；p3选自0、1或2；
Figure PCTCN2021073314-appb-000042
k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2；
Figure PCTCN2021073314-appb-000043
k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8；
Figure PCTCN2021073314-appb-000044
k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8；p3选自0、1或2；
Figure PCTCN2021073314-appb-000045
k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8；
Figure PCTCN2021073314-appb-000046
k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2；
Figure PCTCN2021073314-appb-000047
k选自1至10，可以为整数，也可以为小数，p2选自2、4、6或8；
Figure PCTCN2021073314-appb-000048
k选自1至10，可以为整数，也可以为小数，p2选自2、4、6或8；
Figure PCTCN2021073314-appb-000049
k选自1至10，可以为整数，也可以为小数，p2选自2、4、6或8；
Figure PCTCN2021073314-appb-000050
k选自1至10，可以为整数，也可以为小数，p2选自2、4、6或8；
Figure PCTCN2021073314-appb-000051
k选自1至10，可以为整数，也可以为小数，p2选自2、4、6或8；
Figure PCTCN2021073314-appb-000052
k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2；
Figure PCTCN2021073314-appb-000053
k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2；
Figure PCTCN2021073314-appb-000054
其中R 8选自氢、卤代烷基或C 3-6环烷基，优选氢，R 9选自氢、卤代烷基或C 3-6环烷基，优选氢，或者R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基，k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
Figure PCTCN2021073314-appb-000055
其中R 8选自氢、卤代烷基或C 3-6环烷基，优选氢，R 9选自氢、卤代烷基或C 3-6环烷基，优选氢，或者R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基；k选自1至10，可以为整数，也可以为小数，p2 选自2-6之间整数；
Figure PCTCN2021073314-appb-000056
其中R 8选自氢、卤代烷基或C 3-6环烷基，优选氢，R 9选自氢、卤代烷基或C 3-6环烷基，优选氢，或者R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基，k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2；
Figure PCTCN2021073314-appb-000057
其中R 8选自氢、卤代烷基或C 3-6环烷基，优选氢，R 9选自氢、卤代烷基或C 3-6环烷基，优选氢，或者R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基，k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2。
在一些所述方案中，所述的抗体-药物偶联物(ADC)，其由下式表示：
Figure PCTCN2021073314-appb-000058
Figure PCTCN2021073314-appb-000059
Figure PCTCN2021073314-appb-000060
Figure PCTCN2021073314-appb-000061
Figure PCTCN2021073314-appb-000062
其中k选自1至10，可以为整数，也可以为小数；进一步地，D中R 1a优选自甲基，R 1b优选自氢。
另一方面，本公开抗体-药物偶联物(ADC)中所述抗体选自鼠源抗体、嵌合抗体、人源化抗体和全人源抗体。
在一些实施方案中，所述抗体-药物偶联物(ADC)中所述抗体或其抗原结合片段选自抗HER2(ErbB2)抗体、抗EGFR抗体、抗B7-H3抗体、抗c-Met抗体、抗HER3(ErbB3)抗体、抗HER4(ErbB4)抗体、抗CD20抗体、抗CD22抗体、抗CD30抗体、抗CD33抗体、抗CD44抗体、抗CD56抗体、抗CD70抗体、抗CD73抗体、抗CD105抗体、抗CEA抗体、抗A33抗体、抗Cripto抗体、抗EphA2抗 体、抗G250抗体、抗MUCl抗体、抗Lewis Y抗体、抗VEGFR抗体、抗GPNMB抗体、抗Integrin抗体、抗PSMA抗体、抗Tenascin-C抗体、抗SLC44A4抗体、抗CD79抗体、抗TROP-2抗体、抗CD79B抗体、抗Mesothelin抗体或其抗原结合片段。
在一些实施方案中，所述抗体-药物偶联物(ADC)中抗体为已知抗体，选自但不限于选自曲妥珠单抗(Trastuzumab)、帕妥珠单抗(Pertuzumab)、尼妥珠单抗(Nimotuzumab)、恩波妥珠单抗(Enoblituzumab)、依玛妥珠单抗(Emibetuzumab)、奥英妥珠单抗(Inotuzumab)、维汀-匹那妥珠单抗(Pinatuzumab)、维布妥昔单抗(Brentuximab)、吉妥单抗(Gemtuzumab)、比伐珠单抗(Bivatuzumab)、莫洛伐妥单抗(Lorvotuzumab)、cBR96和Glematumamab或其抗原结合片段。
在另一些实施方案中，所述抗体-药物偶联物(ADC)中抗体选自抗CD79B抗体或其抗原结合片段，其包含抗体重链可变区和/或抗体轻链可变区，其中：
抗体重链可变区，包含：
1)分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的HCDR1、HCDR2和HCDR3；或
2)分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的HCDR1、HCDR2和HCDR3；
和/或抗体轻链可变区，包含：
1)分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的LCDR1、LCDR2和LCDR3；或
2)分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，所述抗体-药物偶联物(ADC)中抗CD79B抗体包含重链可变区和轻链可变区，包含选自以下(I)至(II)中的任一项：
1)重链可变区，其包含分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的LCDR1、LCDR2和LCDR3；
2)重链可变区，其包含分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3。
重链CDR1 GSSFTSY(SEQ ID NO:7) GYTFTTY(SEQ ID NO:13)
重链CDR2 FPRSGN(SEQ ID NO:8) YPRSGN(SEQ ID NO:14)
重链CDR3 GDLGDFDY(SEQ ID NO:9) GSDYDGDFAY(SEQ ID NO:15)
轻链CDR1 RSSQSIVHSDGNTYFE(SEQ RSSQSIVHHDGNTYLE(SEQ
  ID NO:10) ID NO:16)
轻链CDR2 KVSNRFS(SEQ ID NO:11) KVSNRFS(SEQ ID NO:17)
轻链CDR3 FQGSHVPWT(SEQ ID NO:12) FQGSHVPWT(SEQ ID NO:18)
在一些实施方案中，所述抗体-药物偶联物(ADC)中抗CD79B抗体包含重链可变区和轻链可变区，其中：
重链可变区，包含：
1)如SEQ ID NO：3所示或与SEQ ID NO：3具有至少90％、95％、98％、99％同一性的序列；或
2)如SEQ ID NO：5所示或与SEQ ID NO：5具有至少90％、95％、98％、99％同一性的序列；
和/或轻链可变区，包含：
1)如SEQ ID NO:4所示或与SEQ ID NO：4具有至少90％、95％、98％、99％同一性的序列；或
2)如SEQ ID NO:6所示或与SEQ ID NO：6具有至少90％、95％、98％、99％同一性的序列；
优选地，所述抗CD79B抗体或抗原结合片段的重链可变区如序列SEQ ID NO：3所示，轻链可变区如序列SEQ ID NO:4所示；或重链可变区如序列SEQ ID NO：5所示，轻链可变区如序列SEQ ID NO:6所示。
>小鼠杂交瘤细胞单克隆抗体mAb015重链可变区：
Figure PCTCN2021073314-appb-000063
>小鼠杂交瘤细胞单克隆抗体mAb015轻链可变区：
Figure PCTCN2021073314-appb-000064
>小鼠杂交瘤细胞单克隆抗体mAb017的重链可变区：
Figure PCTCN2021073314-appb-000065
>小鼠杂交瘤细胞单克隆抗体mAb017的轻链可变区：
Figure PCTCN2021073314-appb-000066
在另一些实施方案中，所述抗体-药物偶联物(ADC)中抗CD79B抗体包含重链可变区和轻链可变区，其中：
重链可变区，包含：
1)如SEQ ID NO：19所示或与SEQ ID NO：19具有至少90％、95％、98％或99％同一性的序列；或
2)如SEQ ID NO：21所示或与SEQ ID NO：21具有至少90％、95％、98％、或99％同一性的序列；
和/或轻链可变区，包含：
1)如SEQ ID NO:20所示或与SEQ ID NO：20具有至少90％、95％、98％或99％同一性的序列；或
2)如SEQ ID NO:22所示或与SEQ ID NO：22具有至少90％、95％、98％或99％同一性的序列；
优选地，所述抗CD79B抗体或抗原结合片段的重链可变区如序列SEQ ID NO：19所示，轻链可变区如序列SEQ ID NO:20所示；或重链可变区如序列SEQ ID NO：21所示，轻链可变区如序列SEQ ID NO:22所示。
>hAb015-10人源化抗体重链序列：
Figure PCTCN2021073314-appb-000067
>hAb015-10人源化抗体轻链序列：
Figure PCTCN2021073314-appb-000068
>hAb017-10人源化抗体重链序列：
Figure PCTCN2021073314-appb-000069
>hAb017-10人源化抗体轻链序列：
Figure PCTCN2021073314-appb-000070
另一方面，人CD79B细胞外区域(ECD)和人Fc区域融合蛋白(human CD79B ECD-hFc)的氨基酸序列：
Figure PCTCN2021073314-appb-000071
>人CD79B细胞外区域(ECD)和His标签的融合蛋白(human CD79B ECD-His)的氨基酸序列：
Figure PCTCN2021073314-appb-000072
另一方面，在一些实施方案中，所述抗体-药物偶联物(ADC)中抗体选自抗TROP-2抗体。在一些实施方案中，所述抗体-药物偶联物(ADC)中抗体包含重链可变区和轻链可变区，其中所述重链可变区包含序列分别如SEQ ID NO：23、SEQ ID NO：24和SEQ ID NO：25所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含序列分别如SEQ ID NO：26、SEQ ID NO：27和SEQ ID NO：28所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，所述抗体-药物偶联物(ADC)中抗TROP-2抗体包含重链可变区和轻链可变区，其中所述重链可变区包含与如SEQ ID NO：29序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：30序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3。
在一些实施方案中，所述抗体-药物偶联物(ADC)中抗TROP-2抗体包含重链可变区和轻链可变区，其中：所述重链可变区氨基酸序列如SEQ ID NO:29所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO:30所示或与其有至少90％同一性。
在另一些实施方案中，所述抗体-药物偶联物(ADC)中抗TROP-2抗体包含序列如SEQ ID NO:29所示的重链可变区和序列如SEQ ID NO：30所示的轻链可变区。
在一些实施方案中，所述抗体-药物偶联物(ADC)中抗TROP-2抗体包含抗体重链恒定区和轻链恒定区；优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；更优选地，所述抗体包含序列如SEQ ID NO:31所示的重链恒定区和序列如SEQ ID NO:32所示的轻链恒定区。
在一些实施方案中，所述抗体-药物偶联物(ADC)中抗TROP-2抗体包含序列如SEQ ID NO:33所示的重链和序列如SEQ ID NO：34所示的轻链。
TROP-2氨基酸序列(Genbank:NP_002344.2)如下：
Figure PCTCN2021073314-appb-000073
TROP-2-His氨基酸序列：
Figure PCTCN2021073314-appb-000074
抗TROP-2抗体PD3重链可变区：
Figure PCTCN2021073314-appb-000075
抗TROP-2抗体PD3轻链可变区：
Figure PCTCN2021073314-appb-000076
抗体 抗TROP-2抗体PD3
重链CDR1 NYGMN(SEQ ID NO：23)
重链CDR2 WINTYTGEPTYTQDFKG(SEQ ID NO：24)
重链CDR3 GGFGSSYWYFDV(SEQ ID NO:25)
轻链CDR1 KASQDVSIAVA(SEQ ID NO:26)
轻链CDR2 SASYRYT(SEQ ID NO:27)
轻链CDR3 QQHYITPLT(SEQ ID NO:28)
抗体(抗TROP-2抗体)的重链恒定区可选自人IgG1、IgG2、IgG4及其变体的恒定区，轻链恒定区可选自人源κ、λ链或其变体的轻链恒定区。示例性的，抗体重链恒定区选自序列如SEQ ID NO：31所示的人IgG1，轻链恒定区选自序列如SEQ ID NO：32所示的人κ链的恒定区。
人IgG1的重链恒定区：
Figure PCTCN2021073314-appb-000077
人源κ轻链恒定区：
Figure PCTCN2021073314-appb-000078
示例性地，将上述轻链/重链恒定区与前述PD3抗体的可变区组合形成完整的抗体，其轻链/重链序列如下：
抗TROP-2抗体(PD3)重链：
Figure PCTCN2021073314-appb-000079
抗TROP-2抗体(PD3)轻链：
Figure PCTCN2021073314-appb-000080
进一步地，本公开所述抗体-药物偶联物(ADC)选自：
Figure PCTCN2021073314-appb-000081
Figure PCTCN2021073314-appb-000082
Figure PCTCN2021073314-appb-000083
Figure PCTCN2021073314-appb-000084
Figure PCTCN2021073314-appb-000085
Figure PCTCN2021073314-appb-000086
Figure PCTCN2021073314-appb-000087
Figure PCTCN2021073314-appb-000088
Figure PCTCN2021073314-appb-000089
Figure PCTCN2021073314-appb-000090
其中k选自1至10，可以为整数，也可以为小数；进一步地，D中R 1a选自甲基，R 1b选自氢。
本公开还提供了式D所示化合物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，
Figure PCTCN2021073314-appb-000091
其中，
R 1a选自氢、烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选甲基；R 1b选自氢、烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个 取代基所取代，优选氢、甲基；或者R 1a与R 1b与其相连接的原子一起形成5至8元杂环烷基，所述杂环烷基任选被烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，且R 1a和R 1b不同时为氢。
在一些实施方案中，式D所示化合物中R 1a与R 1b各自独立选自C 1-6烷基，包括但不限于甲基、乙基、异丙基。在一些实施方案中，式D所示化合物中R 1a选自C 1-6烷基，包括但不限于甲基、乙基、异丙基；R 1b选自氢。
在一些实施方案中，式D所示化合物中R 1a与R 1b与其相连接的原子一起形成5至8元杂环烷基。
在一些实施方案中，式D所示化合物为：
Figure PCTCN2021073314-appb-000092
在一些实施方案中，式D所示化合物为：
Figure PCTCN2021073314-appb-000093
在一些实施方案中，式D所示化合物为：
Figure PCTCN2021073314-appb-000094
本公开还提供式DZ所示化合物，或其互变异构体、内消旋体、外消旋体、对 映异构体、非对映异构体、或其混合物形式，或其可药用的盐，
Figure PCTCN2021073314-appb-000095
其中，R 1a选自氢、烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选甲基；
R 1b选自氢、烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选氢；
或者R 1a与R 1b与其相连接的原子一起形成5-8元杂环烷基，所述杂环烷基任选被烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代；且R 1a和R 1b不同时为氢；
Y选自-O(CR aR b) m2-CR 8R 9-C(O)-、-NH-(CR aR b) m2-CR 8R 9-C(O)-、-O-CR 8R 9(CR aR b) m2-、-OCR 8R 9-C(O)-、-O(CR aR b) m2C(O)-或-S-(CR aR b) m2-CR 8R 9-C(O)-，其中R a和R b相同或不同，且各自独立地选自氢、氘原子、卤素或烷基；
R 8选自氢、C 3-6环烷基烷基或C 3-6环烷基；
R 9选自氢、卤代烷基或C 3-6环烷基，优选氢；
或者R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基；
m2选自0、1、2或3。
在一些实施方案中，式DZ所示化合物中R 1a与R 1b各自独立选自C 1-6烷基，包括但不限于甲基、乙基、异丙基。
在一些实施方案中，式DZ所示化合物中R 1a选自C 1-6烷基，包括但不限于甲基、乙基、异丙基；R 1b选自氢。
在一些实施方案中，式DZ所示化合物中R 1a与R 1b与其相连接的碳原子一起形成5至8元杂环烷基。
在一些实施方案中，式DZ所示的化合物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其为式DZ-1所示的化合物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐：
Figure PCTCN2021073314-appb-000096
其中，R 8选自氢、C 3-6环烷基烷基或C 3-6环烷基；R 9选自氢、卤代烷基或C 3-6环烷基，优选氢；或者，R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基；m2选自0、1、2或3。
在一些实施方案中，DZ所示化合物选自：
Figure PCTCN2021073314-appb-000097
Figure PCTCN2021073314-appb-000098
另一方面，一些实施方案提供的DZ所示化合物可以含有一个或多个不对称中心，如
Figure PCTCN2021073314-appb-000099
可以为
Figure PCTCN2021073314-appb-000100
另一方面，本公开还提供一种药物组合物，其含有治疗有效量的前述抗体-药物偶联物，以及药学上可接受的药用载体、稀释剂或赋形剂。
本公开还提供一种前述抗体-药物偶联物或前述药物组合物在制备用于治疗或预防肿瘤的药物中的用途。在一些实施方案中，所述的肿瘤为与HER2、HER3、B7H3或EGFR表达相关的癌症。
本公开还提供一种前述抗体-药物偶联物或前述药物组合物在制备治疗和/或预防癌症的药物中的用途。在一些实施方案中，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、结肠癌、直肠癌、结直肠癌、白血病、骨癌、皮肤癌、甲状腺癌、胰腺癌和淋巴瘤。
本公开还提供一种治疗或预防肿瘤的方法，其包括向有需要的患者施用治疗有效量的前述抗体-药物偶联物或前述药物组合物；其中所述肿瘤优选为与HER2、HER3、B7H3或EGFR表达相关的癌症。
本公开还提供一种治疗和和/或预防癌症的方法，其包括向有需要的患者施用治疗有效量的前述抗体-药物偶联物或前述药物组合物；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、结肠癌、直肠癌、结直肠癌、白血病、骨癌、皮肤癌、甲状腺癌、胰腺癌和淋巴瘤。
本公开另外提供一种前述抗体-药物偶联物或前述药物组合物，其用于治疗或预防肿瘤；其中所述肿瘤优选为与HER2、HER3、B7H3或EGFR表达相关的癌症。
本公开另外提供一种前述抗体-药物偶联物或前述药物组合物，其用于治疗和和/或预防癌症；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、结肠癌、直肠癌、结直肠癌、白血病、骨癌、皮肤癌、甲状腺癌、胰腺癌和淋巴瘤。
可将活性化合物制成适合于通过任何适当途径给药的形式，活性化合物优选是以单位剂量的方式，或者是以患者可以以单剂自我给药的方式。本公开化合物或组合物的单位剂量的表现方式可以是片剂、胶囊、扁囊剂、瓶装药水、药粉、颗粒剂、锭剂、栓剂、再生药粉或液体制剂。
本公开治疗方法中所用化合物或组合物的剂量通常将随疾病的严重性、患者的体重和化合物的相对功效而改变。不过，作为一般性指导，合适的单位剂量可以是0.1～1000mg。
本公开的药物组合物除活性化合物外，可含有一种或多种辅料，所述辅料选自以下成分：填充剂(稀释剂)、粘合剂、润湿剂、崩解剂或赋形剂等。根据给药方法的不同，组合物可含有0.1至99重量％的活性化合物。
发明的详细说明
除非另有限定，本公开所用的所有技术和科学术语均与本公开所属领域普通技术人员的通常理解一致。虽然也可采用与本公开所述相似或等同的任何方法和材料实施或测试本公开，但本公开描述了优选的方法和材料。描述和要求保护本公开时，依据以下定义使用下列术语。
当本公开中使用商品名时，申请人旨在包括该商品名产品的制剂、该商品名产品的非专利药和活性药物部分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“药物”是指细胞毒性药物或免疫调节剂。细胞毒性药物能在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。该术语包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素(例如At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32和Lu的放射性同位素)，毒性药物，化疗药物，抗生素和核溶酶。免疫调节剂是免疫关卡分子的抑制剂。本公开的一些实施方案中，药物表示为D，属于免疫调节剂，特别是TLR8激动剂。
术语“连接子”、“连接单元”、“接头单元”、“接头”或“连接片段”是指一端与配体连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与药物相连。
接头可以包含一种或多种接头构件。例示性的接头构件包括6-马来酰亚氨基己酰基(MC)、马来酰亚氨基丙酰基(MP)、缬氨酸-瓜氨酸(Val-Cit或vc)、丙氨酸-苯丙氨酸(ala-phe)、对氨基苄氧羰基(PAB)，及那些源自与接头试剂的偶联的：N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1羧酸酯(SMCC，在本文中也称作MCC)和N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)。接头可以包括拉伸单元、间隔单元、氨基酸单元和延伸单元。可以通过本领域已知方法合成，诸如US2005-0238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等,Cancer Research 52:127-131(1992)；美国专利No.5,208,020)。
术语“拉伸单元”指一端通过碳原子与抗体共价连接而另一端与氨基酸单元、二硫化物部分、磺酰胺部分或非肽化学部分相连的化学结构片段。
术语“间隔单元”是一种双功能化合结构片段，可用于偶联氨基酸单元和细胞毒性药物最终形成抗体-药物偶联物，这种偶联方式可以将细胞毒性药物选择性的连接到氨基酸单元上。
术语“氨基酸”是指分子结构中含有氨基和羧基，并且氨基和羧基都直接连接在-CH-结构上的有机化合物。通式是H 2NCHRCOOH，R为H、取代或未取代烷基等。根据氨基连结在羧酸中碳原子的位置，可分为α、β、γ、δ、ε……-氨基酸。在生物界中，构成天然蛋白质的氨基酸具有其特定的结构特点，即其氨基直接连接在α-碳原子上，即α-氨基酸，包括甘氨酸(Glycine)、丙氨酸(Alanine)、缬氨酸(Valine)、亮氨酸(Leucine)、异亮氨酸(Isoleucine)、苯丙氨酸(Phenylalanine)、色氨酸(Tryptophan)、酪氨酸(Tyrosine)、天冬氨酸(Aspartic acid)、组氨酸(Histidine)、天冬酰胺(Asparagine)、谷氨酸(Glutamic acid)、赖氨酸(Lysine)、谷氨酰胺(Glutamine)、甲硫氨酸(Methionine)、精氨酸(Arginine)、丝氨酸(Serine)、苏氨酸(Threonine)、半胱氨酸(Cysteine)、脯氨酸(Proline)等。非天然氨基酸如瓜氨酸。如本领域技术人员所公知的，非天然氨基酸并不构成天然蛋白质，因此也不参与本公开中抗体的合成。本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
Figure PCTCN2021073314-appb-000101
Figure PCTCN2021073314-appb-000102
Figure PCTCN2021073314-appb-000103
本公开中间隔单元为PAB，结构如对氨基苯甲氧羰基片段，其结构如式(VI)所示，连接在D上,
Figure PCTCN2021073314-appb-000104
接头组件包括但不限于：
MC＝6-马来酰亚氨基己酰基，结构如下：
Figure PCTCN2021073314-appb-000105
Val-Cit或“vc”＝缬氨酸-瓜氨酸(蛋白酶可切割接头中的例示二肽)；
瓜氨酸＝2-氨基-5-脲基戊酸；
Me-Val-Cit＝N-甲基-缬氨酸-瓜氨酸(其中接头肽键已经修饰以防止其受到组织蛋白酶B的切割)；
MC(PEG) 6-OH＝马来酰亚氨基己酰基-聚乙二醇(可附着于抗体半胱氨酸)；
SPP＝N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯；
SPDP＝N-琥珀酰亚氨基3-(2-吡啶基二硫代)丙酸酯；
SMCC＝琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯；
IT＝亚氨基硫烷；
PBS＝磷酸缓冲盐溶液。
术语“抗体-药物偶联物”，指配体通过稳定的连接单元与具有生物活性的药物相连。在本公开中“抗体-药物偶联物”(antibody drug conjugate，ADC)，指把单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的毒性药物相连。
术语“载药量”可以表示为药物量和抗体量的比值。载药量的范围可以是每个抗体(Ab)连接1-20个，优选1-10个细胞毒性药物(D)。在本公开的实施方式中，载药量表示为k，示例性的可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或任意两数值之间数值的均值。优选1-10，更优选1-8，或2-8，或2-7，或3-8，或3-7，或3-6，或4-7，或4-6，或4-5的均值。可用常规方法如UV/可见光光谱法、质谱、ELISA试验、单抗分子大小变异体测定法(CE-SDS)和HPLC特征鉴定偶联反应后每个ADC分子的药物平均数 量。
本公开单抗分子大小变异体测定法(CE-SDS)可采用十二烷基硫酸钠毛细管电泳(CE-SDS)紫外检测方法，在还原和非还原条件下，依据分子量大小，按毛电泳法(2015年版《中国药典》0542)，定量测定重组单克隆抗体产品的纯度。
本公开的一个实施方式中，细胞毒性药物通过连接单元偶联在配体的N端氨基和/或赖氨酸残基的ε-氨基上，一般地，偶联反应中能与抗体偶联的药物分子数将小于理论上的最大值。
可以用以下非限制性方法控制抗体-药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。本公开所述的抗体优选为针对靶细胞上细胞表面抗原的特异性抗体，非限制性实施例为以下抗体：抗HER2(ErbB2)抗体、抗EGFR抗体、抗B7-H3抗体、抗c-Met抗体、抗HER3(ErbB3)抗体、抗HER4(ErbB4)抗体、抗CD20抗体、抗CD22抗体、抗CD30抗体、抗CD33抗体、抗CD44抗体、抗CD56抗体、抗CD70抗体、抗CD73抗体、抗CD105抗体、抗CEA抗体、抗A33抗体、抗Cripto抗体、抗EphA2抗体、抗G250抗体、抗MUCl抗体、抗Lewis Y抗体、抗VEGFR抗体、抗GPNMB抗体、抗Integrin抗体、抗PSMA抗体、抗Tenascin-C抗体、抗SLC44A4抗体、抗CD79抗体、抗TROP-2抗体、抗CD79B抗体、抗Mesothelin抗体或其抗原结合片段。在一些实施方案中，抗体选自曲妥珠单抗(Trastuzumab，商品名Herceptin)、帕妥珠单抗(Pertuzumab，也被称作2C4，商品名Perjeta)、尼妥珠单抗(Nimotuzumab，商品名泰欣生)、恩波妥珠单抗(Enoblituzumab)、依玛妥珠单抗(Emibetuzumab)、奥英妥珠单抗(Inotuzumab)、维汀-匹那妥珠单抗(Pinatuzumab)、维布妥昔单抗(Brentuximab)、吉妥单抗(Gemtuzumab)、比伐珠单抗(Bivatuzumab)、莫洛伐妥单抗(Lorvotuzumab)、cBR96和Glematumamab。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR) 由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体和全人源抗体，优选人源化抗体和全人源抗体。
术语“鼠源抗体”在本公开中为根据本领域知识和技能用鼠制备抗体。制备时用特定抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网 www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。进一步描述参与人源化可使用小鼠抗体的方法的文献包括，例如Queen等，Proc.，Natl.Acad.Sci.USA，88，2869，1991和Winter及其同事的方法[Jones等，Nature，321，522(1986)，Riechmann，等，Nature，332，323-327(1988)，Verhoeyen，等，Science，239，1534(1988)]。
术语“全人源抗体”、“全人抗体”或“完全人源抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。本公开为全人源单克隆抗体。全人抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。
术语“抗原结合片段”是指抗体的保持特异性结合抗原的能力的一个或多个 片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM抗体。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和 Roovers等人(2001)，Cancer Immunol.描述。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的CDR1、CDR2和CDR3(CDR L1、CDR L2、CDR L3或L1、L2、L3)，以及重链可变结构域的CDR2和CDR3(CDR H2、CDR H3或H2、H3)。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(参见Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003))等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。
术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位。表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸(参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996))。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如：大约小于10 -8M、10 -9M或10 -10M或更小的亲和力(KD)结合。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链 的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
氨基酸序列“同一性”指在比对氨基酸序列及必要时引入间隙，以达成最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，第一序列中与第二序列中的氨基酸残基同一的氨基酸残基的百分比。为测定氨基酸序列同 一性百分比的目的，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
术语“肽”是指介于氨基酸和蛋白质之间的化合物片段，由2个或2个以上氨基酸分子通过肽键相互连接而成，是蛋白质的结构与功能片段，如激素、酶类等本质上都是肽。
术语“糖”是指由C、H、O三种元素组成的生物大分子，可分为单糖、二糖和多糖等。
术语“荧光探针”是指在紫外-可见-近红外区有特征荧光，并且其荧光性质(激发和发射波长、强度、寿命和偏振等)可随所处环境的性质，如极性、折射率、粘度等改变而灵敏地改变的一类荧光性分子，其与核酸(DNA或RNA)、蛋白质或其他大分子结构非共价相互作用而使一种或几种荧光性质发生改变，可用于研究大分子物质的性质和行为。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、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、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、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、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷 硫基、杂环烷硫基、氧代基。
术语“杂烷基”指含有一个或多个选自N、O或S的杂原子的烷基，其中烷基如上所定义。
“一价基团”是指一个化合物从“形式上”消除一个单价的原子或基团。“亚基”则是指化合物从“形式上”消除两个单价或一个双价形成的原子或原子团。示例“烷基”是指由烷烃分子中去除1个氢原子后余下的部分，包括1至20个碳原子的直链和支链一价基团。含有1至6个碳原子的烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基及其各种支链异构体等。
术语“亚烷基”指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子，更优选含有1至6个碳原子的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH 2-)、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)和1,5-亚丁基(-CH 2CH 2CH 2CH 2CH 2-)等。亚烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基中的一个或多个。同理，“亚烯基”如前所定义。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基或环烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“杂环烷基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选环烷基环包含3至10个环原子。单环杂环烷基的非限制性实例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、 硫代吗啉基、高哌嗪基等。多环杂环烷基包括螺环、稠环和桥环的杂环烷基。
术语“螺杂环烷基”指5至20元的单环之间共用一个原子(称螺原子)的多环杂环烷基团，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺杂环烷基分为单螺杂环烷基、双螺杂环烷基或多螺杂环烷基，优选为单螺杂环烷基和双螺杂环烷基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环烷基。螺杂环烷基的非限制性实例包括：
Figure PCTCN2021073314-appb-000106
术语“稠杂环烷基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对原子的多环杂环烷基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环稠杂环烷基，优选为双环或三环，更优选为5元/5元或5元/6元双环稠杂环烷基。稠杂环烷基的非限制性实例包括：
Figure PCTCN2021073314-appb-000107
术语“桥杂环烷基”指5至14元，任意两个环共用两个不直接连接的原子的多环杂环烷基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环桥杂环烷基，优选为双环、三环或四环，更优选为双环或三环。桥杂环烷基的非限制性实例包括：
Figure PCTCN2021073314-appb-000108
所述杂环烷基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环烷基，其非限制性实例包括：
Figure PCTCN2021073314-appb-000109
等。
杂环烷基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至10元，例如苯基和萘基，优选苯基。所述芳基环可以稠合于杂芳基、杂环烷基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性实例包括：
Figure PCTCN2021073314-appb-000110
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环烷基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性实例包括：
Figure PCTCN2021073314-appb-000111
Figure PCTCN2021073314-appb-000112
杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“氨基保护基”是为了使分子其它部位进行反应时氨基保持不变，用易于脱去的基团对氨基进行保护。非限制性实施例包含9-芴甲氧羰基、叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基等。这些基团可任选地被选自卤素、烷氧基或硝基中的1-3个取代基所取代。所述氨基保护基优选为9-芴甲氧羰基。
术语“氨基杂环烷基”指杂环烷基被一个或多个氨基取代，优选被一个氨基取代，其中杂环烷基如上所定义，其中“氨基”指-NH 2。本公开的代表性实施例如下：
Figure PCTCN2021073314-appb-000113
术语“杂环烷基氨基”指氨基被一个或多个杂环烷基取代，优选被一个杂环烷基取代，其中氨基如上所定义，其中杂环烷基如上所定义。本公开的代表性实施例如下：
Figure PCTCN2021073314-appb-000114
术语“环烷基氨基”指氨基被一个或多个环烷基取代，优选被一个环烷基取代，其中氨基如上所定义，其中环烷基如上所定义。本公开的代表性实施例如下：
Figure PCTCN2021073314-appb-000115
术语“环烷基烷基”指烷基被一个或多个环烷基取代，优选被一个环烷基取代，其中烷基如上所定义，其中环烷基如上所定义。
术语“卤代烷基”指烷基被一个或多个卤素取代，其中烷基如上所定义。
术语“氘代烷基”指烷基被一个或多个氘原子取代，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH 2。
术语“硝基”指-NO 2。
化学式中简称“Me”为甲基。
本公开还包括各种氘化形式的式(I)化合物。与碳原子连接的各个可用的氢原子可独立地被氘原子替换。本领域技术人员能够参考相关文献合成氘化形式的式(I)化合物。在制备氘代形式的式(I)化合物时可使用市售的氘代起始物质，或它们可使用常规技术采用氘代试剂合成，氘代试剂包括但不限于氘代硼烷、三氘代硼烷四氢呋喃溶液、氘代氢化锂铝、氘代碘乙烷和氘代碘甲烷等。
另一方面，本公开所述化合物的官能团中氢被氘代，获得相应氘代化合物，氘代化合物保留了与氢衍生物相当的选择性和潜力；氘键更稳定，使得“ADME”即“毒药物动力学”不同，从而提供临床上有益效果。
毒药物动力学，指机体对外源化学物的吸收(absorption)、分布(distribution)、代谢(metabolism)及排泄(excretion)过程。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环烷基团”意味着烷基可以但不必须存在，该说明包括杂环烷基团被烷基取代的情形和杂环烷基团不被烷基取代的情形。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
术语“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的盐”或“可药用盐”是指本公开抗体-药物偶联物的盐，或本公开中所述的化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性。本公开抗体-药物偶联物至少含有一个氨基，因此可以与酸形成盐，可药用盐的非限制性实例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
术语“溶剂合物”指本公开的配体-药物偶联化合物与一种或多种溶剂分子形成可药用的溶剂合物，溶剂分子的非限制性实例包括水、乙醇、乙腈、异丙醇、DMSO、乙酸乙酯。
术语“药物载体”用于本公开的药物，是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的实例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
术语“赋形剂”是在药物制剂中除主药以外的附加物，也可称为辅料。如片剂中的粘合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。
本公开中化合物可以含有一个或多个不对称中心，因此可以产生对映异构体、非对映异构体，并且其可以根据绝对立体化学定义为(R)-或(S)-或用于氨基酸的(D)-或(L)-的其它立体异构形式。本公开包括所有可能异构体以及其外消旋和光学纯的形式。光学活性的(+)和(-)、(R)-和(S)-或(D)-和(L)-异构体可以使用手性合成子或手性试剂制备，或者可以使用常规方法例如色谱法和分级结晶制备。用于制备/分离各个对映体的常规方法包括从合适的的光学纯前体手性合成或使用例如手性高效液相色谱法(HPLC)的外消旋物(或盐或衍生物的外消旋物)拆分。当本文描述的化合物含有烯双键或其它几何不对称性中心，除非另有说明，否则其意味着所述化合物包括E和Z几何异构体。而且，所有的互变异构形式也意味着包括在内。
本公开所述化合物的化学结构中，键
Figure PCTCN2021073314-appb-000116
表示未指定构型，即如果化学结构中存在手性异构体，键
Figure PCTCN2021073314-appb-000117
可以为
Figure PCTCN2021073314-appb-000118
或
Figure PCTCN2021073314-appb-000119
或者同时包含
Figure PCTCN2021073314-appb-000120
和
Figure PCTCN2021073314-appb-000121
两种构型。
“立体异构体”指通过相同的键键合但具有不同的三维结构的相同原子组成的化合物，其不可互换。本公开中预期各种立体异构体及其混合物，并且包括“对映异构体”，其指其分子彼此为不能重叠的镜像的两种立体异构体。
“互变异构体”指质子从分子的一个原子转移到同一分子的另一个原子。本公开中包括任何所述化合物的互变异构体。
附图说明
图1：测试例7中空白溶媒组与不同测试给药组的动物体重变化趋势图(横坐标：天数，纵坐标：体重)。
图2：溶媒与不同测试药给药组的动物肿瘤体积变化趋势图(横坐标：天数，纵坐标：肿瘤体积)。
具体实施方式
以下结合实施例进一步描述本公开，但这些实施例并非限制本公开的范围。
本公开实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR位移(δ)以10 -6(ppm)的单位给出。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d 6)、氘代氯仿(CDCl 3)、氘代甲醇(CD 3OD)，内标为四甲基硅烷(TMS)。
MS的测定用Agilent 1200/1290 DAD-6110/6120 Quadrupole MS液质联用仪(生产商：Agilent，MS型号：6110/6120 Quadrupole MS)，waters ACQuity UPLC-QD/SQD(生产商：waters，MS型号：waters ACQuity Qda Detector/waters SQ Detector)，THERMO Ultimate 3000-Q Exactive(生产商：THERMO，MS型号：THERMO Q Exactive)。
高效液相色谱法(HPLC)分析使用Agilent HPLC 1200DAD、Agilent HPLC 1200VWD或Waters HPLC e2695-2489高压液相色谱仪。
手性HPLC分析测定使用Agilent 1260 DAD高效液相色谱仪。
高效液相制备使用Waters 2545-2767、Waters 2767-SQ Detecor2、Shimadzu LC-20AP和Gilson GX-281制备型色谱仪。
手性制备使用Shimadzu LC-20AP制备型色谱仪。
CombiFlash快速制备仪使用Combiflash Rf200(TELEDYNE ISCO)。
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm～0.2mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm。
硅胶柱色谱法一般使用烟台黄海硅胶200～300目硅胶为载体。
本公开的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCR GmbH&Co.KG、Acros Organics、Aldrich Chemical Company、韶远化学科技(Accela ChemBio Inc)、达瑞化学品等公司。
实施例中无特殊说明，反应能够均在氩气氛或氮气氛下进行。
氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
氢气氛是指反应瓶连接一个约1L容积的氢气气球。
加压氢化反应使用Parr 3916EKX型氢化仪和清蓝QL-500型氢气发生器或 HC2-SS型氢化仪。
氢化反应通常抽真空，充入氢气，反复操作3次。
微波反应使用CEM Discover-S 908860型微波反应器。
实施例中无特殊说明，溶液是指水溶液。
实施例中无特殊说明，反应的温度为室温，为20℃～30℃。
实施例中的反应进程的监测采用薄层色谱法(TLC)，反应所使用的展开剂，纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂体系，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和醋酸等碱性或酸性试剂进行调节。
本公开抗体药物偶联物参见WO2020063676A相关的化合物合成及测试全文引用至本公开。其中的非限制性实施例合成如下：
一、抗体的制备
实施例1-1.蛋白抗原的克隆表达
抗体(包含轻、重链)和抗原用本领域公知的重叠延伸PCR方法构建，将重叠延伸PCR得到的DNA片段用HindIII/BstBI这两个酶切位点插入到表达载体pEE6.4(Lonza Biologics)中，在293F细胞(Invitrogen，Cat#R790-07)中表达得到。所得重组蛋白用于免疫或筛选。人CD79B基因序列来源于NCBI(NP_000617.1)，其细胞外区域(ECD)包含159个氨基酸(Met1-Asp159)。
人CD79B细胞外区域(ECD)和人Fc区域融合蛋白(human CD79B ECD-hFc)的氨基酸序列如SEQ ID NO:1所示：
Figure PCTCN2021073314-appb-000122
人CD79B细胞外区域(ECD)和His标签的融合蛋白(human CD79B ECD-His)的氨基酸序列如SEQ ID NO:2所示：
Figure PCTCN2021073314-appb-000123
实施例1-2.小鼠单克隆抗体的制备
1、小鼠免疫和血清效价检测
以人CD79B细胞外区域(ECD)和人Fc区域融合蛋白(human CD79B ECD-hFc)以及人CD79B细胞外区域(ECD)和His标签的融合蛋白(human CD79B ECD-His)分别作为免疫原，通过腹腔注射法对Balb/c和SJL小鼠进行免疫，刺激小鼠体内产生针对人CD79B细胞外区域(ECD)的抗体。同时，以猴CD79B细胞外区域(ECD)和His标签的融合蛋白(cyno CD79B ECD-His)作为免疫原，通过腹腔注射法对SJL小鼠进行免疫，刺激小鼠体内产生针对猴CD79B细胞外区域(ECD)的抗体。
实验步骤如下：
1)腹腔注射免疫法：根据免疫程序计算出该次免疫所需的抗原量。蛋白抗原按照要求用PBS稀释抗原到相应浓度，随后进行抗原的乳化。将乳化好的抗原和佐剂混合物转移到2.0ml无菌注射器中，注意排空气泡。右手抓住小鼠尾巴，左手的大拇指和食指轻轻抓住小鼠的头颈部皮肤，腹腔向上，用75％酒精棉球擦拭小鼠右侧腹部注射部位。将事先吸好抗原药物的注射器针尖斜面向上，小鼠头部向下，平行刺入皮内后，注射器与腹腔呈45度角刺入小鼠腹腔，缓慢注入抗原和佐剂混合物，免疫完成后进行至少2h观察。
2)小鼠血清的收集：将每只小鼠对应的血清管号做好标记，检查小鼠耳钉号，单手抓取小鼠，通过小鼠面部颌下静脉采取大约100ul全血，将收集的全血样品室温静置大约2h后离心收集离心管上部血清。一周内可将血清存放于4℃冰箱，用于抗体效价等相关实验检测。若长期保存血清可置于-80℃冰箱，避免反复冻融。
3)免疫小鼠ELISA血清效价测定：实验开始前将96孔板做好相应的标记，以1ug/ml抗原浓度，每孔50ul，4℃冰箱过夜包被。次日，将前天包被好的抗原板取出，洗板机清洗一次(清洗液：1x PBST)。清洗后以1X PBST配置的1％BSA封闭液37℃封闭1小时。1x PBST清洗液洗板3次后，加入不同稀释浓度的待检血清37℃温箱，孵育1小时。1x PBST清洗液洗板3次后，加入100ul 1:5000稀释的羊抗鼠二抗，37℃温箱孵育0.5小时洗板后，取TMB显色液A液和B液1:1比例混合后显色。15分钟用1N盐酸终止显色反应。在Spectra Max M5多功能读板机上，检测450nm的荧光值。
4)免疫小鼠FACS血清效价测定：DoHH2细胞或猴外周血单核细胞悬液离心后以含0.1％BSA的PBS重悬细胞后计数，加入各组免疫小鼠的待测血清，室温孵育60分钟后清洗细胞三次后加入Anti-Mouse IgG(Fc specific)-FITC二抗，避光室温孵育30分钟后清洗细胞三次，用含0.1％BSA的PBS轻轻重悬细胞，上机检测。
通过以上试验使免疫的小鼠中产生了针对CD79B的特异性抗体，以上小鼠可用于细胞融合以产生能够分泌针对CD79B的特异性抗体的杂交瘤细胞系。
2、杂交瘤制备和抗体筛选
细胞融合是在自发或人工诱导下，促使小鼠的淋巴细胞与骨髓瘤细胞SP2/0 (ATCC，CCL-121 TM)融合成杂交瘤细胞，杂交瘤细胞既具有抗体分泌功能又能无限增殖。利用电融合方法对免疫组小鼠的淋巴细胞和骨髓瘤细胞进行融合，用于后续抗体筛选。
1)电融合实验：融合前一周将SP2/0细胞于10％DMEM培养基中进行扩大培养。将已处死的小鼠于生物安全柜中摘取脾脏和淋巴结于培养皿中进行冲洗研磨，收集淋巴细胞。将SP2/0和淋巴细胞按比例混合，启动电融合仪，设置程序，进行融合。融合后细胞铺于96孔板中，37℃，5％CO 2培养箱中培养，每天观察细胞状态，融合后5天统计细胞融合率。融合后9-14天对融合的杂交瘤细胞进行筛选，挑选阳性孔细胞于24孔板中扩大培养。
2)有限稀释法进行亚克隆：将需要亚克隆的细胞株自24孔培养孔中重悬起来，进行计数。稀释每株细胞株的细胞浓度至5-10个/ml，将稀释好的细胞悬液加入15cm一次性培养皿中，于96孔培养板中每孔加0.2ml，每孔含细胞为1-2个。将铺好细胞的96孔板放进37℃，5％CO 2培养箱中培养。7-10天后根据细胞的生长情况对亚克隆板进行检测筛选，并挑选阳性克隆至24孔进行进一步阳性确认。
3)ELISA筛选：实验开始前将96孔板做好相应的标记，以1ug/ml抗原浓度，每孔50ul，4℃冰箱过夜包被。次日，将前天包被好的抗原板取出，洗板机清洗一次(清洗液：1x PBST)。清洗后以1X PBST配置的1％BSA封闭液37℃封闭1小时。1x PBST清洗液洗板3次后，加入50ul待检细胞上清37℃温箱，孵育1小时。1x PBST清洗液洗板3次后，加入100ul 1:5000稀释的羊抗鼠二抗，37℃温箱孵育0.5小时洗板后，取TMB显色液A液和B液1：1比例混合后显色。15分钟用1N盐酸终止显色反应。在Spectra Max M5多功能读板机上，检测450nm的荧光值。
4)FACS筛选：DOHH2细胞悬液离心后以含0.1％BSA的PBS重悬细胞后计数，加入待测细胞上清，室温孵育60分钟后清洗细胞三次后加入Anti-Mouse IgG(Fc specific)-FITC二抗，避光室温孵育30分钟后清洗细胞三次，用含0.1％BSA的PBS轻轻重悬细胞，上机检测。
5)杂交瘤阳性克隆鉴定：通过对小鼠脾细胞的融合和亚克隆筛选后，我们获得多个针对人CD79B抗原的特异性抗体，对其中ELISA和FACS结合力最好的17株杂交瘤进行抗体生产纯化。抗人CD79B杂交瘤阳性克隆细胞培养上清ELISA检测结果见表1。抗人CD79B杂交瘤阳性克隆细胞培养上清FACS检测结果见表2。同时也获得了针对猴CD79B抗原的特异性抗体，对其中ELISA和FACS结合力最好的4株杂交瘤进行抗体生产纯化。均使用mIgG作为阴性对照。
表1.抗人CD79B杂交瘤阳性克隆ELISA检测结果
抗体编号 克隆号 检测结果(OD450)
阴性对照 mIgG 0.05
mAb015 83B2G2 3.41
mAb017 86F11F6 3.80
表2.抗人CD79B杂交瘤阳性克隆FACS检测结果
抗体编号 克隆号 平均荧光值
阴性对照 mIgG 58
mAb015 83B2G2 10036
mAb017 86F11F6 8132
3、小鼠单克隆抗体生产纯化及鉴定
1)小鼠单克隆抗体生产和纯化：显微镜下观察需要抗体生产的杂交瘤细胞，长至≥70％以上且细胞状态良好，收集细胞并用Countstar IC1000型细胞计数仪进行计数。用配制好的培养基将细胞浓度调至1～5×10 5个/ml，转移至Roller Bottle。将转好细胞的Roller Bottle送到滚瓶培养箱中37℃培养10-15天，每天观察细胞生长状况，待培养液变橙黄且透明，取出待纯化。细胞上清通过Protein A柱子进行抗体纯化，按照常规方法操作。
2)抗人CD79B小鼠单克隆抗体ELISA检测：实验开始前将96孔板做好相应的标记，以1ug/ml抗原浓度，每孔50ul，4℃冰箱过夜包被。次日，将前天包被好的抗原板取出，洗板机清洗一次(清洗液：1x PBST)。清洗后以1X PBST配置的1％BSA封闭液37℃封闭1小时。1x PBST清洗液洗板3次后，加入50ul按照100nM，1:10稀释过的抗体，放入37℃温箱，孵育1小时。1x PBST清洗液洗板3次后，加入100ul 1:5000稀释的羊抗鼠二抗，37℃温箱孵育0.5小时。洗板后，取TMB显色液A液和B液1：1比例混合后显色。15分钟用1N盐酸终止显色反应。在Spectra Max M5多功能读板机上，检测450nm的荧光值。其中有4个抗人CD79B小鼠单克隆抗体的ELISA结合力最强，包括mAb015和mAb017。
3)抗人CD79B小鼠单克隆抗体FACS检测：DOHH2细胞悬液离心后以含0.1％BSA的PBS重悬细胞后计数，加入100ul按照100nM，1:10稀释过的抗体，室温孵育1小时。清洗细胞三次后加入Anti-Mouse IgG(Fc specific)-FITC二抗，避光室温孵育30分钟后清洗细胞三次，用含0.1％BSA的PBS轻轻重悬细胞，上机检测。其中有4个抗人CD79B小鼠单克隆抗体的FACS结合力最强，包括mAb015和mAb017。
4)抗人CD79B小鼠单克隆抗体SPR检测：采用表面等离子共振技术(surface plasmon resonance，SPR)检测抗人CD79B抗体与其抗原人CD79B-His之间的亲和力。将抗原人CD79B-His蛋白固定化至CM5芯片。偶联水平设定在100RU。运行缓冲液为HBS-EP+(10mM HEPES，150mM NaCl，3mM EDTA，0.05％surfactant P20)。将稀释好的抗体在30μl/min的流速下流过实验通道和对比通道3分钟，解离5分钟。然后再生缓冲液(10mM Glycine，pH1.5)在30μl/min的流速下运行30秒。数据用Biacore 8K evaluation software软件进行分析。
实施例1-3.小鼠单克隆抗体可变区氨基酸序列测定
将实施例1-2中得到的高亲和力的杂交瘤单克隆细胞株进行可变区氨基酸序列测定，然后重组表达出人鼠嵌合抗体(chimeric antibody，cAb)，再进行进一步的抗体鉴定。用逆转录PCR扩增抗体基因的重链和轻链可变区，连接到载体测序得到单克隆抗体轻重链序列。首先采用RNA纯化试剂盒(Qiagen公司，货号74134，步骤见此说明书)提取实施例2中活性好的单细胞株的细胞总RNA。然后使用Invitrogen公司的货号为18080-051 cDNA合成试剂盒制备cDNA单链，即Oligo-dT primers cDNA反转录。以此为模版，采用PCR方法合成抗体轻重链可变区序列，PCR产物克隆到TA载体pMD-18T，然后送去测序。将得到的抗体轻重链序列分别克隆到表达载体(方法见实施例1-1)，表达重组单克隆抗体，验证活性(方法见实施例1-2)后，进行人源化工作。
抗人CD79B抗体的VH/VL CDR的氨基酸残基由Chothia编号系统确定并注释。
小鼠杂交瘤细胞单克隆抗体mAb015的序列：
重链可变区：
Figure PCTCN2021073314-appb-000124
轻链可变区：
Figure PCTCN2021073314-appb-000125
小鼠杂交瘤细胞单克隆抗体mAb017的序列：
重链可变区：
Figure PCTCN2021073314-appb-000126
轻链可变区：
Figure PCTCN2021073314-appb-000127
鼠源的CDR序列如表3所示：
表3.鼠源抗人CD79B抗体的CDR序列
Figure PCTCN2021073314-appb-000128
实施例1-4.抗人CD79B抗体人源化
将实施例1-3得到的鼠源抗CD79B单克隆抗体轻重链序列在抗体数据库里进行同源性比较后，建立人源化抗体模型，根据模型选择回复突变筛选最优的人源化抗CD79B单克隆抗体为优选分子。该方法从已经发表的小鼠Fab晶体结构模型数据库(比如PDB数据库)中查找与所得鼠源候选分子同源性相似的晶体结构开始，挑取高分辨率(比如
Figure PCTCN2021073314-appb-000129
)的Fab晶体结构，建立小鼠Fab模型。将鼠源抗体轻重链序列和模型中的序列比对，保留和模型中和鼠源抗体序列一致的序列，得到鼠抗体的结构模型，其中不一致氨基酸为可能的回复突变位点。用Swiss-pdb viewer软件运行鼠抗体结构模型，优化能量(最小化)。将模型中除CDR外的不同氨基酸位点进行回复突变，将所得的突变抗体(人源化)和人源化之前抗体对比进行活性检测。保留活性好的人源化抗体。之后，对CDR区域优化，包括避免糖基化、脱酰胺化、氧化位点等。用基因克隆、重组表达的方法分别克隆、表达、纯化上述抗体，经ELISA、FACS和SPR等检测，最终选出活性保持最好的人源化抗体hAb015-10和hAb017-10。
人源化抗体hAb015-10和hAb017-10的序列见下。
hAb015-10人源化抗体重链：
Figure PCTCN2021073314-appb-000130
hAb015-10人源化抗体轻链：
Figure PCTCN2021073314-appb-000131
hAb017-10人源化抗体重链：
Figure PCTCN2021073314-appb-000132
hAb017-10人源化抗体轻链：
Figure PCTCN2021073314-appb-000133
实施例1-5：TROP-2高表达细胞株构建
将pCDH-hTROP-2慢病毒表达载体质粒与pVSV-G，pCMV-dR8.91慢病毒系统包装载体用Lipofectamine 3000转染试剂转染至病毒包装细胞293T中，收集含有病毒的培养基上清，过滤并进行超高速离心，使用浓缩后的病毒感染中国仓鼠卵巢细胞CHO-K1，经puromycin筛选两至三周，再进行FACS单细胞分选。
通过FACS检测慢病毒感染的CHO-K1细胞表面的TROP-2表达量，挑选出TROP-2表达量高的CHO-K1/hTROP-2单克隆细胞株。
TROP-2氨基酸序列(Genbank:NP_002344.2)如下：
Figure PCTCN2021073314-appb-000134
Figure PCTCN2021073314-appb-000135
TROP-2-His氨基酸序列：
Figure PCTCN2021073314-appb-000136
实施例1-6：抗人TROP-2单克隆抗体的制备
本公开中的抗人TROP-2单克隆抗体依照WO03074566专利中公开的方法制备，以hRS7的抗体可变区基因为模板利用计算机软件针对CDR进行点突变改造设计。通过分子克隆插入至蛋白表达载体Phr-IgG(带信号肽及恒定区基因(CH1-Fc/CL)片段))中，在HEK293和Expi-CHO-S细胞中进行表达。按照常规方法进行抗体纯化。利用过表达huTROP-2蛋白的CHO-K1细胞和huTROP-2蛋白(His27-Thr274Accession#NP_002344.2)进行活性验证，挑选出靶点结合活性较好的抗体，其中PD3可变区序列如下：
PD3重链可变区：
Figure PCTCN2021073314-appb-000137
PD3轻链可变区：
Figure PCTCN2021073314-appb-000138
注：下划线部分为依照Kabat编号规则确定的CDR区。
表4.PD3抗体的CDR区
抗体 PD3
重链CDR1 NYGMN(SEQ ID NO：23)
重链CDR2 WINTYTGEPTYTQDFKG(SEQ ID NO：24)
重链CDR3 GGFGSSYWYFDV(SEQ ID NO：25)
轻链CDR1 KASQDVSIAVA(SEQ ID NO：26)
轻链CDR2 SASYRYT(SEQ ID NO：27)
轻链CDR3 QQHYITPLT(SEQ ID NO：28)
抗体的重链恒定区可选自人IgG1、IgG2、IgG4及其变体的恒定区，轻链恒定区可选自人源κ、λ链或其变体的轻链恒定区。示例性的，抗体重链恒定区选自序列如SEQ ID NO：11所示的人IgG1，轻链恒定区选自序列如SEQ ID NO：12所示的人κ链的恒定区。
人IgG1的重链恒定区：
Figure PCTCN2021073314-appb-000139
人源κ轻链恒定区：
Figure PCTCN2021073314-appb-000140
示例性地，将上述轻链/重链恒定区与前述PD3抗体的可变区组合形成完整的抗体，其轻链/重链序列如下：
PD3重链：
Figure PCTCN2021073314-appb-000141
PD3轻链：
Figure PCTCN2021073314-appb-000142
二、化合物的制备
实施例2-1：化合物L-1的合成
Figure PCTCN2021073314-appb-000143
步骤1：化合物2的制备
冰水浴下，将化合物1(50mg，.08mmol，参照WO2017151979中方法制备而得)溶解于1.5mL的N,N-二甲基甲酰胺中，然后加入DIPEA(N,N-二异丙基乙胺，18mg,0.14mmol)，接着加入二(对硝基苯)碳酸酯(49mg，0.16mmol)。然后在室温搅拌，加入20mL甲基叔丁基醚，搅拌20分钟，过滤，干燥得到36mg固体化合物2。
LC/MS(ESI)：m/z 784.1[M+H] +。
步骤2：化合物D-1b的制备
冰水浴下，将化合物D-1a(艾日布林，参照ZL201010236637.2方法制备获得) (72.91mg，0.1mmol)溶于10mL的四氢呋喃中，加入Fmoc-OSu(芴甲氧羰酰琥珀酰亚胺，41mg，0.12mmol)，然后在室温搅拌至反应完全。减压浓缩得粗品，直接用于下一步反应。
步骤3：化合物D-1c的制备
将上步所得化合物D-1b的粗品溶于10mL无水乙醚中，加入氧化银(34.8mg，0.15mmol)，接着加入碘甲烷(28.4mg,0.2mmol)，然后于室温搅拌至反应完全，过滤，然后减压浓缩得粗品，直接进行下一步反应。
步骤4：化合物D-1的制备
将上步所得化合物D-1c粗品溶于10mL的四氢呋喃中，加入2mL的二乙胺，然后在室温搅拌至反应完全，减压浓缩得粗品，经硅胶柱层析色谱法(洗脱剂：二氯甲烷/乙酸乙酯/石油醚)纯化，得3mg目标产物化合物D-1。
LC/MS(ESI)：m/z 744.2[M+H] +。
步骤5：化合物L-1的制备
将化合物D-1(13.5mg,0.018mmol)溶于1.5mL的DMF中，加入DIPEA(7mg，0.054mmol)，然后分批加入化合物2(18mg，1.3mmol)，搅拌至反应基本完全，浓缩得粗品。经HPLC制备分离(色谱柱：Welch XTimate C18(5.0μm*30.0*150mm)；流动相:A-水(0.1％formic acid)：B-乙腈，梯度洗脱＝70:30-5:95(16min，流速：30.0mL/min)得6.5mg化合物L-1，纯度96.95％。
LC/MS(ESI)：m/z 1388.3[M+H] +。
1HNMR(CDCl 3,400M)δ0.85～0.90(m,3H),0.93～1.00(m,3H),1.08～1.10(m,3H),1.20～1.50(m,15H),1.75～2.04(m,6H),2.13～2.55(m,16H),2.70～2.77(m,1H),2.80～2.96(m,2H),3.16～3.97(m,20H),3.99～4.39(m,8H),4.60～4.80(m,6H),4.88～5.10(m,5H),5.24～5.37(m,4H),6.71(s,2H),7.03(d,J＝6.8Hz,1H),7.18～7.30(m,3H),7.63(d,J＝8.0Hz,2H),8.92(bs,1H)。
实施例2-2 化合物D-2的合成
Figure PCTCN2021073314-appb-000144
在反应瓶中加入(R)-2-环丙基-2-羟基乙酸(4.7mg，0.04mmol，1.5eq)，加入THF搅拌溶解，冰水浴降温。然后加入EDCI HCl(8.0mg，0.04mmol，1.5eq，1-乙基-3(3-二甲基丙胺)碳二亚胺盐酸盐)、HOBT(5.4mg，0.04mmol，1.50eq，1-羟基苯并三氮唑)，接着加入化合物D-1a(20mg,0.027mmol，1.0eq)，最后加入DIPEA (10.5mg,0.08mmol,3.0eq)。加料完毕后，自然升到室温(20℃)搅拌至反应基本完全，加入2ml水淬灭反应，用乙酸乙酯萃取(2×5ml)，合并有机相，用无水硫酸钠干燥，过滤，减压浓缩，得到粗品经硅胶柱层析色谱法(洗脱剂：乙酸乙酯/石油醚)纯化得到6.0mg，纯度98％。
MS：827.8[M+H] +。
1H NMR(400MHz,CDCl 3)δ6.82(s,1H),5.08(s,1H),4.93(s,1H),4.89(s,1H),4.81(s,1H),4.70(t,J＝4.4Hz,1H),4.61(t,J＝4.4Hz,1H),4.42–4.25(m,3H),4.23–4.16(m,1H),4.12(s,1H),4.03(d,J＝9.3Hz,3H),4.02–3.87(m,3H),3.82(d,J＝9.4Hz,1H),3.78–3.70(m,3H),3.58(dd,J＝50.7,8.9Hz,4H),3.43(s,3H),3.32–3.23(m,2H),2.88(d,J＝9.5Hz,2H),2.72(dd,J＝16.0,10.0Hz,1H),2.46(d,J＝13.9Hz,4H),2.33(d,J＝13.8Hz,3H),2.19(dd,J＝21.4,14.3Hz,4H),2.08(s,1H),1.97(ddd,J＝13.6,9.4,4.7Hz,5H),1.44(d,J＝11.5Hz,3H),1.27(d,J＝12.2Hz,4H),1.10(d,J＝6.2Hz,3H),0.68–0.45(m,4H)。
实施例2-3 化合物D-3的合成
Figure PCTCN2021073314-appb-000145
于室温，在氮气氛下，用2mL的DCM溶解化合物D-1(22mg，0.03mmol)和(R)-2-环丙基-2-羟基乙酸(7.0mg，0.06mmol)，再依次加入Et 3N(21uL，0.15mmol)和DMTMM(20.3mg，0.069mmol，4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐)，室温搅拌过夜。反应完后加入H 2O(2mL)淬灭，DCM(2mL X 3)萃取，有机相减压浓缩(浴温30℃)。残余物经硅胶柱层析色谱法(洗脱剂：乙酸乙酯/石油醚)纯化得化合物D-3(18mg，纯度95％)。
MS：863.8[M+Na] +。
实施例2-4 化合物D-4的合成
Figure PCTCN2021073314-appb-000146
用羟基乙酸代替(R)-2-环丙基-2-羟基乙酸参照实施例2-2方法制得化合物D-4。
MS：787.82[M+H] +。
1HNMR(CDCl 3,400M):0.86～0.90(m,1H),1.04～1.13(m,4H),1.22～1.41(m,4H),1.71～1.74(m,3H),1.94～2.00(m,5H),2.15～2.22(m,8H),2.48(S,3H),2.71～2.75(m,2H),2.87～2.89(m,2H),3.26～3.31(m,2H),3.43(S,3H),3.53～3.55(m,1H),3.64～3.70(m,2H),3.74(s,1H),3.80～3.84(m,1H),3.90～4.05(m,4H),4.12(s,3H),4.18～4.20(m,1H),4.26～4.39(m,3H),4.61(t,J＝4.8Hz,1H),4.619(t,J＝4.8Hz,1H),4.82(s,1H),4.82(s,1H),4.88(s,1H),4.93(s,1H),5.08(s,1H),6.89(m,1H)。
实施例2-5 化合物D-5的制备
Figure PCTCN2021073314-appb-000147
用1-羟基环丙烷-1-羧酸代替(R)-2-环丙基-2-羟基乙酸参照实施例2-2方法制得化合物D-5。
MS：835.7[M+Na] +。
实施例2-6 化合物D-6的合成
Figure PCTCN2021073314-appb-000148
利用起始物料(R)-2-环丙基-2-羟基乙酸和E1-30(根据文献Bioorg.Med.Chem.Lett.14(2004)5551–5554.合成得到)参照实施例2-2方法制备式D-6化合物。
MS：850.64[M+Na] +。
实施例2-7 化合物D-7的合成
Figure PCTCN2021073314-appb-000149
利用起始物料1-羟基环丙烷-1-羧酸和E1-30(根据文献Bioorg.Med.Chem.Lett.14(2004)5551–5554.合成得到)参照实施例2-2方法制备式D-7化合物。
MS：836.73[M+Na] +。
实施例2-8 化合物D-8的合成
Figure PCTCN2021073314-appb-000150
利用起始物料对羟基乙基苯甲酸和E1-30参照实施例2-2方法制备式D-8化合物。
MS：886.75[M+Na] +。
测试例1：体外细胞毒活性筛选
1.1.实验原理及方法
本实验利用CTG检测ATP含量，反映肿瘤细胞的存活情况。首先通过种植不同密度的细胞并培养3天和5天，根据IC 50和最大抑制率确定最终培养条件。然后按照此条件检测毒素分子的杀伤作用。
1.2.细胞株的选择
根据实验目的，选择乳腺癌、NSCLC两种疾病模型，选出SKBR3肿瘤细胞(HER2+，ATCC，货号HTB-30)、MDA-MB-468(HER2-，ATCC，货号HTB-132)和A549(人非小细胞肺癌细胞，ATCC，货号CCL-185)三株细胞用于筛选实验。
1.3.细胞培养条件的确定
1)细胞培养：A549、SK-BR-3和MDA-MB-468细胞分别用含10％FBS(Gibco，10099-141)的Ham’s F-12K(Kaighn’s)培养基(Gibco，21127030)和McCoy's 5A培养基(ThermoFisher，货号16600108)培养及Leibovitz's L-15培养基(ThermoFisher，货号11415-114)进行培养。
2)细胞铺板：将A549用胰酶消化后，用上述培养基终止，计数后分别取 4.3×10 5、7.2×10 5、11.5×10 5个细胞加培养液使终体积均为26ml。在96孔板(康宁，货号3903)第2列到第11列每孔加180ul细胞悬液，使得细胞密度分别为每孔3K、5K、8K。第12列为200ul培养液，剩余孔用PBS填充。SKBR3和MDA-MB-468细胞重复上述操作。平行两份。
3)药物配制：在圆底96孔板(康宁，货号3788)中，用DMSO配制阳性对照艾日布林和本公开化合物储备液。在配药板1的第1列配制2mM(用DMSO将储液稀释10倍)，此后到第10列梯度10倍稀释于DMSO中，第11列为DMSO。配药板2第2列至第11列每孔加入相应培养液95ul，从配药板1的第2列至第11列吸取5ul溶液至配药板2，混匀后，吸取20ul加入铺好的细胞中，继续培养3天和5天。
4)CTG检测(Cell Titer-GloTM，发光细胞生存能力检测，Promega公司)：分别在第3天和第5天取出细胞板，平衡至室温。每孔加入90ul CTG，避光室温反应10min，酶标仪读取发光值并计算IC 50。
1.4.数据结果
表5
Figure PCTCN2021073314-appb-000151
结论：化合物D-1在三种肿瘤细胞系中都有很好的杀伤效果，且明显优于阳性药艾日布林。
实施例2-9：化合物D-9的合成
Figure PCTCN2021073314-appb-000152
步骤1：化合物D-9a的制备
于室温，取0.3mL的1,4-二氧六环和0.3mL的化合物E-305(31mg，0.042mmol，根据文献Bioorg.Med.Chem.Lett.14(2004)5551–5554.合成得到)，再依次加入芴甲氧羰酰琥珀酰亚胺(Fmoc-OSu)(17mg，0.050mmol)和碳酸钠固体(18mg，0.168mmol)。室温搅拌过夜，检测原料基本转化完全。反应中加入水淬灭，乙酸乙酯萃取，减压浓缩，残余物经硅胶柱层析色谱法(洗脱剂：乙酸乙酯/石油醚)纯化后得15mg产物。
LC/MS(ESI)：m/z 965.64[M+H] +。
步骤2：化合物D-9b的制备
于室温，用二氯甲烷(0.5mL)溶解化合物D-9a(7mg，0.007mmol)，再依次加入4A分子筛(10mg)、三甲基氧鎓四氟硼酸(11mg，0.07mmol)和质子海绵(16mg，0.07mmol)，室温搅拌1小时。检测原料基本转化完全，反应中加入水淬灭，甲基叔丁基醚萃取，再用1N稀盐酸洗涤，减压浓缩，残余物经硅胶柱层析色谱法(洗脱剂:乙酸乙酯/石油醚＝1:1)纯化后得7mg产物。
LC/MS(ESI)：m/z 979.68[M+H] +。
步骤3：化合物D-9的制备
冰水浴下，取1mL四氢呋喃溶解化合物D-9b(10mg，0.01mmol)，再滴加入DBU(6uL，0.04mmol)，搅拌至反应完全。反应中加入水淬灭，二氯甲烷萃取，减压浓缩，残余物经HPLC制备分离(色谱柱：Welch Xtimate C18(10*150mm*5um)；流动相:A-水(20mM NH 4HCO 3)：B-乙腈，梯度洗脱＝30％B至95％B)得5mg化合物D-9。
LC/MS(ESI)：m/z 757.85[M+H] +。
实施例2-10化合物D-10的合成
Figure PCTCN2021073314-appb-000153
步骤1：化合物D-10b的制备
冰水浴下，取2mL的四氢呋喃溶解化合物D-10a(6mg，0.008mmol，根据文献Bioorg.Med.Chem.Lett.21(2011)1639–1643.合成得到)，再滴加入四氢锂铝溶液(80uL，1M在THF中，0.08mmol)，搅拌并将反应温度缓慢升至40℃。LCMS检测原料基本转化完全，反应用十水合硫酸钠淬灭，冰水浴下搅拌半小时，过滤，滤液减压浓缩得粗品，直接用于下一步反应。
LC/MS(ESI)：m/z 758.4[M+H] +。
步骤2：化合物D-10c的制备
于室温，取0.5mL的1,4-二氧六环和0.5mL的水溶解上步所得化合物D-10b，再依次加入芴甲氧羰酰琥珀酰亚胺(6.5mg，0.019mmol)和碳酸钠(6.8mg，0.064mmol)，室温搅拌过夜，检测原料基本转化完全。反应中加入水淬灭，乙酸乙酯萃取，减压浓缩，残余物经硅胶柱层析色谱法(洗脱剂：乙酸乙酯/石油醚＝3:2)纯化后得14mg化合物D-10c。
LC/MS(ESI)：m/z 980.4[M+H] +。
步骤3：化合物D-10d的制备
冰水浴下，取1mL的二氯甲烷溶解上步所得化合物D-10c(14mg，0.014mmol)，再加入戴斯-马丁氧化剂(18.2mg，0.042mmol)，搅拌并允许反应缓慢升至室温，搅拌至LCMS检测原料基本转化完全。加入碳酸氢钠水溶液淬灭反应，二氯甲烷萃取，减压浓缩，残余物经硅胶柱层析色谱法(洗脱剂：乙酸乙酯/石油醚＝3:2)纯化后得8mg化合物D-10d。
LC/MS(ESI)：m/z 978.4[M+H] +。
步骤4：化合物D-10的制备
冰水浴下，取1mL的四氢呋喃溶解上步所得化合物D-10d(8mg，0.008mmol)，再滴加入DBU(6uL，0.032mmol)，搅拌1小时，TLC检测原料基本转化完全，反应中加入水淬灭，二氯甲烷萃取，减压浓缩，残余物经HPLC制备分离(色谱柱：Welch Boltimate C18Core-Shell(4.6*50mm*2.7um)；流动相:A-水(20mM NH 4HCO 3)得目标产物化合物D-10(1.3mg)。
LC/MS(ESI)：m/z 755.93[M+H] +。
测试例2：体外细胞毒活性筛选
2.1.实验原理及方法
本实验利用CTG检测ATP含量，反映肿瘤细胞的存活情况。
2.2.细胞培养条件的确定
1)细胞培养：A549、SK-BR-3和MDA-MB-468细胞分别用含10％FBS(Gibco，10099-141)的Ham’s F-12K(Kaighn’s)培养基(Gibco，21127030)和McCoy's 5A培养基(ThermoFisher，货号16600108)培养及Leibovitz's L-15培养基(ThermoFisher，货号11415-114)进行培养。
将A549、SKBR3和MDA-MB-468用胰酶消化后，各自用培养液重悬计数，将细胞密度调整为2.2×10 4个/ml，在96孔板的列2至列11每孔加入135μl细胞悬液，列12为空白对照。在5％CO 2的37℃培养箱中培养24h。
2)药物配制：
a)储液准备：用DMSO将待测化合物和阳性对照药物溶解，使储液浓度为5mM。
b)配药板1：起始列1将储液稀释40倍，列2至列11依次3倍梯度稀释。列12为DMSO。
c)配药板2：在列2至列11加入196ul相应培养液，从配药板1的列3至列12吸4ul至配药板2的列2到列11。混匀。
2.3.处理细胞
从配药板2吸取15ul加入上述铺好的细胞中。在5％CO 2的37℃培养箱中继 续培养5天。
2.4.CTG检测(Cell Titer-GloTM，发光细胞生存能力检测)：取出细胞板，平衡至室温。每孔加入75ul CTG，避光室温反应10min，酶标仪读取发光值并计算IC 50。
2.5.数据结果
表6
Figure PCTCN2021073314-appb-000154
阳性对照药物化合物E-305和化合物E1-30的结构式如下所示，其制备方法参见Bioorganic&Medicinal Chemistry Letters 14(2004)5551–5554中方法制备：
Figure PCTCN2021073314-appb-000155
实施例2-11：化合物L-2的合成
Figure PCTCN2021073314-appb-000156
Figure PCTCN2021073314-appb-000157
冰水浴下，将化合物D-1a(9mg，0.012mmol)溶于0.3mL DMF中，加入DIPEA(3.5mg,0.028mmol)，然后分批加入化合物2(7.8mg,0.011mmol)，搅拌至反应基本完全，减压浓缩得粗品，经HPLC制备分离(色谱柱：XBridge Prep C18 OBD 5um19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱)得4.95mg化合物L-2，纯度97％。
LC/MS(ESI)：m/z 1374.3[M+H] +。
实施例2-12：化合物L-3的合成
Figure PCTCN2021073314-appb-000158
Figure PCTCN2021073314-appb-000159
步骤1：化合物4的制备
将化合物4a(1.3g，采用WO2013106717公开方法制备而得)溶于50ml乙腈中，依次加入碳酸钾(6.2g)、溴化苄(1.35ml)和四丁基碘化铵(415mg)。室温搅拌反应至基本完全，过滤，浓缩，以石油醚/乙酸乙酯为展开剂，用硅胶柱色谱法纯化得化合物4b。
将化合物4b(121mg)和4c(180mg)加入反应瓶中，加入4ml四氢呋喃。氮气氛下，冰水浴降温至0℃左右，加入叔丁醇钾(109mg，0.98mmol)，升至室温搅拌40分钟。加入10mL冰水，用乙酸乙酯(20mL×2)和氯仿(10mL×5)萃取，合并有机相并浓缩。所得残余物溶于4mL二氧六环中，加入2mL水，加入碳酸氢钠(49.2mg，0.586mmol)和氯甲酸-9-芴甲酯(126mg，0.49mmol)，室温搅拌2小时。加入20mL水，用乙酸乙酯(10mL×3)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。以石油醚/乙酸乙酯为展开剂，用硅胶柱色谱法纯化得到化合物4d，MS m/z(ESI):515.0[M+1] +。
将化合物4d(20mg，0.038mmol)溶于4.5mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中，加入钯碳(12mg，含量10％，干型)，氢气置换三次，室温搅拌反应1小时。反应液用硅藻土过滤，滤饼用乙酸乙酯淋洗，滤液浓缩，得到粗品标题化合物4(13mg)，产品不经纯化直接进行下一步反应。
MS m/z(ESI)：424.9[M+1]。
步骤2：化合物DZ-1a的制备
称取化合物4(13.4mg，0.0316mmol，1.7eq)和化合物D-1a的甲磺酸盐(15mg，0.0182mmol，1eq)溶于DMF(0.5ml)中，冰浴冷却下加入三乙胺(10mg，0.0988mmol，5.4eq)、DMTMM(9.8mg，0.0332mmol，1.8eq)，反应液自然升 至室温搅拌至基本完成。加入水(2ml)和乙酸乙酯(3ml)稀释分液，乙酸乙酯萃取水相，合并有机相用无水硫酸钠干燥，过滤，滤液减压浓缩，残余物经制备薄层色谱法(乙酸乙酯/石油醚)纯化得到16mg化合物DZ-1a，收率86.7％。
LC/MS(ESI):m/z 1136.3[M+H] +。
步骤3：化合物DZ-1b的制备
在冰浴冷却下，称取上步所得化合物DZ-1a(16mg，0.0141mmol，1eq)溶于THF中(0.4ml)，加入三乙胺(4.2mg,0.057mmol,4eq)，保持冰浴下搅拌至基本完成。加入二氯甲烷(5ml)稀释，水(2ml X 3)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩，得到粗品直接用于下一步。
LC/MS(ESI)：m/z 914.3[M+H] +。
步骤4：化合物L-3的制备
称取上步所得化合物DZ-1b粗品(16mg，0.0175mmol，1eq)和化合物6(11.6mg，0.0246mmol，1.4eq，采用EP2907824中方法制备而得)溶于DMF中(0.5ml)，加入HATU(9.9mg，0.026mmol，1.5eq)和N,N-二异丙基乙胺(DIPEA)(5.5mg，0.0426mmol，2.4eq)，保持冰浴下搅拌至基本完成。加入水(2ml)和乙酸乙酯(3ml)稀释分液，乙酸乙酯萃取水相，合并有机相用无水硫酸钠干燥，过滤，滤液减压浓缩，残余物经制备HPLC(色谱柱：XBridge Prep C18 OBD 5um 19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱)纯化得到10mg化合物L-3。
LC/MS(ESI)：m/z 1368.3[M+H] +。
实施例2-13：化合物L-4的合成
Figure PCTCN2021073314-appb-000160
Figure PCTCN2021073314-appb-000161
步骤1：化合物DZ-2a的制备
称取化合物4(11.6mg，0.0273mmol，1.5eq)和化合物D-1(13.5mg，0.0181mmol，1eq)溶于N,N-二甲基甲酰胺(0.5ml)中，冰浴冷却下加入DMTMM(10.1mg，0.0343mmol，1.3eq)，搅拌至基本反应完成。加入水(2ml)和乙酸乙酯(3ml)中止和稀释反应，分液，乙酸乙酯萃取水相，合并有机相用无水硫酸钠干燥，过滤，滤液减压浓缩，残余物经制备薄层色谱法(乙酸乙酯/石油醚)纯化得到10mg，收率47.9％。
LC/MS(ESI)：m/z 1150.2[M+H] +。
步骤2：化合物DZ-2b的制备
称取上步产物化合物DZ-2a(10mg，0.0087mmol，1eq)溶于THF(1ml)中，加入DBU(1,8-二氮杂二环十一碳-7-烯)(5.2mg，0.034mmol，4eq)，冰浴下搅拌至基本反应完成。加入二氯甲烷(5ml)稀释，水(2ml X 3)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩，得到粗品直接用于下一步。
LC/MS(ESI)：m/z 928.2[M+H] +。
步骤3：化合物L-4的制备
称取上步产物化合物DZ-2b(16mg，0.0087mmol，1eq)和化合物6(7.8mg，0.0165mmol，1.9eq)溶于DMF中(0.5ml)，加入HATU(6.2mg，0.0163mmol，1.9eq)和DIEA(5.7mg，0.0441mmol，5eq)，冰浴下搅拌至反应基本完成。加入水(2ml)和乙酸乙酯(3ml)稀释分液，乙酸乙酯萃取水相，合并有机相用无水硫酸钠干燥，过滤，滤液减压浓缩，残余物经制备HPLC(色谱柱：XBridge Prep C18 OBD 5um 19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱) 纯化得到3.5mg化合物L-4，两步收率29.1％。
LC/MS(ESI):m/z 1382.2[M+H] +。
实施例2-14：抗体药物偶联物ADC-1的制备
Figure PCTCN2021073314-appb-000162
于37℃，向抗体PD3的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/ml，0.9mL，60.6nmol)加入配制好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，15.2uL，152nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物L-3(0.83mg，606nmol)溶解于50ul DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-1的PBS缓冲液(0.76mg/mL，10mL)，于4℃冷冻储存。
十二烷基硫酸钠毛细管电泳(CE-SDS)紫外检测方法计算平均值：k＝3.87。
实施例2-15：抗体药物偶联物ADC-2的制备
Figure PCTCN2021073314-appb-000163
在37℃，向抗体PD3的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/ml，1.14mL，77.2nmol)加入配制好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，19.3uL，193nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物L-2(0.83mg，772nmol)溶解于50ul DMSO中，加入到上述反应液 中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-2的PBS缓冲液(0.71mg/mL，12mL)，于4℃冷冻储存。
CE-SDS计算平均值：k＝3.88。
实施例2-16：抗体药物偶联物ADC-3的制备
Figure PCTCN2021073314-appb-000164
在37℃，向抗体PD3的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/ml，0.9mL，60.6nmol)加入配制好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，15.2uL，152nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物L-1(0.84mg，605nmol)溶解于50ul DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-3的PBS缓冲液(0.77mg/mL，10.2mL)，于4℃冷冻储存。
CE-SDS计算平均值：k＝3.81。
实施例2-17：抗体药物偶联物ADC-4的制备
Figure PCTCN2021073314-appb-000165
在37℃，向抗体PD3的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/ml，0.9mL，60.6nmol)加入配制好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，15.2uL，152nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反 应。将反应液用水浴降温至25℃。
将化合物L-4(0.84mg，608nmol)溶解于50ul DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-4的PBS缓冲液(0.64mg/mL，13.5mL)，于4℃冷冻储存。
CE-SDS计算平均值：k＝3.88。
实施例2-18：抗体药物偶联物ADC-5的制备
Figure PCTCN2021073314-appb-000166
在37℃，向CD79B抗体hAb015-10的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/ml，1.0mL，67.3nmol)加入配制好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，16.8uL，168nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物L-1(0.93mg，673nmol)溶解于50ul DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-5的PBS缓冲液(0.68mg/mL，9.6mL)，于4℃冷冻储存。
CE-SDS计算平均值：k＝4.07。
测试例3：评价并比较ADC-5、Polivy对人弥漫性大B细胞淋巴瘤WSU-DLCL2裸小鼠皮下移植瘤的疗效。
3.1药物信息
空白组：hIgG1；
ADC-5：无色澄明液体，浓度0.68mg/mL，纯度98.00％，遮光，2-8℃密闭保存；
Polivy(polatuzumab)：无色澄明液体，浓度5.83mg/mL，纯度97.69％，遮光，2-8℃密闭保存。
3.2药物配制
均用生理盐水稀释至所需浓度。
生理盐水，规格10ml:0.09g，购自中国大冢制药有限公司。
3.3细胞
人弥漫性大B细胞淋巴瘤WSU-DLCL2细胞购自DSMZ。WSU-DLCL2细胞用10-cm培养皿培养，培养条件为RPMI 1640培养基(Gibco)中加10％胎牛血清以及青、链霉素(GIBCO，货号15070-063)，于37℃、含5％的CO2空气的培养箱中培养。一周2-3次传代，当细胞呈指数生长期时，收集细胞，计数，接种。
3.4实验动物
BALB/c-nu裸小鼠，28-35日，雌性，购自北京华阜康生物科技股份有限公司。生产许可证号：SCXK(京)2019-0008，动物合格证号No.1103221911012510。饲养环境：SPF级。
3.5实验步骤
每只裸小鼠皮下接种2.0×10 7WSU-DLCL2细胞，待肿瘤生长至～100mm 3后，根据肿瘤体积将动物分组(D0)。小鼠静脉注射(IV)给药，给药体积10mL/kg；具体给药剂量和给药方案见表3。每周测2次肿瘤体积，称小鼠体重，记录数据。
本实验动物的使用及福利遵照“国际实验动物评估和认可委员会(AAALAC)”的规定执行。每天监测动物的健康状况及死亡情况，例行检查包括观察受试物和药物对动物日常行为表现的影响如行为活动，体重变化，外观体征等。
3.6实验指标
实验指标为考察药物对肿瘤生长的影响，具体指标为T/C％或抑瘤率TGI(％)。
每周二次用游标卡尺测量肿瘤直径，肿瘤体积(V)计算公式为：
V＝1/2×a×b 2其中a、b分别表示长、宽。
T/C(％)＝(T-T0)/(C-C0)×100其中T、C为实验结束时的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
抑瘤率(TGI)(％)＝100-T/C(％)。
当肿瘤出现消退时，抑瘤率(TGI)(％)＝100-(T-T0)/T0×100
如果肿瘤比起始体积缩小，即T<T0或C<C0时，即定义为肿瘤部分消退(PR)；如果肿瘤完全消失，即定义为肿瘤完全消退(CR)。
实验结束、达到实验终点、或溶剂组平均肿瘤体积达到1500mm 3，CO 2麻醉处死动物，随后解剖取瘤并拍照。
3.7统计学分析
除非特别说明，两组肿瘤体积之间比较采用双尾学生t检验，P<0.05定义为有统计学显著性差异。
3.8结果
ADC-5、Polivy(IV；D0，1mg/kg)对人弥漫性大B细胞淋巴瘤WSU-DLCL2裸小鼠皮下移植瘤的抑瘤率分别为53％和37％；荷瘤小鼠对以上药物均能很好耐受，没有明显体重减轻等症状发生。
表7
Figure PCTCN2021073314-appb-000167
注：IV静脉注射
结论
抗体-药物偶联物ADC-5和Polivy(阳性对照组)对人弥漫性大B细胞淋巴瘤WSU-DLCL2裸小鼠皮下移植瘤的抑瘤率分别为53％和37％，具有显著抗肿瘤活性；荷瘤小鼠对以上药物均能很好耐受。
测试例4：细胞杀伤实验
4.1实验目的
本实验的目的是为了检测本公开中的抗TROP-2抗体(PD3)-药物偶联物，对不同肿瘤细胞系：Miapaca2肿瘤细胞(人胰腺癌细胞，南京科佰，货号CBP60544)，Fadu肿瘤细胞(人鳞状细胞癌，ATCC，货号HTB-43)、SK-OV-3(人卵巢癌细胞，ATCC，货号HTB-77)、K562(人慢性粒细胞白血病细胞，ATCC，货号CCL-243)、HCC827(人肺癌细胞，ATCC，货号CRL-2868)和BXPC3(人胰腺癌细胞，ATCC，货号CRL-1687)的增殖抑制活性。以不同浓度的抗体药物偶联物体外处理细胞，经6天培养后，采用CTG(
Figure PCTCN2021073314-appb-000168
Luminescent Cell Viability Assay，Promega，货号：G7573)试剂对细胞的增值进行检测，根据IC 50值来评价抗体药物偶联物的体外活性。
4.2实验方法
1)细胞培养：MiaPaCa2、Fadu、SK-OV-3、K562、HCC827和BXPC3细胞分别用含10％FBS(Gibco，10099-141)的DMEM/高葡萄糖培养基(GE，SH30243.01)、MEM培养基(Gibco，11095080)、McCoy’S 5a培养基(Gibco，16600108)、IMDM培养基(ThermoFisher，12440061)、RPIM1640培养基(Gibco，11875119)培养。
2)细胞铺板：实验当天，细胞分别用胰酶(0.25％Trypsin-EDTA(1x)，Life Technologies，货号25200-072)消化后，使用相应的培养基分别将MiaPaCa2、Fadu、SK-OV-3、K562、HCC827和BXPC3制成细胞悬液，使密度为3.7×10 3个/mL，在96孔板(corning，货号3903)中每孔加入135uL悬液，使每孔500个细胞，于37℃，培养24h。
3)药物配制：将待测ADC的母液调整首浓度为4uM加在配药板(corning， 货号3599)首列。在配药板的第2列至第9列梯度稀释5倍，第10列为PBS。每孔取15uL加入到细胞培养板中。
4)CTG检测：于37℃培养6天后，取出细胞培养板，平衡至室温。每孔加入75uL CTG，避光室温反应10min，酶标仪(BMG labtech，PHERAstar FS)读取发光值。
4.3数据分析
用Graphpad Prism 5对数据进行处理分析。结果参见下表8。
表8
编号 Miapaca K562 SKOV3 HCC827 BXPC3 Fadu
ADC-1 64.05 73.21 54.14 0.30 0.16 0.96
ADC-2 24.59 27.95 0.27 0.11 0.05 0.11
ADC-3 31.22 44.10 10.42 0.01 0.09 0.60
ADC-4 29.95 39.44 18.73 1.37 1.38 4.04
E1-30 0.45 0.39 0.17 0.31 0.23 0.32
测试例5：旁观杀伤实验
5.1实验目的
检测本公开中的抗体药物偶联物在TROP-2阳性细胞BXPC3(人原位胰腺腺癌细胞，普诺赛)和TROP-2阴性细胞MiaPaCa2(人胰腺腺癌细胞，普诺赛)共培养时，选取抗体药物偶联物在杀伤实验中对TROP-2阳性的BXPC3细胞有杀伤而对TROP-2阴性的细胞MiaPaCa2无杀伤作用的浓度5nM进行实验，考察在两者共培养的体系内抗体药物偶联物是否具有对TROP-2阴性的细胞Miapaca2的旁观杀伤作用。
5.2实验方法
1)细胞培养：MiaPaCa2和BXPC3细胞分别用10％FBS(Gibco，10099-141)的DMEM/高葡萄糖培养基(GE，SH30243.01)、RPIM1640培养基(Gibco,11875119)培养。
2)细胞铺板：实验当天，细胞分别用胰酶(0.25％Trypsin-EDTA(1x)，Life Technologies，货号25200-072)消化后，用新鲜RPIM1640(含10％FBS)培养基中和，1000rpm离心3min，弃上清，细胞用RPMI1640+10％FBS重悬。细胞计数后，将BXPC3细胞密度调整为6×10 4个/mL，将MiaPaCa2细胞密度调整为1.5×10 4个/mL。12孔板中板1中每孔加入500uL的BXPC3细胞和500uL的MiaPaCa2细胞。12孔板板2中加入500uL的MiaPaCa2细胞和500uL的RPMI1640+10％FBS的培养液。5％二氧化碳，37℃，培养24小时。
3)抗体药物偶联物配制：将抗体药物偶联物ADC-1、ADC-2、ADC-3和ADC-4分别用RPMI1640稀释成浓度为600nM，从中取50uL加培液100uL稀释成200nM (40X，终浓度5nM)。取25uL加入细胞培养板中。另设置PBS溶剂对照组，继续培养6天。
4)流式分析：12孔板(板1和板2)中的细胞用胰酶消化，使用新鲜培养基中和，1000rpm离心3min，弃上清，用1mL的FACS缓冲液(PBS+2.5％FBS)重悬，取20uL的细胞加入20uL的台盼蓝(Sigma，T8154-100ML)，计数。板1中的细胞，1000rpm离心3min，弃上清，用100uL的FACS缓冲液重悬，加入2uL的TROP-2(EGP-1)单克隆抗体(MR54)(ThermoFisher，货号12-6024-42)，冰上孵育30min。然后在4℃，2000rpm离心1min，加入150uL FACS缓冲液重悬细胞，重复上述操作两次。用流式细胞仪(BD，FACSVerse)检测。
5.3数据分析
用Flowjo 10.0对数据进行处理分析。
结论：所有抗体偶联物在试验中都显示旁观者效应。
测试例6：药代动力学测试
1、概述
以
Figure PCTCN2021073314-appb-000169
的比格犬为受试动物，应用LC/MS/MS法测定了比格犬静脉注射给予化合物D-1和艾日布林后不同时刻血浆中的药物浓度。研究本公开化合物在犬体内的药代动力学行为，评价其药动学特征。
2、试验方案
2.1试验药品
化合物D-1和艾日布林
2.2试验动物
比格犬6只，雄性，平均分为2组，由美迪西普亚医药科技(上海)有限公司采购并进行动物给药实验。
2.3药物配制
称取化合物D-1，加5％体积的DMSO，20％PG和20％PEG400使其溶解，然后加入55％生理盐水配制成0.25mg/ml无色澄明溶液。
称取艾日布林，加5％体积的DMSO，20％PG和20％PEG400使其溶解，然后加入55％生理盐水配制成0.25mg/ml无色澄明溶液。
2.4给药
一组犬静脉注射给药化合物D-1，给药剂量均为0.5mg/kg，给药体积均为2ml/kg。
另一组犬静脉注射给药艾日布林，给药剂量均为0.5mg/kg，给药体积均为2ml/kg。
3、操作
犬注射给药化合物D-1，于给药前及给药后5分钟、0.25、0.5、1.0、2.0、4.0、 8.0、12.0、24.0小时采血1ml，将收集的血液样本置于EDTA-K2抗凝型采血管中，将收集到的全血放在冰上，1小时内离心分离血浆(离心力2200g，离心10min，2-8℃)。血浆样本在检测前存放于-80℃冰箱内。
犬注射给药艾日布林化合物，于给药前及给药后5分钟、0.25、0.5、1.0、2.0、4.0、8.0、12.0、24.0小时采血1ml，将收集的血液样本置于EDTA-K2抗凝型采血管中，将收集到的全血放在冰上，1小时内离心分离血浆(离心力2200g，离心10min，2-8℃)。血浆样本在检测前存放于-80℃冰箱内。
测定药物注射给药后犬血浆中的待测化合物含量：取给药后各时刻的犬血浆25μl，加入内标溶液喜树碱(中国生物制品检定所)50μl(100ng/mL)和乙腈200μl，涡旋混合5分钟，离心10分钟(3700转/分钟)，血浆样品取上清液3-4μl进行LC/MS/MS(API4000三重四极杆串联质谱仪(No.2)，美国Applied Biosystems公司；Shimadzu LC-30AD超高效液相色谱系统，日本Shimadzu公司。)分析。
4、药代动力学参数结果
本公开化合物的药代动力学参数如下表9所示。
表9
Figure PCTCN2021073314-appb-000170
测试例7：ADC-2和ADC-3对人咽鳞癌FaDu细胞株BALB/c裸小鼠皮下移植瘤中的药效作用
(1)细胞培养
用于本实验的人咽鳞癌Fadu细胞株(ScienCell实验室，ml-cs-0374)在MEM培养基(添加10％(v/v)胎牛血清(FBS)(GIBCO，货号10099-141)和0.1％磷酸缓冲液)中于含5％CO 2的37℃培养箱中培养。接种前用3-4％异氟烷将小鼠麻醉。细胞连续培养十代之前，将5×10 6Fadu细胞的培养基100μL和等体积Matrigel(基底胶，solarbio)混匀后，通过皮下注射接种于小鼠背部右边靠近腋下。
(2)动物分组和给药方案
当肿瘤生长到平均约100～150mm 3左右时，小鼠根据肿瘤体积和体重被随机分组，每组8只。分组给药当天定义为第0天。分组情况和给药方案如下表10所示：
表10分组和给药方案
Figure PCTCN2021073314-appb-000171
给药体积：动物的给药体积按照10μL/g体重进行调整
肿瘤体积：分组后每周测量肿瘤体积两次，连续4周。肿瘤体积(V)的计算方法如下：V＝(长×宽 2)/2。每只小鼠相对肿瘤体积(RTV)的计算方法是：RTV＝Vt/V0，其中Vt为每次的测量体积，V0为治疗开始时的体积。
动物体重：分组后每周测量并记录老鼠体重两次。
动物状态观察：本实验中给予溶媒或测试药的动物并未出现异常现象。实验终点，部分动物出现溃疡。
(3)实验停药和恢复给药标准
在实验过程中，小鼠体重下降≥15％时，即停止给药，停药期应足够长让小鼠恢复体重。仅单只小鼠停药，其余小鼠正常给药；停药时小鼠体重恢复参照以下标准将继续实验：体重下降≤10％。
(4)实验终点.
体内实验结束，所有动物将被CO 2窒息，然后脱颈椎处死。采集肿瘤，称重并拍照。体内实验结束前，已死亡的荷瘤动物将不进行样品收集。
(5)统计分析
结果将以平均值±S.E.M的方式呈现。两组间比较将用Dunnett’s multi-comparison test进行检验。如果p<0.05则认为有统计学显著性差异，记为*，p<0.01记为**，p<0.001记为***。
(6)结果
体重：溶媒与不同测试药给药组的动物体重变化趋势如图1所示。动物体重随实验进程正常增长。
肿瘤体积：
实验周期内，空白溶媒组别(G1组)的动物接种分组开始给药后，肿瘤缓慢增长；直至第14天，肿瘤体积快速增加。实验第0天，G1组的肿瘤体积均值为：125.05±3.66mm 3；第25天，肿瘤体积均值为：1854.48±99.50mm 3。由此肿瘤体积 和相对肿瘤体积实验数据，说明人咽鳞癌Fadu细胞株在BALB/c nude小鼠皮下移植瘤模型成功建立。
ADC-3高剂量(3mg/kg)组别(G2组)的动物在第0天的肿瘤体积均值为：121.40±3.18mm 3；第25天，肿瘤体积均值为：721.56±169.15mm 3。实验周期内，与空白溶媒组相比，ADC-3高剂量组能显著地抑制肿瘤的增长。
相对肿瘤增殖率和瘤重抑制率：
相对肿瘤增殖率(T/C％)：用于评价药物抗肿瘤活性的作用，相对肿瘤增殖率T/C(％)＝治疗组(T)RTV平均值/阴性对照组(C)RTV平均值×100％。各个时间点各组相对肿瘤增值率见表11，趋势图见图2。
表11各组各时间点相对肿瘤增值率
Figure PCTCN2021073314-appb-000172
结论：
ADC-3组给药后，高剂量组的肿瘤体积相比模型组显著降低，低剂量组的肿瘤体积相比模型组有降低趋势，但没有统计差异。该药显示出了有剂量依赖性的抑制肿瘤生长的作用。同时，3mg/kg剂量给药，ADC-3组在25天时的体内药效优于ADC-2组，二者具有显著性差异。
虽然以上描述了本公开的具体实施方案，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方案做出多种变更或修改。因此，本公开的保护范围由所附权利要求书限定。

Claims (46)

  1. 一种抗体-药物偶联物，其具有式(I)所示结构或其药学上可接受的盐或溶剂化物：
    Ab-(L-D) k
    (I)
    其中，
    Ab为抗体或其抗原结合片段，
    L为将Ab共价连接于D的连接子，且k为1至20，
    -D如下式所示：
    Figure PCTCN2021073314-appb-100001
    其中R 1a选自氢、烷基、环烷基、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选甲基；R 1b选自氢、烷基、烷氧基、环烷基、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选氢；或者R 1a与R 1b与其相连接的原子一起形成5至8元杂环烷基，所述杂环烷基任选被烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，且R 1a和R 1b不同时为氢。
  2. 根据权利要求1所述的抗体-药物偶联物，其中k选自1至10，可以为整数，也可以为小数。
  3. 根据权利要求1或2所述的抗体-药物偶联物，其中所述连接子包含可裂解肽部分。
  4. 根据权利要求3所述的抗体-药物偶联物，其中所述可裂解肽部分能够由酶裂解，优选能够由组织蛋白酶裂解，进一步的所述组织蛋白酶优选组织蛋白酶B。
  5. 根据权利要求3或4所述的抗体-药物偶联物，其中所述连接子包含氨基酸单元，所述氨基酸单元优选包含由2至7个选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸的氨基酸构成的肽残基，更优选缬氨酸-瓜氨酸(Val-Cit)、丙氨酸-丙氨酸-天冬酰胺(Ala-Ala-Asn)、甘氨酸-甘氨酸-赖氨酸(Gly-Gly-lys)、缬氨酸-赖氨酸(Val-lys)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-苯丙氨酸(Val-Phe)或甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)。
  6. 根据权利要求1或2所述的抗体-药物偶联物，其中所述连接子包含可裂解磺酰胺部分。
  7. 根据权利要求1或2所述的抗体-药物偶联物，其中所述连接子包含可裂解二硫化物部分。
  8. 根据权利要求6或7所述的抗体-药物偶联物，其中所述连接子在还原条件下能够裂解。
  9. 根据权利要求1-8任一项所述的抗体-药物偶联物，其中所述连接子包含连接于D的间隔单元。
  10. 根据权利要求9所述的抗体-药物偶联物，其中所述间隔单元包含对氨基苯甲氧基羰基(PAB)。
  11. 根据权利要求9所述的抗体-药物偶联物，其中所述间隔单元包含
    Figure PCTCN2021073314-appb-100002
    其中，Z 1～Z 5各自独立地选自碳原子或氮原子；R 14选自烷基、环烷基、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代；R 11和R 12各自独立选自氢、氘、C 1-6烷基、C 3-6环烷基，优选氢；或者，R 11与R 12与其相连接的碳原子一起形成C 3-6环烷基；X选自-O-或-NH-；L选自1-4之间整数；
    Q为V-E-，V-E-提供了可被位于胞内的糖苷酶切割的糖苷键，E选自-O-、-S- 或-NR 13-，R 13选自氢或甲基；进一步地，V选自
    Figure PCTCN2021073314-appb-100003
    其中R 15选自-COOH或CH 2OH。
  12. 根据权利要求9所述的抗体-药物偶联物，其中所述间隔单元包含选自以下的部分：
    -(CR aR b) m1-O(CR aR b) m2-CR 8R 9-C(O)-、-(CR aR b) m1NH-(CR aR b) m2-CR 8R 9-C(O)-、-(CR aR b) m1O-CR 8R 9(CR aR b) m2-、-(CR aR b) m1OCR 8R 9-C(O)-、-(CR aR b) m1-O-(CR aR b) m2C(O)-和-(CR aR b) m1-S-(CR aR b) m2-CR 8R 9-C(O)-，其中R a和R b相同或不同，且各自独立地选自氢、氘原子、卤素或烷基；R 8选自氢、C 3-6环烷基烷基或C 3-6环烷基；R 9选自氢、卤代烷基或C 3-6环烷基，优选氢；或者，R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基；m1和m2各自独立选自0、1、2或3。
  13. 根据权利要求12所述的抗体-药物偶联物，其中所述间隔单元包含选自以下的部分：
    -(CH 2) 3-C(O)-、-CH 2-O-CH 2-C(O)-、-(CH 2) 2-O-CH 2-C(O)-、
    Figure PCTCN2021073314-appb-100004
    Figure PCTCN2021073314-appb-100005
  14. 根据权利要求1-13任一项所述的抗体-药物偶联物，其中L-D是由下式表示的化学部分：
    -Str-(Pep)-Sp-D
    Str是与Ab共价连接的拉伸单元，
    Sp为间隔单元，
    Pep选自氨基酸单元、二硫化物部分、磺酰胺部分或以下非肽化学部分：
    Figure PCTCN2021073314-appb-100006
    其中，W是-NH-杂环烷基-或杂环烷基；Y是杂芳基、芳基、-C(O)C 1-6亚烷基、C 2-6亚烯基、C 1-6亚烷基或-C 1-6亚烷基-NH-；每个R 2独立选自C 1-10烷基、C 2-10烯基、C 1-6亚烷基-NH 2、-(C 1-10亚烷基)NHC(NH)NH 2或-(C 1-10亚烷基)NHC(O)NH 2；R 3和R 4各自独立为H、C 1-10烷基、C 2-10烯基、芳基烷基、杂芳基烷基，或R 3和R 4一起可形成C 3-7环烷基； R 5和R 6各自独立为C 1-10烷基、C 2-10烯基、芳基烷基、杂芳基烷基、(C 1-10烷基)OCH 2-，或R 5和R 6一起可形成C 3-7环烷基环。
  15. 根据权利要求14所述的抗体-药物偶联物，其中Y选自以下部分：
    Figure PCTCN2021073314-appb-100007
  16. 根据权利要求14所述的抗体-药物偶联物，其中Str选自下式表示的化学部分：
    Figure PCTCN2021073314-appb-100008
    其中R 7选自-W1-C(O)-、-C(O)-W1-C(O)-、-(CH 2CH 2O) p1C(O)-、-(CH 2CH 2O) p1CH 2C(O)-、-(CH 2CH 2O) p1CH 2CH 2C(O)-，其中W1选自C 1-8亚烷基、C 1-8亚烷基-环烷基或1至8个原子的直链杂烷基，所述杂烷基包含1至3个选自N、O或S的杂原子，其中所述的C 1-8亚烷基、环烷基和直链杂烷基各自独立地任选进一步被选自卤素、氘、羟基、氰基、氨基、烷基、卤代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
    L 1选自-NR 10(CH 2CH 2O) p1CH 2CH 2C(O)-、-NR 10(CH 2CH 2O) p1CH 2C(O)-、-S(CH 2) p1C(O)-、-(CH 2) p1C(O)-或化学键，优选化学键；
    其中，p1为1至20的整数，R 10选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基。
  17. 根据权利要求16所述的抗体-药物偶联物，其中R 7选自-C 1-6亚烷基-C(O)-、-(CH 2-CH 2O) 2C(O)-、-(CH 2-CH 2O) 2CH 2C(O)-、-(CH 2-CH 2O) 2CH 2CH 2C(O)-、-(CH 2-CH 2O) 3C(O)-和-(CH 2-CH 2O) 4C(O)-。
  18. 根据权利要求16所述的抗体-药物偶联物，其中R 7选自-C 1-8亚烷基-环烷基-C(O)-、-(CH 2-CH 2O) 4CH 2C(O)-和-(CH 2-CH 2O) 6CH 2C(O)-。
  19. 根据权利要求16-18任一项所述的抗体-药物偶联物，其中连接子L包含：顺丁烯二酰亚胺-(PEG) 2-Val-Cit、顺丁烯二酰亚胺-(PEG) 6-Val-Cit、顺丁烯二酰亚胺-(PEG) 8-Val-Cit、顺丁烯二酰亚胺-(PEG) 4-CH 2CH 2C(O)-Val-lys、顺丁烯二酰亚胺-(CH 2) 5-Val-Cit、顺丁烯二酰亚胺-(CH 2) 5-Val-lys、顺丁烯二酰亚胺-(CH 2) 5-Gly-Gly-Phe-Gly、顺丁烯二酰亚胺-(PEG) 2-Ala-Ala-Asn、顺丁烯二酰亚胺-(PEG) 6-Ala-Ala-Asn、顺丁烯二酰亚胺-(PEG) 8-Ala-Ala-Asn、顺丁烯二酰亚胺-(PEG) 4-三唑-(PEG) 3-磺酰胺、顺丁烯二酰亚胺-(PEG) 2-CH 2CH 2C(O)-Val-lys、顺丁 烯二酰亚胺-(PEG) 4-三唑-(PEG) 3-磺酰胺或Mal-(PEG) 4-三唑-(PEG) 3-二硫化物。
  20. 根据权利要求16-18任一项所述的抗体-药物偶联物，其中连接子L包含：顺丁烯二酰亚胺-(PEG) 4-CH 2C(O)-Gly-Gly-Phe-Gly、顺丁烯二酰亚胺-(PEG) 2-CH 2CH 2C(O)-Gly-Gly-Phe-Gly、顺丁烯二酰亚胺-(PEG) 6-CH 2C(O)-Gly-Gly-Phe-Gly-、顺丁烯二酰亚胺-(CH 2) 5C(O)-Gly-Gly-Phe-Gly-、顺丁烯二酰亚胺-C 1-8亚烷基-环烷基-C(O)-NH(CH 2CH 2O) 4CH 2C(O)-Gly-Gly-Phe-Gly-、顺丁烯二酰亚胺-(PEG) 2-CH 2C(O)-Gly-Gly-Phe-Gly-、顺丁烯二酰亚胺-(PEG) 2-CH 2CH 2C(O)-Val-Cit-、顺丁烯二酰亚胺-(PEG) 2-Gly-Gly-Phe-Gly-、顺丁烯二酰亚胺-(PEG) 2-CH 2C(O)-Val-Cit-、顺丁烯二酰亚胺-(PEG) 4-CH 2C(O)-Val-Cit-和顺丁烯二酰亚胺-(PEG) 6-CH 2C(O)-Val-Cit-。
  21. 根据权利要求14所述的抗体-药物偶联物，其中Str选自下式表示的化学部分：
    Figure PCTCN2021073314-appb-100009
    其中R 8选自C 1-10亚烷基、C 2-10亚烯基、(C 1-10亚烷基)O-、N(R d)-(C 2-6亚烷基)-N(R d)和N(R d)-(C 2-6亚烷基)，且每个R d独立为H或C 1-C 6烷基。
  22. 根据权利要求14或15所述的抗体-药物偶联物，其中L-D由选自以下的式表示：
    Figure PCTCN2021073314-appb-100010
    其中R 2是C 1-6烷基、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2；
    Figure PCTCN2021073314-appb-100011
    其中R 2是C 1-6烷基、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2；
    Figure PCTCN2021073314-appb-100012
    其中R 2是C 1-6烷基、C 2-6亚烯基、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2；
    Figure PCTCN2021073314-appb-100013
    其中R 2是C 1-6烷基、C 2-6亚烯基、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2；
    Figure PCTCN2021073314-appb-100014
    其中R 2是C 1-6烷基、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2，且R 5和R 6一起形成C 3-7环烷基环；
    Figure PCTCN2021073314-appb-100015
    其中R 2是C 1-6烷基、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2，且R 5和R 6一起形成C 3-7环烷基环；
    W和Str如权利要求14中所定义，D如权利要求1中所定义。
  23. 根据权利要求22所述的抗体-药物偶联物，其由下式表示：
    Figure PCTCN2021073314-appb-100016
    其中R 2选自C 1-6亚烷基-NH 2、(C 1- 6亚烷基)NHC(NH)NH 2或(C 1- 6亚烷基)NHC(O)NH 2，k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数，Y、R 3、R 4如权利要求14所定义；
    Figure PCTCN2021073314-appb-100017
    其中R 2选自C 1-6亚烷基-NH 2、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2，k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数，Y、R 3、R 4如权利要求14所定义；
    Figure PCTCN2021073314-appb-100018
    其中R 2是C 1-6亚烷基-NH 2、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2，且R 5和R 6形成C 3-7环烷基环，k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
    Figure PCTCN2021073314-appb-100019
    其中R 2是C 1-6亚烷基-NH 2、(C 1-6亚烷基)NHC(NH)NH 2或(C 1-6亚烷基)NHC(O)NH 2，且R 5和R 6形成C 3-7环烷基环，k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
    Ab、D如权利要求1中所定义。
  24. 根据权利要求12-20任一项所述的抗体-药物偶联物，其由下式表示：
    Figure PCTCN2021073314-appb-100020
    其中R 8选自氢、C 3-6环烷基烷基或C 3-6环烷基，优选氢；R 9选自氢、卤代烷基或C 3-6环烷基，优选氢，或者R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基，k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
    Figure PCTCN2021073314-appb-100021
    其中R 8选自氢、C 3-6环烷基烷基或C 3-6环烷基，优选氢；R 9选自氢、卤代烷基或C 3-6环烷基，优选氢；或者R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基；k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2；
    Figure PCTCN2021073314-appb-100022
    k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
    Figure PCTCN2021073314-appb-100023
    k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
    Figure PCTCN2021073314-appb-100024
    k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2；
    Figure PCTCN2021073314-appb-100025
    k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8；p3选自0、1或2；
    Figure PCTCN2021073314-appb-100026
    k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8；
    Figure PCTCN2021073314-appb-100027
    k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8；p3选自0、1或2；
    Figure PCTCN2021073314-appb-100028
    k选自1至10，可以为整数，也 可以为小数，p1选自2、4、6或8；
    Figure PCTCN2021073314-appb-100029
    k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2；
    Figure PCTCN2021073314-appb-100030
    k选自1至10，可以为整数，也可以为小数，p2选自2、4、6或8；
    Figure PCTCN2021073314-appb-100031
    k选自1至10，可以为整数，也可以为小数，p2选自2、4、6或8；
    Figure PCTCN2021073314-appb-100032
    k选自1至10，可以为整数，也可以为小数，p2选自2、4、6或8；
    Figure PCTCN2021073314-appb-100033
    k选自1至10，可以为整数，也可以为小数，p2选自2、4、6或8；
    Figure PCTCN2021073314-appb-100034
    k选自1至10，可以为整数，也可以为小数，p2选自2、4、6或8；
    Figure PCTCN2021073314-appb-100035
    k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2；
    Figure PCTCN2021073314-appb-100036
    k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2；
    Figure PCTCN2021073314-appb-100037
    其中R 8选自氢、卤代烷基或C 3-6环烷基，优选氢，R 9选自氢、卤代烷基或C 3-6环烷基，优选氢，或者，R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基，k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
    Figure PCTCN2021073314-appb-100038
    其中R 8选自氢、卤代烷基或C 3-6环烷基，优选氢，R 9选自氢、卤代烷基或C 3-6环烷基，优选氢，或者，R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基，k选自1至10，可以为整数，也可以为小数，p2选自2-6之间整数；
    Figure PCTCN2021073314-appb-100039
    其中R 8选自氢、卤代烷基或C 3-6环烷基，优选氢，R 9选自氢、卤代烷基或C 3-6环烷基，优选氢，或者R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基，k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2；
    Figure PCTCN2021073314-appb-100040
    其中R 8选自氢、卤代烷基或C 3-6环烷基，优选氢，R 9选自氢、卤代烷基或C 3-6环烷基，优选氢，或者，R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基，k选自1至10，可以为整数，也可以为小数，p1选自2、4、6或8，p3选自0、1或2；
    Ab、D如权利要求1中所定义。
  25. 根据权利要求1所述的抗体-药物偶联物，其由下式表示：
    Figure PCTCN2021073314-appb-100041
    Figure PCTCN2021073314-appb-100042
    Figure PCTCN2021073314-appb-100043
    Figure PCTCN2021073314-appb-100044
    Figure PCTCN2021073314-appb-100045
    其中k选自1至10，可以为整数，也可以为小数；Ab、D如权利要求1中所定义；进一步地，D中R 1a优选自甲基，R 1b优选自氢。
  26. 根据权利要求1所述的抗体-药物偶联物，其中所述抗体选自鼠源抗体、嵌合抗体、人源化抗体和全人源抗体。
  27. 根据权利要求1所述的抗体-药物偶联物，其中所述的抗体或其抗原结合片段选自抗HER2(ErbB2)抗体、抗EGFR抗体、抗B7-H3抗体、抗c-Met抗体、抗HER3(ErbB3)抗体、抗HER4(ErbB4)抗体、抗CD20抗体、抗CD22抗体、抗CD30抗体、抗CD33抗体、抗CD44抗体、抗CD56抗体、抗CD70抗体、抗CD73抗体、抗CD105抗体、抗CEA抗体、抗A33抗体、抗Cripto抗体、抗EphA2抗体、抗G250抗体、抗MUCl抗体、抗Lewis Y抗体、抗VEGFR抗体、抗GPNMB抗体、抗Integrin抗体、抗PSMA抗体、抗Tenascin-C抗体、抗SLC44A4抗体、抗CD79抗体、抗TROP-2抗体、抗CD79B抗体、抗Mesothelin抗体或其抗原结合片段。
  28. 根据权利要求1-27任一项所述的抗体-药物偶联物，其中所述的抗体或其抗原结合片段选自曲妥珠单抗、帕妥珠单抗、尼妥珠单抗、恩波妥珠单抗、依玛妥珠单抗、奥英妥珠单抗、维汀-匹那妥珠单抗、维布妥昔单抗、吉妥单抗、比伐珠单抗、莫洛伐妥单抗、cBR96和Glematumamab或其抗原结合片段。
  29. 根据权利要求1-27任一项所述的抗体-药物偶联物，其中所述的抗体选自抗CD79B抗体或其抗原结合片段，其包含抗体重链可变区和/或抗体轻链可变区，抗体重链可变区，包含：
    1)分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的HCDR1、HCDR2和HCDR3；或
    2)分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的HCDR1、HCDR2和HCDR3；
    和/或抗体轻链可变区，包含：
    1)分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的LCDR1、LCDR2和LCDR3；或
    2)分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3。
  30. 根据权利要求29所述的抗体-药物偶联物，其中所述抗CD79B抗体包含重链可变区和轻链可变区，包含选自以下1)至2)中的任一项：
    1)重链可变区，其包含分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的LCDR1、LCDR2和LCDR3；
    2)重链可变区，其包含分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的LCDR1、LCDR2和LCDR3。
  31. 根据权利要求29或30所述的抗体-药物偶联物，其中所述抗CD79B抗体包含重链可变区和轻链可变区，其中：
    重链可变区，包含：
    1)如SEQ ID NO：3所示或与SEQ ID NO：3具有至少90％、95％、98％、99％同一性的序列；或
    2)如SEQ ID NO：5所示或与SEQ ID NO：5具有至少90％、95％、98％、99％同一性的序列；
    和/或轻链可变区，包含：
    1)如SEQ ID NO:4所示或与SEQ ID NO：4具有至少90％、95％、98％、99％同一性的序列；或
    2)如SEQ ID NO:6所示或与SEQ ID NO：6具有至少90％、95％、98％、99％ 同一性的序列；
    优选地，所述抗CD79B抗体或抗原结合片段的重链可变区如序列SEQ ID NO：3所示，轻链可变区如序列SEQ ID NO:4所示；或重链可变区如序列SEQ ID NO：5所示，轻链可变区如序列SEQ ID NO:6所示。
  32. 根据权利要求29-31任一项所述的抗体-药物偶联物，其中所述抗CD79B抗体包含重链可变区和轻链可变区，其中：
    重链可变区，包含：
    1)如SEQ ID NO：19所示或与SEQ ID NO：19具有至少90％、95％、98％或99％同一性的序列；或
    2)如SEQ ID NO：21所示或与SEQ ID NO：21具有至少90％、95％、98％、或99％同一性的序列；
    和/或轻链可变区，包含：
    1)如SEQ ID NO:20所示或与SEQ ID NO：20具有至少90％、95％、98％或99％同一性的序列；或
    2)如SEQ ID NO:22所示或与SEQ ID NO：22具有至少90％、95％、98％或99％同一性的序列；
    优选地，所述抗CD79B抗体或抗原结合片段的重链可变区如序列SEQ ID NO：19所示，轻链可变区如序列SEQ ID NO:20所示；或重链可变区如序列SEQ ID NO：21所示，轻链可变区如序列SEQ ID NO:22所示。
  33. 根据权利要求1-27任一项所述的抗体-药物偶联物，其中所述抗TROP-2抗体包含重链可变区和轻链可变区，其中所述重链可变区包含与如SEQ ID NO：29序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：30序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3。
  34. 根据权利要求33所述的抗体-药物偶联物，其中所述抗体选自抗TROP-2抗体，其包含重链可变区和轻链可变区，其中所述重链可变区包含序列分别如SEQ ID NO：23、SEQ ID NO：24和SEQ ID NO：25所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含序列分别如SEQ ID NO：26、SEQ ID NO：27和SEQ ID NO：28所示的LCDR1、LCDR2和LCDR3。
  35. 根据权利要求33或34所述的抗体-药物偶联物，其所述抗TROP-2抗体包含重链可变区和轻链可变区，其中：所述重链可变区氨基酸序列如SEQ ID NO:29所示或与其有至少90％同一性，和所述轻链可变区氨基酸序列如SEQ ID NO: 30所示或与其有至少90％同一性。
  36. 根据权利要求33-35任一项所述的抗体-药物偶联物，其所述抗TROP-2抗体包含序列如SEQ ID NO:29所示的重链可变区和序列如SEQ ID NO：30所示的轻链可变区。
  37. 根据权利要求33-36任一项所述的抗体-药物偶联物，其所述抗TROP-2抗体包含抗体重链恒定区和轻链恒定区；优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；更优选地，所述抗体包含序列如SEQ ID NO:31所示的重链恒定区和序列如SEQ ID NO:32所示的轻链恒定区。
  38. 根据权利要求33-37任一项所述的抗体-药物偶联物，其所述抗TROP-2抗体包含序列如SEQ ID NO:33所示的重链和序列如SEQ ID NO：34所示的轻链。
  39. 根据权利要求1所述的抗体-药物偶联物，其选自以下结构式：
    Figure PCTCN2021073314-appb-100046
    Figure PCTCN2021073314-appb-100047
    Figure PCTCN2021073314-appb-100048
    Figure PCTCN2021073314-appb-100049
    Figure PCTCN2021073314-appb-100050
    Figure PCTCN2021073314-appb-100051
    Figure PCTCN2021073314-appb-100052
    Figure PCTCN2021073314-appb-100053
    Figure PCTCN2021073314-appb-100054
    Figure PCTCN2021073314-appb-100055
    其中：k选自1至10，可以为整数，也可以为小数，进一步地，D中R 1a选自甲基，R 1b选自氢。
  40. 式D所示的化合物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，
    Figure PCTCN2021073314-appb-100056
    其中，R 1a选自氢、烷基、环烷基、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选甲基；R 1b选自氢、烷基、烷氧基、环烷基、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选氢；或者R 1a与R 1b与其相连接的原子一起形成5-8元杂环烷基，所述杂环烷基任选被烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，且R 1a和R 1b不同时为氢。
  41. 式DZ所示的化合物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，
    Figure PCTCN2021073314-appb-100057
    其中，R 1a选自氢、烷基、环烷基、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选甲基；
    R 1b选自氢、烷基、烷氧基、环烷基、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，优选氢；
    或者R 1a与R 1b与其相连接的原子一起形成5-8元杂环烷基，所述杂环烷基任选 被烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环烷基、芳基和杂芳基中的一个或多个取代基所取代，且R 1a和R 1b不同时为氢；
    Y选自-O(CR aR b) m2-CR 8R 9-C(O)-、-NH-(CR aR b) m2-CR 8R 9-C(O)-、-O-CR 8R 9(CR aR b) m2-、-OCR 8R 9-C(O)-、-O(CR aR b) m2C(O)-或-S-(CR aR b) m2-CR 8R 9-C(O)-，其中R a和R b相同或不同，且各自独立地选自氢、氘原子、卤素或烷基；
    R 8选自氢、C 3-6环烷基烷基或C 3-6环烷基；
    R 9选自氢、卤代烷基或C 3-6环烷基，优选氢；
    或者R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基；
    m2选自0、1、2或3。
  42. 根据权利要求41所述的式DZ所示的化合物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其为式DZ-1所示的化合物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐：
    Figure PCTCN2021073314-appb-100058
    其中，R 8选自氢、C 3-6环烷基烷基或C 3-6环烷基；R 9选自氢、卤代烷基或C 3-6环烷基，优选氢；或者，R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基；m2选自0、1、2或3。
  43. 根据权利要求41或42所述的式DZ所示的化合物，或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其选自：
    Figure PCTCN2021073314-appb-100059
    Figure PCTCN2021073314-appb-100060
  44. 一种药物组合物，其含有治疗有效量的根据权利要求1至39任一项所述的抗体-药物偶联物，以及药学上可接受的药用载体、稀释剂或赋形剂。
  45. 根据权利要求1至39任一项所述的抗体-药物偶联物或权利要求44所述药物组合物在制备用于治疗或预防肿瘤的药物中的用途，所述肿瘤优选与HER2、HER3、B7H3或EGFR表达相关的癌症。
  46. 根据权利要求1至39任一项所述的抗体-药物偶联物或权利要求44所述药物组合物在制备治疗和/或预防癌症的药物中的用途，其中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、结肠癌、直肠癌、结直肠癌、白血病、骨癌、皮肤癌、甲状腺癌、胰腺癌和淋巴瘤。
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WO2023124963A1 (zh) * 2021-12-27 2023-07-06 昆山新蕴达生物科技有限公司 一种可逆反应减少的抗体-药物偶联物，其制备方法及应用
WO2023143319A1 (zh) * 2022-01-28 2023-08-03 启德医药科技（苏州）有限公司 抗体药物偶联物、药物组合物及用途
WO2023143347A1 (zh) * 2022-01-26 2023-08-03 上海迈晋生物医药科技有限公司 一种包含抗CD79b抗体药物偶联物的药物组合物及其用途
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WO2023024949A1 (zh) * 2021-08-24 2023-03-02 昆山新蕴达生物科技有限公司 一种由可断裂连接子偶联的抗体偶联药物
WO2023041007A1 (zh) * 2021-09-16 2023-03-23 正大天晴药业集团股份有限公司 抗her2抗体药物偶联物及其组合物和用途
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WO2023143319A1 (zh) * 2022-01-28 2023-08-03 启德医药科技（苏州）有限公司 抗体药物偶联物、药物组合物及用途
WO2023143320A1 (zh) * 2022-01-28 2023-08-03 博瑞生物医药（苏州）股份有限公司 用于制备抗体药物偶联物的连接子、化合物及用途
WO2023143351A1 (zh) * 2022-01-29 2023-08-03 上海盛迪医药有限公司 糖皮质激素的药物偶联物
WO2023155808A1 (zh) * 2022-02-16 2023-08-24 苏州宜联生物医药有限公司 抗体-艾日布林或其衍生物的偶联物、其中间体、制备方法、药物组合物和用途

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