WO2023041007A1 - 抗her2抗体药物偶联物及其组合物和用途 - Google Patents

抗her2抗体药物偶联物及其组合物和用途 Download PDF

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WO2023041007A1
WO2023041007A1 PCT/CN2022/119215 CN2022119215W WO2023041007A1 WO 2023041007 A1 WO2023041007 A1 WO 2023041007A1 CN 2022119215 W CN2022119215 W CN 2022119215W WO 2023041007 A1 WO2023041007 A1 WO 2023041007A1
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cancer
antibody
drug conjugate
optionally substituted
pharmaceutically acceptable
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PCT/CN2022/119215
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English (en)
French (fr)
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陈天玺
徐同杰
唐小齐
丰巍伟
张正平
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正大天晴药业集团股份有限公司
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Priority to CN202280049991.5A priority Critical patent/CN117642186A/zh
Publication of WO2023041007A1 publication Critical patent/WO2023041007A1/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
    • 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
    • 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

Definitions

  • the present invention relates to antibody drug conjugates comprising linked antibody moieties, intermediate linker moieties and cytotoxic drug moieties.
  • the present invention also relates to the use of the antibody-drug conjugate in the preparation of medicines for treating cancer.
  • HER2 human epidermal growth factor receptor 2, human epidermal growth factor receptor 2, also known as ErbB-2 (Receptor tyrosine-protein kinase erbB-2), is the human epidermal growth factor receptor (HER/EGFR/ERBB) family Members of this family include EGFR (ErbB-1), HER2/c-neu (ErbB-2), HER3 (ErbB-3) and HER4 (ErbB-4).
  • the HER2 protein has an extracellular ligand-binding domain (domain I-IV), a transmembrane domain, and an intracellular domain.
  • HER2 forms heterodimers with any of the other three receptors (ErbB-1, ErbB-3, ErbB-4), and dimerization results in autophosphorylation of tyrosine residues within the receptor cytoplasmic domain , and activate a variety of signaling pathways, including MAPK (mitogen-activated protein kinase), PI3K/Akt (phosphoinositide 3-kinase), PKC (protein kinase C), STAT (Signal transducer and activator of transcription). Amplification or overexpression of the HER2 gene plays an important role in the occurrence and development of some invasive breast cancers. 15%-30% of breast cancer patients are HER2 positive. HER2 is an important biomarker and therapeutic target for breast cancer patients. In addition, 7%-34% of gastric cancer patients overexpress HER2, and 30% of salivary duct carcinoma HER2 overexpresses.
  • Antibody-Drug Conjugate is a class of drugs that combines the high specificity of therapeutic antibodies and the high killing activity of cytotoxic drugs, in which the therapeutic antibody part and the cytotoxic drug part pass through the middle linker Partial connection.
  • ADC drugs there are at least ten ADC drugs on the market worldwide.
  • Cytotoxic drugs brentuximab vedotin, polatuzumab vedotin, enfortumab vedotin, and belantamab mafodotin all use auristatins that act on microtubules, and Trastuzumab emtansine uses maytansinoid toxin molecules that act on microtubules.
  • Gemtuzumab ozogamicin and inotuzumab ozogamicin use calicheamicin toxin molecules that act on DNA
  • trastuzumab deruxtecan and sacituzumab govitecan both use camptothecin toxoid molecules
  • loncastuximab tesirine uses PBD dimers that act on DNA.
  • Eribulin (Formula I below) is a synthetic analogue of the natural marine product halichondrin B, which inhibits the growth phase of microtubules, which acts through a tubulin-based anti-mitotic mechanism, resulting in G2/M cell Arrest of the cycle, disruption of the mitotic spindle, and ultimately apoptosis after prolonged mitotic arrest. Eribulin is currently approved for the treatment of metastatic breast cancer and soft tissue sarcoma.
  • ADC drugs combine the dual advantages of high potency of cytotoxic small molecules and high selectivity of antibodies to specific tumor cells.
  • ADC Antibody Drug Conjugate
  • the present invention provides an antibody-drug conjugate, wherein the antibody or its antigen-binding fragment is coupled to the cytotoxic drug eribulin or its derivative; preferably, the antibody or its antigen-binding fragment specifically binds to HER2.
  • the present invention provides an antibody drug conjugate having the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof, wherein Ab represents an antibody moiety, L represents a linker moiety, and U represents For the cytotoxic drug part, n is an integer or decimal selected from 1 to 10.
  • Ab represents an antibody moiety
  • L represents a linker moiety
  • U represents For the cytotoxic drug part
  • n is an integer or decimal selected from 1 to 10.
  • the antibody portion Ab is linked to the linker portion through a specific functional group, and the antibody portion can specifically bind to an antigen.
  • the present invention provides an antibody drug conjugate having the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug moiety U is coupled to the antibody moiety Ab through a linker moiety L couplet.
  • the linker part L of the present invention can be connected to the antibody part by any method known in the art, preferably the linker part and the antibody part are connected through sulfhydryl and/or amino groups.
  • the linker moiety of the invention is linked to the antibody moiety via a sulfhydryl group.
  • the present invention provides an antibody drug conjugate having the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug moiety U is coupled to the antibody moiety Ab via a linker moiety L , the linker portion may be a cleavable linker or a non-cleavable linker.
  • the linker moiety of the present invention is a cleavable linker, such as a low pH-dependent degradable type (including hydrazone bonds, carbonate bonds, etc.), a proteolytic type (including peptidyl bonds), or a high glutathione-dependent degradable type. Glycopeptide concentration degradation type (including disulfide bonds), etc.
  • the cleavable linker can be broken within the target cell, thereby releasing the cytotoxic drug.
  • the linker moiety of the present invention is a non-cleavable linker, such as maleimidocaproyl and the like.
  • the present invention provides an antibody drug conjugate of the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody portion Ab is combined with one or more cytotoxic drugs Part U coupling
  • cytotoxic drugs can be selected from alkaloids, anti-metabolites, antitumor antibiotics, alkylating agents and platinums, etc.
  • preferred cytotoxic drugs are microtubule inhibitors cytotoxic drugs (including Maytansinoids, orlistatins, eribulins, etc.) or cytotoxic drugs that act on DNA (including calicheamicins, duocarmycins, PBD (pyrrolobenzodiazepines), topoisomerase I inhibitors classes, etc.).
  • the cytotoxic drug moiety U of the antibody-drug conjugate having the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof provided by the present invention is a microtubule inhibitor.
  • the cytotoxic drug moiety U of the antibody-drug conjugate of the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof provided by the present invention is eribulin or its derivative.
  • the cytotoxic drug moiety is connected to the linker moiety through a functional group, and the cytotoxic drug molecule will be dissociated in tumor cells, thereby exerting an anti-tumor effect.
  • the present invention provides an antibody drug conjugate having the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof, wherein Ab represents an antibody moiety, L represents a linker moiety, and U represents Cytotoxic drug moiety, n is an integer or decimal selected from 1 to 10, wherein the antibody drug conjugate comprises the structure shown in the following formula IIa:
  • R a is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R b is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R a and R b together with the atoms to which they are attached form an optionally substituted 5- to 8-membered heterocyclic group.
  • each of R a and R b is independently selected from a hydrogen atom, methyl, ethyl, propyl or isopropyl.
  • the R a and R b are hydrogen atoms.
  • the antibody drug conjugate comprises the structure described in Formula IIa-1 below:
  • the present invention provides an antibody drug conjugate having the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof, wherein Ab represents an antibody moiety, L represents a linker moiety, U represents a cytotoxic drug moiety, n is an integer or decimal selected from 1 to 10, wherein -U is a structure shown in formula IIa,
  • R a is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R b is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R a and R b together with the atoms to which they are attached form an optionally substituted 5- to 8-membered heterocyclic group.
  • each of R a and R b is independently selected from a hydrogen atom, methyl, ethyl, propyl or isopropyl.
  • the R a and R b are hydrogen atoms.
  • the -U is the structure shown in Formula IIa-1.
  • the antibody-drug conjugate of the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof provided by the present invention comprises the structure shown in the following formula IIIa:
  • R a is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R b is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R a and R b together with the atoms to which they are attached form an optionally substituted 5- to 8-membered heterocyclic group.
  • each of R a and R b is independently selected from a hydrogen atom, methyl, ethyl, propyl or isopropyl.
  • the R a and R b are hydrogen atoms.
  • the antibody drug conjugate comprises the structure described in Formula IIIa-1 below:
  • the antibody-drug conjugate of the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof provided by the present invention has the structure shown in the following formula IV:
  • Ab represents the antibody portion
  • n is an integer or decimal selected from 1-10;
  • R a is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R b is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R a and R b together with the atoms to which they are attached form an optionally substituted 5- to 8-membered heterocyclic group.
  • the R a and R b are each independently selected from a hydrogen atom or a C 1-5 alkyl group (preferably a C 1-4 alkyl group, such as a C 1-3 alkyl group).
  • each of R a and R b is independently selected from a hydrogen atom, methyl, ethyl, propyl or isopropyl.
  • said R a and R b are hydrogen atoms.
  • the present invention provides an antibody drug conjugate represented by the following formula IV-1 or a pharmaceutically acceptable salt or solvate thereof,
  • n is an integer or a decimal selected from 1 to 10.
  • the n is 2-4.5, 2.2-4.5, 2.2-2.7, 2.5-4.5, 2.7-4.5 , 3.3-4.5, 3.4-4.5, 3.5-4.5, 3.9-4.5, 3.3-4, 3.3-3.9, 3.4-3.9, 7-8, 7-7.5, 7-7.8, 7.1-7.5, 7.1-7.8, 7.5 -7.8, or 7.5-8.
  • the n is about 2.2, about 2.7, about 3.3, about 3.4, about 3.9, about 4, about 7, about 7.1, about 7.5, about 7.8, or about 8.
  • the amount of the cytotoxic drug conjugated with the antibody part in the antibody drug conjugate (ADC) of the present invention can vary, so that the antibody drug conjugate provided in the present invention or its pharmaceutically acceptable salt or solvate can be Heterogeneous, that is, the antibody-drug conjugates of the present invention or pharmaceutically acceptable salts or solvates thereof include antibodies or antigen-binding fragments thereof conjugated with different amounts of cytotoxic drugs, such as 1 molecule of antibody or antigen-binding fragments thereof There are 0 (ie no cytotoxic drugs), 1, 2, 3, 4, 5, 6, 7, 8 or other more molecular cytotoxic drugs coupled.
  • DAR drug-to-antibody ratios
  • DAR is about 3.9 or "n is about 3.9” refers to an antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof comprising each molecule of antibody or antigen-binding fragment thereof conjugated with different A heterogeneous mixture of cytotoxic drugs (e.g., 0, 1, 2, 3, 4, 5, 6, 7, or 8 cytotoxic drugs conjugated to each antibody or antigen-binding fragment thereof), but the cytotoxic drugs are The average molar ratio of antibodies or antigen-binding fragments thereof is about 3.9.
  • cytotoxic drugs e.g., 0, 1, 2, 3, 4, 5, 6, 7, or 8 cytotoxic drugs conjugated to each antibody or antigen-binding fragment thereof
  • the cytotoxic drugs are The average molar ratio of antibodies or antigen-binding fragments thereof is about 3.9.
  • “DAR is about 8" or “n is about 8” means that the average molar ratio of cytotoxic drug to antibody or antigen-binding fragment thereof in the ADC is about 8.
  • the present invention provides an antibody drug conjugate having the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof, wherein Ab (antibody part) can specifically bind to a tumor antigen
  • the tumor antigen can be selected from any tumor therapy target known in the art, non-limiting examples of the target include HER2, HER3, EGFR, CD20, CD30, CD33, CD47, CD79b, VEGF, VEGFR, MET , RET, PD-1 or PD-L1.
  • the present invention provides an antibody drug conjugate represented by formula IV-1 or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab (antibody part) can specifically bind to a tumor antigen, so
  • the tumor antigen can be selected from any tumor treatment target known in the art, non-limiting examples of the target include HER2, HER3, EGFR, CD20, CD30, CD33, CD47, CD79b, VEGF, VEGFR, MET, RET, PD-1, or PD-L1.
  • Ab in the antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof, is an anti-HER2 antibody or an antigen-binding fragment thereof.
  • the Ab is an anti-HER2 antibody or an antigen-binding fragment thereof
  • the anti-HER2 antibody or an antigen-binding fragment thereof comprises heavy chain CDR (HCDR) 1 of the amino acid sequence shown in SEQ ID NO: 1, comprising SEQ ID NO: 1 HCDR2 of the amino acid sequence shown in ID NO: 2, HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, light chain CDR (LCDR) 1 comprising the amino acid sequence shown in SEQ ID NO: 4, comprising SEQ ID NO: 5 LCDR2 showing the amino acid sequence, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:6.
  • HCDR heavy chain CDR
  • HCDR2 of the amino acid sequence shown in SEQ ID NO: 2
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3
  • light chain CDR (LCDR) 1 comprising the amino acid sequence shown in SEQ ID NO: 4
  • LCDR2 showing the amino acid sequence
  • LCDR3 comprising the amino acid sequence shown in
  • the CDR amino acid sequences of the anti-HER2 antibodies or antigen-binding fragments thereof are provided in Table S1 below.
  • the antibody moiety of the antibody-drug conjugate of the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof provided by the present invention is Trastuzumab (Trastuzumab), which Has the sequence shown in Table S1 below.
  • the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises at least 80% of the amino acid sequence shown in SEQ ID NO:7. %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence of 97%, 98%, 99% or 100% identity
  • said light chain variable region comprising at least 80%, 81%, 82%, 83%, 84% of the amino acid sequence shown in SEQ ID NO:8 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical Sexual amino acid sequence.
  • the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, the light chain The variable region comprises the amino acid sequence shown in SEQ ID NO:8.
  • the amino acid sequence of the heavy chain variable region of the anti-HER2 antibody or an antigen-binding fragment thereof is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8 .
  • the anti-HER2 antibody or antigen-binding fragment thereof may further comprise a constant region of an immunoglobulin, or a fragment, analog, variant or derivative of said constant region.
  • the constant region is from a human immunoglobulin heavy chain, such as IgGl, IgG2, IgG3, and IgG4 or heavy chains of other classes of immunoglobulins, preferably IgGl.
  • the constant region may comprise any of the modifications described herein, such as insertions, deletions, substitutions or chemical modifications of amino acids.
  • the constant region comprises mutations that alter effector function.
  • any amino acid residue of the constant region may be substituted with an amino acid residue of any allotype.
  • the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain comprising at least 80%, 81%, 82%, 83% of the amino acid sequence shown in SEQ ID NO: 9 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or An amino acid sequence with 100% identity, said light chain comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% of the amino acid sequence shown in SEQ ID NO: 10 Amino acid sequences that are %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
  • the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO: 9, and the light chain comprising the amino acid sequence set forth in SEQ ID NO: 10 The amino acid sequence shown.
  • the amino acid sequence of the heavy chain of the anti-HER2 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain is shown in SEQ ID NO: 10.
  • the anti-HER2 antibody is selected from the group consisting of Trastuzumab, Pertuzumab, Margetuximab, Initux Monoclonal antibody (Inetetamab), HLX-22, GB-235, HK001, SSGJ-612, ZW25 (Zanidatamab), KN026 (Anbenitamab), BCD-147, MBS-301 or KM257.
  • the anti-HER2 antibody is trastuzumab.
  • the anti-HER2 antibody or antigen-binding fragment thereof is selected from monoclonal antibodies, multispecific antibodies, Fab fragments, Fab' fragments, F(ab)'2 fragments, Fd fragments, Fv fragments, dAb fragments , isolated CDR regions, scFv, Nanobody or fusion protein.
  • the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof has the general formula Ab-(LU) n , wherein Ab can be modified, for example, to include one or more Amino acid sequence changes, additions or subtractions.
  • Ab can be modified, for example, to include one or more Amino acid sequence changes, additions or subtractions.
  • the modified Ab still retains the activity of specifically binding to its corresponding antigen.
  • the antibody drug conjugate provided by the present invention or a pharmaceutically acceptable salt or solvate thereof has the structure shown below:
  • the n is 2-4.5, 2.2-4.5, 2.2-2.7, 2.5-4.5, 2.7-4.5, 3.3-4.5, 3.4-4.5, 3.5-4.5, 3.9-4.5, 3.3- 4, 3.3-3.9, 3.4-3.9, 7-8, 7-7.5, 7-7.8, 7.1-7.5, 7.1-7.8, 7.5-7.8, or 7.5-8.
  • the n is about 2.2, about 2.7, about 3.3, about 3.4, about 3.9, about 4, about 7, about 7.1, about 7.5, about 7.8 or about 8.
  • the antibody-drug conjugate having the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof provided by the present invention exhibits a combination of one or more of the following properties:
  • the antibody drug conjugate, or a pharmaceutically acceptable salt or solvate thereof binds human HER2.
  • the antibody-drug conjugates provided by the present invention or pharmaceutically acceptable salts or solvates thereof show strong endocytosis in cells with different HER2 expression levels, and at the same time, they can increase with endocytosis time. Increases the amount of ADC that is accumulatively endocytosed.
  • the present invention provides a pharmaceutical composition comprising the antibody drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides a pharmaceutical composition, which comprises the antibody drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers or other agents.
  • the present invention provides the use of the antibody drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for treating cancer.
  • the present invention provides the use of a pharmaceutical composition comprising the antibody drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier in the preparation of a drug for treating cancer.
  • the present invention provides the use of the pharmaceutical composition of the present invention in the manufacture of a medicament for the treatment of cancer.
  • the present invention provides an antibody-drug conjugate for treating cancer or a pharmaceutically acceptable salt or solvate thereof, or the above-mentioned pharmaceutical composition.
  • the present invention provides a method of treating cancer, the method comprising administering a therapeutically effective amount of the antibody drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof or comprising the present invention to a patient in need thereof.
  • the present invention provides a method of treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention.
  • the method comprises contacting tumor cells with the antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition, thereby killing tumor cells or inhibiting tumor cell growth .
  • administering a therapeutically effective amount of the antibody drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of the present invention to a patient can kill tumor cells or inhibit the growth of tumor cells .
  • the present invention provides the use of the antibody drug conjugate of the present application or a pharmaceutically acceptable salt or solvate thereof to treat cancer.
  • the present invention provides the application of the pharmaceutical composition comprising the antibody drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier for treating cancer.
  • the present invention also provides the use of the pharmaceutical composition of the present invention for treating cancer.
  • the cancer in the above method or use, is a HER2 positive cancer.
  • administering a therapeutically effective amount of the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition of the present invention to a patient can kill HER2-expressing tumor cells or inhibit HER2 express tumor cell growth.
  • cancers include, but are not limited to: biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, endometrial cancer Carcinoma, Uterine Cancer, Melanoma, Pharyngeal Cancer, Oral Cancer, Skin Cancer, Lung Cancer, Urethral Cancer, Prostate Cancer, Bladder Cancer, Gastrointestinal Stromal Tumor, Squamous Cell Carcinoma, Peritoneal Cancer, Liver Cancer, Uterine Cancer, Salivary Gland Cancer , vulvar, thyroid, penile, leukemia, malignant lymphoma, plasmacytoma, or myeloma.
  • the antibody drug conjugates of the present invention or pharmaceutically acceptable salts or solvates thereof can be used to treat HER2-positive cancers, HER2-negative cancers (including triple-negative breast cancer) and immunohistochemical detection methods Cancers that test for HER2 expression as IHC2+.
  • the present invention provides a linker-drug intermediate compound of the structure shown in the following formula III:
  • R a is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R b is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R a and R b form an optionally substituted 5- to 8-membered heterocyclic group together with the atoms they are connected to.
  • the linker-drug intermediate compound of the structure shown in formula III wherein, R a and R b are each independently selected from a hydrogen atom, methyl, ethyl, propyl or isopropyl.
  • the present invention provides a linker-drug intermediate compound having the structure shown in the following formula III-1,
  • the linker structure adopted in the present invention connects the anti-tumor compound eribulin or its derivatives with the antibody or its antigen-binding fragment, and the provided antibody-drug conjugate achieves excellent anti-tumor effect and/or safety.
  • the antitumor effect and/or safety of the antibody drug conjugate is better than that of eribulin.
  • the provided anti-HER2 antibody drug conjugate exhibits good killing activity on tumor cells, and is effective against various tumor cells and/or cells with different expression levels of HER2 (high and/or medium and/or low) exhibited good killing activity.
  • the provided anti-HER2 antibody drug conjugate exhibits good killing activity on trastuzumab drug-resistant tumor cells.
  • provided anti-HER2 antibody drug conjugates have excellent safety profile. In some embodiments, provided antibody drug conjugates, such as anti-HER2 antibody drug conjugates, are not prone to aggregation. In some experiments, it was found that using the linker structure of the present invention to link the anti-tumor compound eribulin or its derivatives with the antibody or its antigen-binding fragment can improve the anti-aggregation property of the antibody-drug conjugate.
  • Figures 1A-1F are the binding activities of trastuzumab-eribulin conjugates, DS-8201a and trastuzumab with different DAR values to cells with different HER2 expression levels;
  • Figure 2A-2D shows the endocytosis of trastuzumab-eribulin conjugates, DS-8201a and trastuzumab with different DAR values in cells with different HER2 expression levels;
  • Figure 3A-3G shows the cell killing rate of trastuzumab-eribulin conjugates with different DAR values and DS-8201a on cells with different HER2 expression levels
  • Figure 4 shows the bystander effect of trastuzumab-eribulin conjugates, DS-8201a and T-DM1 with different DAR values
  • Figure 5 is the effect of trastuzumab-eribulin conjugates with different DAR values and vehicle control on the change of mouse tumor volume in the nude mouse subcutaneous xenograft tumor model of JIMT-1 human breast cancer cells;
  • Figure 6 shows the effect of trastuzumab-eribulin conjugates with different DAR values and vehicle controls on the tumor weight in mice in the subcutaneous xenograft tumor model of JIMT-1 human breast cancer cells;
  • Figure 7 shows the effect of trastuzumab-eribulin conjugates with different DAR values and vehicle control on the change of mouse body weight in nude mice subcutaneously transplanted tumor model of JIMT-1 human breast cancer cells.
  • substituted means that any one or more hydrogen atoms on the specified atom are replaced by a substituent, as long as the valence of the specified atom is normal and the substituted compound is stable.
  • optionally substituted means substituted or unsubstituted, for example, the ethyl group is "optionally” substituted by halogen, meaning that the ethyl group can be unsubstituted (CH 2 CH 3 ), monosubstituted (such as CH 2 CH 2 F), polysubstituted (eg CHFCH 2 F, CH 2 CHF 2 etc.) or fully substituted (CF 2 CF 3 ). It will be appreciated by those skilled in the art that for any group containing one or more substituents, no sterically impossible and/or synthetically impossible substitution or substitution pattern is introduced.
  • C mn herein, is that the moiety has an integer or fractional number of carbon atoms in the given range.
  • C 1-6 means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.
  • variable e.g, R
  • R any variable
  • its definition is independent at each occurrence. So, for example, if a group is substituted by 2 R's, each R has independent options.
  • linking group When the number of a linking group is 0, such as -(CH 2 ) 0 -, it means that the linking group is a covalent bond.
  • halo or halogen refers to fluorine, chlorine, bromine and iodine.
  • hydroxyl refers to a -OH group.
  • cyano refers to a -CN group.
  • mercapto refers to a -SH group.
  • amino refers to a -NH2 group.
  • nitro refers to a -NO2 group.
  • alkyl refers to a hydrocarbon group of the general formula CnH2n +1 .
  • the alkyl group may be linear or branched.
  • C 1-6 alkyl refers to an alkyl group containing 1 to 6 carbon atoms (such as methyl, ethyl, n - propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc.).
  • the alkyl portion (ie, alkyl group) of alkoxy, alkylamino, dialkylamino, alkylsulfonyl and alkylthio has the same definition as above.
  • alkoxy refers to -O-alkyl
  • alkylamino refers to -NH-alkyl
  • dialkylamino refers to -N(alkyl) 2 .
  • alkylsulfonyl refers to -SO2 -alkyl.
  • alkylthio refers to -S-alkyl.
  • alkenyl refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond.
  • alkenyl include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, and the like.
  • alkynyl refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, having at least one triple bond.
  • alkynyl include, but are not limited to, ethynyl (-C ⁇ CH), 1-propynyl (-C ⁇ C- CH3 ), 2-propynyl (-CH2 - C ⁇ CH), 1,3-Butadiynyl (-C ⁇ CC ⁇ CH), etc.
  • cycloalkyl refers to a carbocyclic ring that is fully saturated and may exist as a monocyclic, bridged or spiro ring. Unless otherwise indicated, the carbocycle is typically a 3 to 10 membered ring.
  • Non-limiting examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl Alkyl etc.
  • cycloalkenyl refers to a non-aromatic carbocyclic ring which is not fully saturated and which may exist as a monocyclic, bridged or spiro ring. Unless otherwise indicated, the carbocycle is typically a 5 to 8 membered ring.
  • Non-limiting examples of cycloalkenyl include, but are not limited to, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadienyl, and the like.
  • heterocyclyl refers to a non-aromatic ring that is fully saturated or partially unsaturated (but not fully unsaturated heteroaromatic) and that can exist as a monocyclic, bridged, or spiro ring.
  • the heterocycle is typically a 3 to 7 membered ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen.
  • heterocyclyl include, but are not limited to, oxiranyl, tetrahydrofuranyl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl , pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothiophenyl, etc.
  • heterocycloalkyl refers to a cyclic group that is fully saturated and can exist as a monocyclic, bridged, or spiro ring. Unless otherwise indicated, the heterocycle is typically a 3 to 7 membered ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen.
  • 3-membered heterocycloalkyl groups include, but are not limited to, oxiranyl, thioethyl, cycloazaethyl
  • 4-membered heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetyl, Cyclic, thiabutanyl
  • 5-membered heterocycloalkyl include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidine , imidazolidinyl, tetrahydropyrazolyl
  • 6-membered heterocycloalkyl include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazin
  • aryl refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated ⁇ -electron system.
  • an aryl group can have 6-20 carbon atoms, 6-14 carbon atoms, or 6-12 carbon atoms.
  • Non-limiting examples of aryl include, but are not limited to, phenyl, naphthyl, anthracenyl, tetralin, and the like.
  • heteroaryl refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O or S, the remaining ring atoms being C, and having at least one aromatic ring.
  • Preferred heteroaryl groups have a single 4 to 8 membered ring, especially a 5 to 8 membered ring, or multiple fused rings comprising 6 to 14, especially 6 to 10 ring atoms.
  • heteroaryl include, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolyl , tetrazolyl, triazolyl, triazinyl, benzofuryl, benzothienyl, indolyl, isoindolyl, etc.
  • Derivative A compound formed by replacing atoms or atomic groups in the molecule of the parent compound with other atoms or atomic groups is called a derivative of the parent compound.
  • tautomer or "tautomeric form” refers to structural isomers of different energies that can interconvert via a low energy barrier.
  • proton tautomers also known as prototropic tautomers
  • proton tautomers include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations.
  • a specific example of a proton tautomer is the imidazole moiety, where a proton can migrate between two ring nitrogens.
  • Valence tautomers include interconversions through recombination of some of the bonding electrons.
  • Compounds of the present application may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers are included, such as enantiomers and diastereomers.
  • the compounds of the present application containing asymmetric carbon atoms can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or reagents.
  • Any atom of the synthesized compound labeled in the present invention may represent any stable isotope of the atom unless otherwise specified. Unless otherwise stated, when a position in a structure is defined as H, hydrogen (H-1), that position contains only the naturally occurring isotope.
  • a position in a structure is defined as D, or deuterium (H-2)
  • the position contains an isotope at least 3340 times greater than the naturally occurring isotope (0.015%) (i.e., at least 50.1% deuterium isotope)
  • the content of the compound shown in the structure can be at least 52.5%, at least 60%, At least 67.5%, at least 75%, at least 82.5%, at least 90%, at least 95%, at least 97%, at least 98.5%, at least 99%, at least 99.5%.
  • the deuterium rate of the labeled synthetic compound in the present invention refers to the ratio of the labeled synthetic isotope content to the naturally occurring isotope amount.
  • the deuterium rate of each specified deuterium atom of the compound marked and synthesized in the present invention can be at least 3500 times (52.5%), at least 4000 times (60%), at least 4500 times (67.5%), at least 5000 times (75%), at least 5500 times (82.5%), at least 6000 times (90%), at least 6333.3 times (95%), at least 6466.7 times (97%), at least 6566.7 times (98.5%), At least 6600 times (99%), at least 6633.3 times (99.5%).
  • Isotopologues in the present invention refer to compounds that differ only in isotopic composition in terms of chemical structure.
  • the compounds labeled and synthesized in the present invention have the same chemical structure, only the isotopic changes in the atomic composition of the molecule. Therefore, the deuterium-containing compound at a specific position synthesized by marking in the present invention will also contain very little hydrogen isotope at this position, and the amount of hydrogen isotope at a certain position in the compound marked and synthesized in the present invention depends on many factors , which includes the deuterium isotope purity of deuterium reagents (D 2 O, D 2 , NaBD 4 , LiAlD 4 , etc.) and the effectiveness of the introduction of deuterium isotope synthesis methods.
  • the amount of such hydrogen isotopologues at a certain position would total less than 49.9% as previously stated.
  • the total amount of hydrogen isotope at a certain position in the compound marked in the present invention will be less than 47.5%, 40%, 32.5%, 25%, 17.5%, 10%, 5%, 3%, 1% or 0.5% %.
  • any atom not designated as deuterium is present in its natural isotopic abundance.
  • bystander effect refers to the effect in which a cytotoxic drug coupled to an antibody or antigen-binding fragment thereof via a cleavable or non-cleavable linker Released has the ability to diffuse across cell membranes and thereby cause killing of adjacent cells.
  • the ability to diffuse across the cell membrane is related to the hydrophobicity of the cytotoxic drug or combination of cytotoxic drug and linker.
  • cytotoxic drugs may eg be eribulin or MMAE.
  • the bystander effect may be desirable.
  • treating means administering the compound or pharmaceutical composition described herein to prevent, improve or eliminate a disease or one or more symptoms associated with the disease, and includes but is not limited to:
  • terapéuticaally effective amount means (i) treating or preventing a particular disease, condition or disorder, (ii) alleviating, ameliorating or eliminating one or more symptoms of a particular disease, condition or disorder, or (iii) preventing or delaying The amount of a compound of the application for the onset of one or more symptoms of a particular disease, condition or disorder described herein.
  • the amount of a compound or pharmaceutical composition of the present application that constitutes a "therapeutically effective amount” may vary according to factors such as the compound or pharmaceutical composition and its ability to elicit a desired response in an individual, the disease state and its severity , the mode of administration and the age, sex and weight of the mammal to be treated.
  • the effective amount can also be routinely determined by those skilled in the art based on their own knowledge and the content of the present invention.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without excessive Toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • salts for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, etc. .
  • solvate refers to a substance formed by the association of a compound with solvent molecules.
  • antibody is used in its broadest sense and encompasses in particular intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (e.g. bispecific antibodies), multifunctional antibodies and antibody fragments, as long as they have the desired biological activity.
  • humanized antibody refers to an antibody comprising CDR regions derived from a non-human antibody and the remainder of the antibody molecule derived from one or more human antibodies.
  • mutant is used to refer to a peptide comprising an amino acid sequence derived from that of the peptide by substitution of one or two or more amino acids, deletion of one or two or more Wild-type amino acid, insertion of one or two or more amino acids not present in the wild-type, and/or addition of amino acids not present in the wild-type to the amino-terminal (N-terminal) and/or carboxy-terminal of the wild-type (C-terminus) (collectively referred to as “mutations").
  • insertion may also be included in “addition”.
  • CDR complementarity determining region
  • hypervariable region refers to each region of an antibody variable domain that is highly variable in sequence and/or forms structurally defined loops.
  • Native four-chain antibodies typically contain six CDRs, three in the heavy chain variable region and three in the light chain variable region.
  • variable region refers to the domain of about 100 to 110 or more amino acids defined by the N-terminal domain of the light or heavy chain of an antibody primarily responsible for antigen recognition.
  • VL light chain variable region
  • VH heavy chain variable region
  • Fab refers to the constant domain (CL) containing the light chain and the first constant domain (CH1) of the heavy chain together with the variable domain VL (light chain variable region) on the light chain and the heavy chain respectively and VH (heavy chain variable region).
  • the variable domains comprise complementarity determining regions (CDRs) involved in antigen binding.
  • scFv includes the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding.
  • ECD extracellular domain.
  • the extracellular domain of HER2 includes four domains, namely ECD1, ECD2, ECD3 and ECD4.
  • antibody moiety refers to the antibody moiety in the antibody drug conjugate. In some specific schemes, it is connected to the intermediate linker part through a specific functional group, and the antibody part can specifically bind to the antigen.
  • linker part refers to the part of the antibody drug conjugate that connects the antibody part to the cytotoxic drug part, which can be cleavable or non-cleavable.
  • the cleavable linker can be broken in the target cell, thereby releasing the cytotoxic drug drug.
  • cytotoxic drug moiety refers to the cytotoxic drug moiety in the antibody drug conjugate. In some specific schemes, it is connected with the intermediate linker part through a functional group, and the cytotoxic drug molecule will be released in the tumor cells, thereby exerting the anti-tumor effect.
  • Trastuzumab with the common name Trastuzumab is a recombinant humanized monoclonal antibody that selectively acts on the ECD4 of human epidermal growth factor receptor-2 (HER2), and can be used to treat HER2-positive Cancer, an example of which is under the trade name Marketed therapeutic monoclonal antibody products.
  • HER2 human epidermal growth factor receptor-2
  • HER2 is the second member of the EGFR family with tyrosine kinase activity, where the expression level of HER2 can be detected by immunohistochemical assay, HER2 positive means IHC3+, HER2 negative means IHC1+/0, for IHC2+ Additional ISH testing should be performed for further clarification.
  • triple negative breast cancer refers to breast cancers that are negative for the expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2.
  • EC50 refers to the effective concentration which elicits 50% of the maximal response of the antigen binding construct. EC50 can be measured by ELISA or FACS analysis or any other method known in the art.
  • identity also called consistency.
  • Percent identity (%) of an amino acid sequence refers to the alignment of the sequences to be compared with the specific amino acid sequences shown herein and after introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without regard to Where any conservative substitutions are considered as part of the sequence identity, the percentage of amino acid residues in the aligned sequences that are identical to the amino acid residues of the particular amino acid sequence shown herein. Alignment of amino acid sequences for identity can be performed by various means within the skill in the art, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the term “subject”, “patient” or “subject” is used interchangeably herein.
  • the term “subject”, “patient” or “subject” is a mammal.
  • the subject, patient or subject is a mouse.
  • the subject, patient or subject is a human.
  • “about” means within a range of acceptable error for a particular value, as judged by one of ordinary skill in the art, which depends in part on how the value is measured or determined, ie, the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean up to ⁇ 5%, for example within ⁇ 2%, within ⁇ 1%, or within ⁇ 0.5% of the particular numerical range given. When a specific value is given in the present application or the patent scope of the invention, unless otherwise stated, the meaning of "about” should be considered as being within the acceptable error range of the specific value.
  • values of step parameters or conditions are modified by "about” by default.
  • the present invention also provides the following specific embodiments, but protection scope of the present invention is not limited thereto:
  • Embodiment 1 An antibody drug conjugate having the general formula Ab-(LU) n or a pharmaceutically acceptable salt or solvate thereof, wherein Ab represents an antibody moiety, L represents a linker moiety, and U represents a cytotoxic drug moiety , n is an integer or decimal selected from 1 to 10, wherein the antibody drug conjugate comprises the structure shown in the following formula IIa:
  • R a is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R b is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R a and R b together with the atoms to which they are attached form an optionally substituted 5- to 8-membered heterocyclic group.
  • Embodiment 2 The antibody-drug conjugate according to embodiment 1 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate comprises the structure shown in the following formula IIIa:
  • Embodiment 3 The antibody-drug conjugate according to embodiment 1 or 2, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate has the structure shown in the following formula IV:
  • n is an integer or decimal selected from 1-10.
  • Embodiment 4 The antibody drug conjugate according to any one of embodiments 1-3, or a pharmaceutically acceptable salt or solvate thereof, wherein R a and R b are each independently selected from a hydrogen atom, a radical, ethyl, propyl or isopropyl.
  • Embodiment 5 The antibody-drug conjugate according to embodiment 1 or 2, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate comprises the structure shown in the following formula IIIa-1:
  • Embodiment 6 The antibody-drug conjugate according to embodiment 3 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate has the structure shown in the following formula IV-1:
  • Embodiment 7 The antibody drug conjugate according to any one of embodiments 1-6, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 2-4.5, 2.2-4.5, 2.2- 2.7, 2.5-4.5, 2.7-4.5, 3.3-4.5, 3.4-4.5, 3.5-4.5, 3.9-4.5, 3.3-4, 3.3-3.9, 3.4-3.9, 7-8, 7-7.5, 7-7.8, 7.1-7.5, 7.1-7.8, 7.5-7.8, or 7.5-8.
  • Embodiment 8 The antibody drug conjugate according to embodiment 7 or a pharmaceutically acceptable salt or solvate thereof, wherein said n is about 2.2, about 2.7, about 3.3, about 3.4, about 3.9, about 4. About 7, about 7.1, about 7.5, about 7.8 or about 8.
  • Embodiment 9 The antibody drug conjugate according to any one of embodiments 1-8, or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab is an anti-HER2 antibody or an antigen-binding fragment thereof.
  • Embodiment 10 The antibody drug conjugate according to embodiment 9 or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:1
  • the HCDR1 comprising the HCDR2 of the amino acid sequence shown in SEQ ID NO:2, the HCDR3 comprising the amino acid sequence shown in SEQ ID NO:3, the LCDR1 comprising the amino acid sequence shown in SEQ ID NO:4, comprising the amino acid sequence shown in SEQ ID NO:5 LCDR2 of amino acid sequence and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:6.
  • Embodiment 11 The antibody drug conjugate according to embodiment 10 or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region A variable region, the heavy chain variable region comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:8 Amino acid sequences of at least 80% identity; or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8.
  • Embodiment 12 The antibody drug conjugate according to embodiment 10 or 11, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, so
  • the heavy chain comprises an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:9
  • the light chain comprises an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:10.
  • Embodiment 13 The antibody drug conjugate according to embodiment 9, or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-HER2 antibody is trastuzumab.
  • Embodiment 14 The antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments 9-13, wherein the antibody drug conjugate or a pharmaceutically acceptable A salt or solvate exhibits one or a combination of the following properties:
  • Embodiment 15 A pharmaceutical composition comprising the antibody drug conjugate of any one of embodiments 1-14 or a pharmaceutically acceptable salt or solvate thereof; optionally, the pharmaceutical composition Also included are pharmaceutically acceptable carriers.
  • Embodiment 16 The antibody drug conjugate described in any one of embodiments 1-14 or a pharmaceutically acceptable salt or solvate thereof or the pharmaceutical composition described in embodiment 15 in the preparation of a medicament for treating cancer
  • the cancer is HER2-positive cancer
  • the cancer is biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer , kidney cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, melanoma, pharyngeal cancer, oral cancer, skin cancer, lung cancer, urethral cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, squamous Cell carcinoma, peritoneal cancer, liver cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, or myeloma.
  • Embodiment 17 A method of treating cancer, comprising administering to a patient in need a therapeutically effective amount of the antibody drug conjugate of any one of embodiments 1-14 or a pharmaceutically acceptable salt or solvate thereof Or the pharmaceutical composition of embodiment 15; preferably, the cancer is a HER2-positive cancer.
  • Embodiment 18 The method according to embodiment 17, comprising contacting tumor cells with the antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition, thereby killing tumor cells or inhibit tumor cell growth.
  • Embodiment 19 The method according to embodiment 17 or 18, wherein the cancer is biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colorectal cancer, Kidney cancer, cervical cancer, ovarian cancer, endometrial cancer, uterine cancer, melanoma, pharyngeal cancer, oral cancer, skin cancer, lung cancer, urethral cancer, prostate cancer, bladder cancer, gastrointestinal stromal tumor, squamous cell cancer, peritoneal cancer, liver cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma or myeloma.
  • the cancer is biliary tract cancer, carcinosarcoma, esophageal cancer, gastroesophageal junction cancer, breast cancer, gastric cancer, pancreatic cancer, head and neck cancer, colore
  • Embodiment 20 A linker-drug intermediate compound having the structure shown in formula III:
  • R a is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R b is selected from hydrogen atom, deuterium atom, optionally substituted C 1-6 alkyl, optionally substituted C 3-7 cycloalkyl, optionally substituted C 3-7 heterocyclyl, optionally Substituted C 6-10 aryl, optionally substituted C 5-12 heteroaryl;
  • R a and R b form an optionally substituted 5- to 8-membered heterocyclic group together with the atoms they are connected to.
  • Embodiment 21 The linker-drug intermediate compound according to embodiment 20, wherein R a and R b are each independently selected from a hydrogen atom, methyl, ethyl, propyl or isopropyl.
  • the trastuzumab (Trastuzumab) used in the examples of the present application was prepared according to the conventional method for antibodies, and the expression vector (including, for example, the pcDNA3.1 vector disclosed in CN107001463A, the pCHO1.0 vector disclosed in CN109422811A, etc.) was constructed first, and then transformed into Expressed after transfection in CHO host cells, and purified by multi-step chromatography; the amino acid sequences of the heavy chain and light chain of trastuzumab are shown in SEQ ID NO: 9 and 10, respectively.
  • DS-8201a is the active ingredient of Enhertu, an ADC drug commercially available from Daiichi Sankyo Co., Ltd.
  • T-DM1 is Trastuzumab-Matansine Union, product name
  • Solution A pH 7.4 PBS buffer
  • Solution B 10mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution
  • Solution D Histidine buffer (containing L-histidine 0.89mg/mL and L-histidine hydrochloride monohydrate 4.04mg/mL)
  • Solution E 700mg/mL sucrose solution (prepared with solution D)
  • Solution F 20mg/mL Tween 80 (prepared with solution D)
  • the DAR value was determined by LC-MS method. Take 50 ⁇ g of ADC sample, add 1 ⁇ L of glycosidase PNGase F (Ruian Biology, China), and incubate at 37°C for 20 hours.
  • the mass spectrometer used in the experiment is a high-resolution Xevo G2-XS (Waters, USA).
  • the sample concentration was adjusted to 5 ⁇ M, and the mass spectrometry data in positive ion mode was collected by direct injection method.
  • the collected non-denaturing mass spectrometry data were analyzed and processed using the software UNIFI 1.8.2.169 (Waters, USA).
  • the protein concentration was detected by the Lowry method. Measure the absorbance value of the sample at OD 650 wavelength with a microplate reader, fit the standard curve, bring the absorbance value of the sample into the standard curve, and calculate the protein concentration.
  • Antibody-drug conjugates of formula IV-1 (including IV-1 (Trastuzumab)) and other antibody-drug conjugates of formula IV series are prepared by the above method:
  • the small molecule cytotoxic compound was diluted to 35000ng/mL-0.0896ng/mL with culture medium, a total of 9 concentrations. Tumor cells in the logarithmic growth phase were collected, adjusted to a density of 1 ⁇ 10 5 cells/mL for plating, 100 ⁇ L was added to each well, and a cell-free blank well was set as a control. Add the above-mentioned serially diluted samples, 50 ⁇ L per well. Cultivate for 5 days in a 37°C, 5% CO 2 carbon dioxide incubator.
  • the antibody-drug conjugate was diluted to 5000ng/mL-0.0128ng/mL with culture medium, a total of 9 concentrations. Take tumor cells in logarithmic growth phase, adjust the density to 2 ⁇ 10 4 cells/mL for plating, add 100 ⁇ L to each well, and set a cell-free blank well as a control. Add the above-mentioned serially diluted samples, 50 ⁇ L per well. Cultured in a 37°C, 5% CO 2 carbon dioxide incubator.
  • Solution G histidine/histidine hydrochloride buffer (L-histidine 1.43mg/mL, L-histidine hydrochloride monohydrate 2.27mg/mL);
  • Solution H 10mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution;
  • Solution I DMSO (dimethyl sulfoxide);
  • Solution J 500mg/mL sucrose solution (prepared with solution G);
  • Solution K 30mg/mL Tween 80 (prepared with solution G);
  • Solution L Solution G in 10% DMSO
  • Solution M 0.3M Na 2 HPO 4 ;
  • Antibody trastuzumab
  • Linker-payload (linker-drug intermediate compound): the compound MC-GGFG-eribulin with the structure shown in Formula III-1 in Example 1.
  • trastuzumab-eribulin conjugates with a DAR of 2.2-2.7 or 7.1-7.5 according to the following experimental procedure, named trastuzumab-eribulin-D2 and trastuzumab respectively Zizumab-eribulin-D8:
  • trastuzumab-eribulin conjugate with a DAR of 3.4-3.9 according to the following experimental procedure, and name it trastuzumab-eribulin-D4:
  • linker-payload Take an appropriate amount of linker-payload and dissolve it with 50% acetone aqueous solution to a final concentration of 10 mg/mL; add an appropriate amount of the above-mentioned linker-payload solution dissolved in acetone aqueous solution to the reaction solution 3, so that the molar weight of linker-payload is N3; The mixture was reacted at 5-10° C. in the dark for 40 min to obtain a reaction solution 4 .
  • ADC Antibody linker-payload N1:N2 N1:N3 Trastuzumab-eribulin-D4 Trastuzumab MC-GGFG-eribulin 1:2.58 1:5.1
  • the components of the trastuzumab-eribulin conjugate prepared in the above example 4 were separated by using a non-porous polystyrene/divinylbenzene (PS/DVB) filler bonded to butyl, and neutral
  • PS/DVB polystyrene/divinylbenzene
  • the high-salt mobile phase improves the hydrophobicity of the protein molecule, thereby combining with the hydrophobic bonded phase in the chromatographic column, and then by gradually reducing the salt concentration and gradually increasing the proportion of isopropanol to elute the substance, the components with low hydrophobicity are first Elution, the most hydrophobic components elute later.
  • the column specifications are Sepax HIC-Butyl, 4.6 ⁇ 100mm, 5 ⁇ m, and the column temperature is 25°C.
  • Mobile phase A is 10mM phosphate buffer-1.5M ammonium sulfate, pH 7.0 (Weigh 1.42g of anhydrous disodium hydrogen phosphate and 198.21g of ammonium sulfate, add about 800mL of ultrapure water, stir until fully dissolved, adjust with phosphoric acid to pH 7.0 ⁇ 0.1, dilute to 1L, mix well and filter through a 0.22 ⁇ m filter membrane).
  • Mobile phase B is 10mM phosphate buffer, pH 7.0 (Weigh 1.42g of anhydrous disodium hydrogen phosphate, add about 800mL ultrapure water, stir until fully dissolved, adjust to pH 7.0 ⁇ 0.1 with phosphoric acid, and dilute to 1L , and filtered through a 0.22 ⁇ m membrane filter).
  • Mobile phase C was 100% isopropanol. Flow rate is 0.5mL/min, gradient elution is 30min, mobile phase parameters are: 0-15min from 75% mobile phase A plus 25% mobile phase B to 75% mobile phase B plus 25% mobile phase C, 15-20min is 75% mobile phase B plus 25% mobile phase C, 20-30min is 75% mobile phase A plus 25% mobile phase B).
  • DAR value (the percentage of ADC peak area containing 0 cytotoxic drugs ⁇ 0 + the percentage of ADC peak area containing 1 cytotoxic drug ⁇ 1 + the percentage of ADC peak area containing 2 cytotoxic drugs ⁇ 2+ ADC peak area percentage with 3 cytotoxic drugs ⁇ 3 + ADC peak area percentage with 4 cytotoxic drugs ⁇ 4 + ADC peak area percentage with 5 cytotoxic drugs ⁇ 5 + ADC with 6 cytotoxic drugs Peak area percentage ⁇ 6+ADC peak area percentage containing 7 cytotoxic drugs ⁇ 7+ADC peak area percentage containing 8 cytotoxic drugs ⁇ 8)/100%.
  • the measured DAR value of trastuzumab-eribulin-D2 is 2.2-2.7; the measured DAR value of trastuzumab-eribulin-D4 is 3.4-3.9; For trastuzumab-eribulin-D8, the measured DAR value is 7.1-7.5.
  • the components of the trastuzumab-eribulin conjugate prepared in Example 4 above were separated by using a gel chromatography column.
  • the neutral pH buffer solution added with 10% isopropanol was used as the mobile phase for elution, and each component was eluted sequentially in descending order of molecular weight.
  • the column is ACQUITY UPLC Protein BEH SEC Column 1.7 ⁇ m, 4.6 ⁇ 300mm gel chromatographic column, the column temperature is 25°C.
  • the mobile phase is 50mM phosphate buffer-200mM sodium chloride-10% isopropanol, pH 7.0 (weigh 12.53g of disodium hydrogen phosphate dodecahydrate, 2.33g of sodium dihydrogen phosphate dihydrate, and 11.69g of sodium chloride, Add about 800mL of ultrapure water, stir until fully dissolved, add ultrapure water to 1000mL for later use, take 100mL of isopropanol and add the above solution to 1000mL, mix well and filter through a 0.22 ⁇ m membrane). Precisely take 20 ⁇ g of trastuzumab-eribulin conjugate and inject it into a liquid chromatograph, and detect it at a wavelength of 280 nm. The flow rate is 0.3mL/min, isocratic elution is 15min.
  • trastuzumab-eribulin conjugate was diluted 5 times with 9 concentration gradients at an initial concentration of 135.14 nM, and diluted in Incubate in FACS buffer (Miltenyi Biotec, product number: 130-091-221) at 4°C for 60 minutes, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, wash with pre-cooled PBS (pH 7.4) for 3 times, add 1:200 (v /v) Diluted goat anti-human IgG Fc ⁇ -PE secondary antibody (Jackson immunoresearch, catalog number: 109-116-170), 100 ⁇ L/well, incubated at 4°C for 30 min.
  • the endocytosis of the trastuzumab-eribulin conjugate prepared in Example 4 above in cells with different HER2 expression levels was analyzed, Including SKBR3 and BT474 cells with high expression level of HER2, JIMT-1 cells with high expression level of HER2 and MCF-7 cells with low expression level of HER2.
  • Sample preparation pre-dilute the trastuzumab-eribulin conjugate to a concentration of 20 ⁇ g/mL, mark it as S1, and then dilute it in a 3-fold concentration gradient to obtain 9 samples S1-S9; Add the sample solution to the cell culture plate, add 50 ⁇ L to each well, and incubate at 4°C for 30 min. After the incubation, the 96-well cell culture plate was taken out, placed at 4°C, centrifuged at 400g for 4min, and the supernatant was discarded.
  • Dilute pHrodo TM Green Maleimide green maleimide; Invitrogen, catalog number: P35370 labeled AffiniPure Goat Anti-Human IgG, Fc ⁇ fragment specific (goat anti-human IgG, Fc fragment specific antibody) according to 1:200 (v/v) ; Jackson Immuno; Cat. No.: 109-005-190), add 50 ⁇ L to each well and incubate at 4°C. After 30 minutes of washing, 50 ⁇ L of cell culture medium was added to each well, mixed well, placed at 37° C. for 2 hours, placed in a flow cytometer (Sartorius, iQUE), and the fluorescence reading value of the BL1 channel was measured.
  • DS-8201a was used as a control, and cells with different expression levels of HER2 were used to detect the killing activity, including NCI-N87, BT474, and SK-OV3 cells with high expression levels of HER2, JIMT-1 and Capan1 cells with high expression levels of HER2, and MCF-7 and KYSE410 cells with low expression levels of HER2.
  • the cells in the logarithmic growth phase were added to a 96-well plate, 100 ⁇ L/well, and the cell density was 1 ⁇ 10 4 cells/mL or 2 ⁇ 10 4 cells/mL. 37°C, 5% CO 2 for overnight culture.
  • Sample preparation The trastuzumab-eribulin conjugate was prepared into a test sample (starting at 5 ⁇ g/mL, 5-fold serial dilution, nine gradients) in basal medium containing 10% FBS.
  • Killing rate (%) (1-luminescence value of experimental group/luminescence value of control group) ⁇ 100%.
  • the processed data was analyzed with Graphpad Prism5, the results are shown in Figures 3A-3G, and the calculated EC50 is shown in Table 5 below.
  • the results showed that the larger the DAR value, the stronger the killing activity of the trastuzumab-eribulin conjugate on cells with high, medium and low expression levels of HER2, and the trastuzumab-eribulin conjugates of each DAR value
  • the killing activity of Bollinger conjugates is better than DS-8201a.
  • Table 5 The killing activity of trastuzumab-eribulin conjugates on cells with different expression levels of HER2
  • T-DM1 and DS-8201a were used as controls, and a blank control group was set up.
  • NCI-N87 was used as positive cells and MDA-MB-468 was used as negative cells.
  • the method based on FACS Tested. Take the NCI-N87 and MDA-MB-468 cells in the logarithmic growth phase, adjust the living cell density of the two kinds of cells to 2 ⁇ 105 cells/mL, add 1mL of the two kinds of cells to each well of the 6-well cell culture plate, Culture overnight in a 37°C, 5% CO 2 cell incubator.
  • the trastuzumab-eribulin conjugate prepared in Example 4 above was pre-diluted to 0.5 ⁇ g/mL (final concentration), and added to the cell plate in sequence. After incubation for 5 days, cells were taken from the 6-well cell culture plate, trypsinized and counted, and the cells in different loading wells were collected, and the cell density was adjusted to 1 ⁇ 10 6 cells/mL with Running buffer (Miltenyi Biotec, catalog number: 130-091-221) , 100 ⁇ L/well was added to a 96-well V-bottom plate.
  • Running buffer Miltenyi Biotec, catalog number: 130-091-221
  • the in vivo efficacy of the trastuzumab-eribulin conjugate prepared in Example 4 was evaluated by using the trastuzumab drug-resistant cell line JIMT-1 human breast cancer cell subcutaneous xenograft model in nude mice.
  • JIMT-1 cells were inoculated subcutaneously in the right axilla of SPF female nude mice (source: Changzhou Cavens Experimental Animal Co., Ltd.), 2 ⁇ 10 6 cells/mouse. When the average volume of the tumor reached 100-300 mm 3 , the animals were divided into 8 groups, 6 animals in each group.
  • the day of grouping was d0 day, and the tail vein was administered on d1 day after grouping.
  • the tumor volume was measured 2-3 times a week, and the mice were weighed at the same time, and the data was recorded; the general performance of the mice was observed and recorded daily. After the experiment, the tumors were stripped, weighed, and photographed.
  • Detection indicators include:
  • Tumor volume TV (mm 3 ) 1/2 ⁇ (a ⁇ b 2 ); wherein, a is the long axis and b is the short axis.
  • Relative tumor volume RTV TV t /TV 0 ; wherein, TV 0 is the tumor volume on day d0, and TV t is the tumor volume at each measurement.
  • T/C (%) T RTV /C RTV ⁇ 100%; wherein, T RTV is the RTV of the treatment group, and C RTV is the RTV of the control group.
  • Tumor growth inhibition rate 1-T/C.
  • Tumor inhibition rate TGI(%) (1-TW/TW 0 ) ⁇ 100%; wherein, TW is the tumor weight of the treatment group, and TW 0 is the tumor weight of the control group.

Abstract

提供了一种抗HER2抗体药物偶联物，具体包含相连接的抗体部分、中间接头部分和细胞毒药物部分。提供的抗体药物偶联物实现了优异的抗肿瘤活性或/和较好的安全性。提供的抗体药物偶联物可以用于癌症的治疗。

Description

抗HER2抗体药物偶联物及其组合物和用途 技术领域
本发明涉及抗体药物偶联物，其包含相连接的抗体部分、中间接头部分和细胞毒药物部分。本发明还涉及所述抗体药物偶联物在制备用于治疗癌症的药物中的用途。
背景技术
HER2(human epidermal growth factor receptor 2，人类表皮生长因子受体2)，也称为ErbB-2(Receptor tyrosine-protein kinase erbB-2)，是人类表皮生长因子受体(HER/EGFR/ERBB)家族的成员，该家族包括EGFR(ErbB-1)、HER2/c-neu(ErbB-2)、HER3(ErbB-3)和HER4(ErbB-4)。HER2蛋白具有细胞外配体结合结构域(domain I-IV)、跨膜结构域和细胞内结构域。HER2与其它三种受体(ErbB-1、ErbB-3、ErbB-4)的任何一种形成异源二聚体，二聚化导致受体胞质结构域内酪氨酸残基的自磷酸化，并激活多种信号通路，包括MAPK(mitogen-activated protein kinase)、PI3K/Akt(phosphoinositide 3-kinase)、PKC(protein kinase C)、STAT(Signal transducer and activator of transcription)。HER2基因的扩增或过表达在某些侵袭性乳腺癌的发生和发展中起重要作用，15％-30％的乳腺癌患者为HER2阳性，HER2是乳腺癌患者重要生物标志物和治疗靶标。此外，7％-34％的胃癌患者HER2过表达、30％的涎腺导管癌HER2过表达。
抗体药物偶联物(Antibody-Drug Conjugate，ADC)是结合了治疗性抗体的高特异性和细胞毒药物的高杀伤活性的一类药物，其中治疗性抗体部分与细胞毒药物部分通过中间的接头部分连接。目前全球范围内已有至少十款ADC药物上市，其中brentuximab vedotin、polatuzumab vedotin与enfortumab vedotin的抗体部分分别针对靶点CD30、CD79b与Nectin-4，Trastuzumab emtansine与Trastuzumab deruxtecan的抗体部分针对HER2靶点，gemtuzumab ozogamicin与inotuzumab ozogamicin的抗体部分分别针对CD33和CD22靶点，sacituzumab govitecan的抗体部分针对TROP2靶点，最新获批的belantamab mafodotin和loncastuximab tesirine分别针对BCMA和CD19靶点。细胞毒药物部分：brentuximab vedotin、polatuzumab vedotin、enfortumab vedotin和belantamab mafodotin均采用作用于微管的奥利斯他汀类(auristatins)，Trastuzumab emtansine采用作用于微管的美登素类(maytansinoid)毒素分子，gemtuzumab ozogamicin与inotuzumab ozogamicin采用作用于DNA的卡奇霉素类(calicheamicins)毒素分子，Trastuzumab deruxtecan和sacituzumab govitecan均采用喜树碱类毒素分子，loncastuximab tesirine则采用作用于DNA的PBD二聚体。中间接头部分：Trastuzumab emtansine、belantamab mafodotin采用不可断裂接头，其余八个分子都采用可断裂的接头。
艾日布林(Eribulin，下式I)为天然海洋产物halichondrin B的一种合成类似物，其可抑制微管生长期，其通过基于微管蛋白的抗有丝分裂机制发挥作用，导致G2/M细胞周期的停滞、有丝分裂纺锤体的破坏，并最终在长期的有丝分裂阻滞后导致细胞凋亡。艾日布林目前已获批用于转移性乳腺癌和软组织肉瘤的治疗。
Figure PCTCN2022119215-appb-000001
ADC类药物结合了细胞毒小分子的高效能和抗体对特定肿瘤细胞的高选择性的双重优点，当前仍然存在开发可以针对更多适应症的高效低毒的ADC药物的需求。
发明内容
抗体药物偶联物(ADC)
本发明提供了一种抗体药物偶联物，其中，抗体或其抗原结合片段与细胞毒药物艾日布林或其衍生物相偶联；优选地，所述抗体或其抗原结合片段特异性结合HER2。
在一个方面，本发明提供了一种具有通式Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中Ab表示抗体部分，L表示接头部分，U表示细胞毒药物部分，n为选自1至10的整数或小数。在某些实施方案中，抗体部分Ab与接头部分通过特定官能团连接，该抗体部分可以与抗原特异性结合。
在一个方面，本发明提供了通式为Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中细胞毒药物部分U与抗体部分Ab通过接头部分L偶联。在一些具体实施方案中，本发明提供的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中每一个细胞毒药物部分U与抗体部分Ab通过一个接头部分L偶联。本发明的接头部分L可以通过本领域已知的任何方法与抗体部分连接，优选接头部分与抗体部分通过巯基和/或氨基连接。在一些更优选的实施方案中，本发明的接头部分通过巯基与抗体部分相连。
在一个方面，本发明提供通式为Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中细胞毒药物部分U与抗体部分Ab通过接头部分L偶联，所述接头部分可以是可切割的接头或者不可切割的接头。在一些实施方案中，本发明的接头部分为可切割的接头，例如可以是依赖低pH值降解型(包括腙键、碳酸酯键等)、蛋白酶解型(包括肽基键)或者依赖高谷胱甘肽浓度降解型(包括二硫键)等。可切割接头可以在靶细胞内断裂，从而释放细胞毒药物。在另一些实施方案中，本发明的接头部分为不可切割的接头，例如可以是马来酰亚氨基己酰基等。
在一个方面，本发明提供了一种通式为Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中抗体部分Ab与一个或更多个细胞毒药物部分U偶联，细胞毒药物例如可以选自生物碱类、抗代谢类、抗肿瘤抗生素类、烷化剂类及铂类等，优选的细胞毒性药物为微管抑制剂类细胞毒药物(包括美登素类、奥利斯他汀类、艾日布林类等)或者作用于DNA的细胞毒药物(包括卡奇霉素类、duocarmycin类、PBD(pyrrolobenzodiazepine)类、拓扑异构酶I抑制剂类等)。
在一些具体实施方案中，本发明提供的通式为Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物的细胞毒药物部分U为微管抑制剂。
在一些具体实施方案中，本发明提供的通式为Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物的细胞毒药物部分U为艾日布林或其衍生物。
在某些实施方案中，所述细胞毒药物部分与接头部分通过官能团连接，在肿瘤细胞内，会游离出细胞毒药物分子，从而发挥抗肿瘤效果。
在一个方面，本发明提供了一种具有通式Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中Ab表示抗体部分，L表示接头部分，U表示细胞毒药物部分，n为选自1至10的整数或小数，其中所述抗体药物偶联物包含以下式IIa所示的结构：
Figure PCTCN2022119215-appb-000002
其中，
R a选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
R b选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
或者，
R a与R b与其相连接的原子一起形成任选被取代的5至8元的杂环基。在一些实施方案中，所述R a与R b各自独立地选自氢原子、甲基、乙基、丙基或异丙基。在一些实施方案中，所述R a和R b为氢原子。
在一些实施方案中，所述抗体药物偶联物包含以下式IIa-1所述的结构：
Figure PCTCN2022119215-appb-000003
在一些实施方案中，本发明提供了一种具有通式Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中Ab表示抗体部分，L表示接头部分，U表示细胞毒药物部分，n为选自1至10的整数或小数，其中所述-U为式IIa所示的结构，
其中，
R a选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
R b选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
或者，
R a与R b与其相连接的原子一起形成任选被取代的5至8元的杂环基。在一些实施方案中，所述R a与R b各自独立地选自氢原子、甲基、乙基、丙基或异丙基。在一些实施方案中，所述R a和R b为氢原子。在一些实施方案中，所述-U为式IIa-1所示的结构。
在一些实施方案中，本发明提供的通式为Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物包含以下式IIIa所示的结构：
Figure PCTCN2022119215-appb-000004
其中，
R a选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环 基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
R b选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
或者，
R a与R b与其相连接的原子一起形成任选被取代的5至8元的杂环基。在一些实施方案中，所述R a与R b各自独立地选自氢原子、甲基、乙基、丙基或异丙基。在一些实施方案中，所述R a和R b为氢原子。
在一些实施方案中，所述抗体药物偶联物包含以下式IIIa-1所述的结构：
Figure PCTCN2022119215-appb-000005
在一些实施方案中，本发明提供的通式为Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物为以下式IV所示的结构：
Figure PCTCN2022119215-appb-000006
其中，Ab表示抗体部分；
n为选自1-10的整数或小数；
R a选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
R b选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
或者，
R a与R b与其相连接的原子一起形成任选被取代的5至8元的杂环基。在一些实施方案中，所述R a与R b各自独立地选自氢原子或C 1-5烷基(优选C 1-4烷基，例如C 1-3烷基)。在一些实施方案中，所述R a与R b各自独立地选自氢原子、甲基、乙基、丙基或异丙基。在一个实施方案中，所述R a和R b为氢原子。
在一个具体实施方案中，本发明提供下式IV-1所示的抗体药物偶联物或其药学上可接受的盐或溶剂化物，
Figure PCTCN2022119215-appb-000007
其中，
Ab为抗体部分，
n为选自1至10的整数或小数。
在一些实施方案中，在上述的抗体药物偶联物或其药学上可接受的盐或溶剂化物中，所述n为2-4.5、2.2-4.5、2.2-2.7、2.5-4.5、2.7-4.5、3.3-4.5、3.4-4.5、3.5-4.5、3.9-4.5、3.3-4、3.3-3.9、3.4-3.9、7-8、7-7.5、7-7.8、7.1-7.5、7.1-7.8、7.5-7.8、或7.5-8。在一些实施方案中，所述n为约2.2、约2.7、约3.3、约3.4、约3.9、约4、约7、约7.1、约7.5、约7.8或约8。
与本发明的抗体药物偶联物(ADC)中抗体部分偶联的细胞毒药物的数量可以变化，使得在本发明提供的抗体药物偶联物或其药学上可接受的盐或溶剂化物可以是异质的，即本发明的抗体药物偶联物或其药学上可接受的盐或溶剂化物包括偶联有不同数量细胞毒药物的抗体或其抗原结合片段，例如1分子抗体或其抗原结合片段偶联有0个(即不含细胞毒药物)、1个、2个、3个、4个、5个、6个、7个、8个或其他更多个分子细胞毒药物。
通过控制上述偶联有不同数量细胞毒药物的抗体或其抗原结合片段的比例，可以产生具有不同药物抗体比率(DAR)的抗体药物偶联物或其药学上可接受的盐或溶剂化物。在本文中，“DAR”与“n”可互换使用。应理解，所述DAR或n是ADC中细胞毒药物与抗体或其抗原结合片段的平均摩尔比值，即每一分子抗体或其抗原结合片段的细胞毒药物平均偶联数。例如，“DAR为约3.9”或“n为约3.9”是指如下的抗体药物偶联物或其药学上可接受的盐或溶剂化物：包含每一分子抗体或其抗原结合片段偶联有不同数量细胞毒药物(例如，每个抗体或其抗原结合片段偶联有0、1、2、3、4、5、6、7或8个细胞毒药物)的异质混合物，但细胞毒药物与抗体或其抗原结合片段的平均摩尔比为约3.9。类似地，“DAR为约8”或“n为约8”是指该ADC中细胞毒药物与抗体或其抗原结合片段的平均摩尔比为约8。
在一个方面，本发明提供的一种具有通式Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中Ab(抗体部分)可以特异性结合肿瘤抗原，所述肿瘤抗原可以选自本领域已知的任何肿瘤治疗的靶点，所述靶点的非限制性实例包括HER2、HER3、EGFR、CD20、CD30、CD33、CD47、CD79b、VEGF、VEGFR、MET、RET、PD-1或PD-L1。在一些实施方案中，本发明提供了一种式IV-1所示的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中Ab(抗体部分)可以特异性结合肿瘤抗原，所述肿瘤抗原可以选自本领域已知的任何肿瘤治疗的靶点，所述靶点的非限制性实例包括HER2、HER3、EGFR、CD20、CD30、CD33、CD47、CD79b、VEGF、VEGFR、MET、RET、PD-1或PD-L1。
在一些实施方案中，所述抗体药物偶联物或其药学上可接受的盐或溶剂化物中，Ab为抗HER2抗体或其抗原结合片段。
在一些实施方案中，所述Ab为抗HER2抗体或其抗原结合片段，所述抗HER2抗体或其抗原结合片段包含SEQ ID NO:1所示氨基酸序列的重链CDR(HCDR)1，包含SEQ ID NO:2所示氨基酸序列的HCDR2，包含SEQ ID NO:3所示氨基酸序列的HCDR3，包含SEQ ID NO:4所示氨基酸序列的轻链CDR(LCDR)1，包含SEQ ID NO:5所示氨基酸序列的LCDR2，和包含SEQ ID NO:6所示氨基酸序列的LCDR3。
所述抗HER2抗体或其抗原结合片段的CDR氨基酸序列提供于下表S1中。
在一些实施方案中，本发明提供的通式为Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物的抗体部分为曲妥珠单抗(Trastuzumab)，其具有下表S1所示的序列。
表S1 曲妥珠单抗的CDR和可变区氨基酸序列
Figure PCTCN2022119215-appb-000008
在一些实施方案中，所述抗HER2抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含与SEQ ID NO:7所示氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，所述轻链可变区包含与SEQ ID NO:8所示氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。在一些实施方案中，所述抗HER2抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:7所示的氨基酸序列，轻链可变区包含SEQ ID NO:8所示的氨基酸序列。在一些实施方案中，所述抗HER2抗体或其抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO:7所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示。
在一些实施方案中，所述抗HER2抗体或其抗原结合片段还可包含免疫球蛋白的恒定区，或所述恒定区的片段、类似物、变体或衍生物。在一些实施方案中，所述恒定区来自人免疫球蛋白重链，例如IgG1、IgG2、IgG3和IgG4或其他类别免疫球蛋白的重链，优选为IgG1的重链。在一些实施方案中，所述恒定区可包含任何文本所述的修饰，例如氨基酸的插入、缺失、取代或化学修饰。在一些实施方案中，所述恒定区包含改变效应功能的突变。在一些实施方案中，所述恒定区的任意氨基酸残基可用任意同种异型(allotype)的氨基酸残基取代。
在一些实施方案中，所述抗HER2抗体或其抗原结合片段包含重链和轻链，所述重链包含与SEQ ID NO:9所示氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，所述轻链包含与SEQ ID NO:10所示氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。在一些实施方案中，所述抗HER2抗体或其抗原结合片段包含重链和轻链，所述重链包含SEQ ID NO:9所示的氨基酸序列，所述轻链包含SEQ ID NO:10所示的氨基酸序列。在一些实施方案中，所述抗HER2抗体或其抗原结合片段的重链的氨基酸序列如SEQ ID NO:9所示，轻链的氨基酸序列如SEQ ID NO:10所示。
Figure PCTCN2022119215-appb-000009
Figure PCTCN2022119215-appb-000010
在一些实施方案中，所述抗HER2抗体选自以下抗体组成的组：曲妥珠单抗(Trastuzumab)、帕妥珠单抗(Pertuzumab)、玛格妥昔单抗(Margetuximab)、伊尼妥单抗(Inetetamab)、HLX-22、GB-235、HK001、SSGJ-612、ZW25(Zanidatamab)、KN026(Anbenitamab)、BCD-147、MBS-301或KM257。在一些具体的实施方案中，所述抗HER2抗体为曲妥珠单抗。
在一些实施方案中，所述抗HER2抗体或其抗原结合片段选自单克隆抗体、多特异性抗体、Fab片段、Fab’片段、F(ab)’2片段、Fd片段、Fv片段、dAb片段、分离的CDR区、scFv、纳米抗体或融合蛋白。
在一些实施方案中，本发明的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其通式为Ab-(L-U) n，其中Ab是可以修饰的，例如包括一个或多个氨基酸序列的改变、增加或减少。在本文中，修饰后的Ab仍然保留了特异性地结合至其相应抗原的活性。
在一些实施方案中，本发明提供的抗体药物偶联物或其药学上可接受的盐或溶剂化物为如下所示的结构：
Figure PCTCN2022119215-appb-000011
在一些这种实施方案中，所述n为2-4.5、2.2-4.5、2.2-2.7、2.5-4.5、2.7-4.5、3.3-4.5、3.4-4.5、3.5-4.5、3.9-4.5、3.3-4、3.3-3.9、3.4-3.9、7-8、7-7.5、7-7.8、7.1-7.5、7.1-7.8、7.5-7.8、或7.5-8。在一些具体的实施方案中，所述n为约2.2、约2.7、约3.3、约3.4、约3.9、约4、约7、约7.1、约7.5、约7.8或约8。
在一个方面，本发明提供的具有通式Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物，展现出下述性质中的一种或多种的组合：
(a)结合HER2；
(b)阻断HER2与配体的结合；
(c)在表达HER2的细胞中显示内吞作用；
(d)对表达HER2的肿瘤细胞具有杀伤活性；
(e)具有旁观者效应。
在一些实施方案中，所述抗体药物偶联物或其药学上可接受的盐或溶剂化物结合人HER2。
在一些实施方案中，本发明提供的抗体药物偶联物或其药学上可接受的盐或溶剂化物在不同HER2表达量细胞中显示了较强的内吞作用，同时能随着内吞时间的增加不断累积内吞的ADC量。
药物组合物
在一个方面，本发明提供了一种药物组合物，其包含本发明的抗体药物偶联物或其药学上可接受的盐或溶剂化物。在一些实施方案中，本发明提供了一种药物组合物，其包含根据本发明的抗体药物偶联物或其药学上可接受的盐或溶剂化物，以及可药用载体。可药用载体包括例如赋形剂、稀释剂、包封材料、填 充剂、缓冲剂或其它试剂。
用途
在一个方面，本发明提供了本发明的抗体药物偶联物或其药学上可接受的盐或溶剂化物在制备用于治疗癌症的药物中的用途。在一个方面，本发明提供了包含本发明的抗体药物偶联物或其药学上可接受的盐或溶剂化物及可药用载体的药物组合物在制备用于治疗癌症的药物中的用途。在一个方面，本发明提供了本发明的药物组合物在制备用于治疗癌症的药物中的用途。
在一个方面，本发明提供了用于治疗癌症的抗体药物偶联物或其药学上可接受的盐或溶剂化物、或上述的药物组合物。
在一个方面，本发明提供了一种治疗癌症的方法，该方法包括向有需要的患者施用治疗有效量的本发明的抗体药物偶联物或其药学上可接受的盐或溶剂化物或者包含本发明的抗体药物偶联物或其药学上可接受的盐或溶剂化物与可药用载体的药物组合物。在一个方面，本发明提供了一种治疗癌症的方法，该方法包括向有需要的患者施用治疗有效量的本发明的药物组合物。在一些实施方案中，所述方法包括使肿瘤细胞与所述抗体药物偶联物或其药学上可接受的盐或溶剂化物、或者所述药物组合物接触，从而杀伤肿瘤细胞或抑制肿瘤细胞生长。
在一些实施方案中，向患者施用治疗有效量的本发明的抗体药物偶联物或其药学上可接受的盐或溶剂化物、或者本发明的药物组合物，可以杀伤肿瘤细胞或抑制肿瘤细胞生长。
在一个方面，本发明提供了本申请的抗体药物偶联物或其药学上可接受的盐或溶剂化物治疗癌症的用途。在一个方面，本发明提供了本申请的包含本发明的抗体药物偶联物或其药学上可接受的盐或溶剂化物、可药用载体的药物组合物治疗癌症的用途。在一个方面，本发明还提供了本发明的药物组合物治疗癌症的用途。
在一些实施方案中，上述的方法或用途中，所述癌症为HER2阳性癌症。在一些实施方案中，向患者施用治疗有效量的本发明的抗体药物偶联物或其药学上可接受的盐或溶剂化物、或者本发明的药物组合物，可以杀伤HER2表达肿瘤细胞或抑制HER2表达肿瘤细胞生长。癌症的实例包括但不限于：胆道癌、癌肉瘤、食管癌、胃食管结合部癌、乳腺癌、胃癌、胰腺癌、头颈癌、结直肠癌、肾癌、宫颈癌、卵巢癌、子宫内膜癌、子宫癌、黑色素瘤、咽癌、口腔癌、皮肤癌、肺癌、尿道癌、前列腺癌、膀胱癌、胃肠道间质瘤、鳞状细胞癌、腹膜癌、肝癌、子宫癌、唾液腺癌、外阴癌、甲状腺癌、阴茎癌、白血病、恶性淋巴瘤、浆细胞瘤或骨髓瘤。
在一些实施方案中，本发明的抗体药物偶联物或其药学上可接受的盐或溶剂化物可以用于治疗HER2阳性癌症、HER2阴性癌症(包括三阴性乳腺癌)以及按照免疫组织化学检测法检测显示HER2表达为IHC2+的癌症。
接头-药物中间体化合物
在一些方面，本发明提供以下式III所示结构的接头-药物中间体化合物：
Figure PCTCN2022119215-appb-000012
其中，
R a选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
R b选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环 基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
或者R a与R b与其相连接的原子一起形成任选被取代的5至8元的杂环基。
在一些实施方案中，所述式III所示结构的接头-药物中间体化合物，其中，R a与R b各自独立地选自氢原子、甲基、乙基、丙基或异丙基。
在一个具体实施方案中，本发明提供以下式III-1所示结构的接头-药物中间体化合物，
Figure PCTCN2022119215-appb-000013
本发明采用的接头结构将接抗肿瘤化合物艾日布林或其衍生物与抗体或其抗原结合片段连接，所提供的抗体药物偶联物实现了优异的抗肿瘤效果及/或安全性。在一些实施方案中，抗体药物偶联物的抗肿瘤效果和/或安全性优于艾日布林。在一些实施方案中，提供的抗HER2抗体药物偶联物表现了对肿瘤细胞良好的杀伤活性，对多种肿瘤细胞和/或不同HER2表达水平(高和/或中和/或低)的细胞表现了良好的杀伤活性。在一些实施方案中，提供的抗HER2抗体药物偶联物对曲妥珠单抗耐药肿瘤细胞表现了良好的杀伤活性。在一些实施方案中，提供的抗HER2抗体药物偶联物具有优异的安全性。在一些实施方案中，提供的抗体药物偶联物如抗HER2抗体药物偶联物不易聚集。在一些实验中发现，采用本发明的接头结构将抗肿瘤化合物艾日布林或其衍生物与抗体或其抗原结合片段连接可以提高抗体药物偶联物的抗聚集性。
附图说明
图1A-1F为不同DAR值的曲妥珠单抗-艾日布林偶联物、DS-8201a和曲妥珠单抗对不同HER2表达水平细胞的结合活性；
图2A-2D为不同DAR值的曲妥珠单抗-艾日布林偶联物、DS-8201a和曲妥珠单抗在不同HER2表达水平细胞中的内吞作用；
图3A-3G为不同DAR值的曲妥珠单抗-艾日布林偶联物、以及DS-8201a对不同HER2表达水平细胞的细胞杀伤率；
图4为不同DAR值的曲妥珠单抗-艾日布林偶联物、DS-8201a和T-DM1的旁观者效应；
图5为不同DAR值的曲妥珠单抗-艾日布林偶联物、以及溶媒对照对JIMT-1人乳腺癌细胞裸小鼠皮下移植瘤模型中小鼠肿瘤体积变化的影响；
图6为不同DAR值的曲妥珠单抗-艾日布林偶联物、以及溶媒对照对JIMT-1人乳腺癌细胞裸小鼠皮下移植瘤模型中小鼠体内瘤重的影响；
图7为不同DAR值的曲妥珠单抗-艾日布林偶联物、以及溶媒对照对JIMT-1人乳腺癌细胞裸小鼠皮下移植瘤模型中小鼠体重变化的影响。
解释和定义
除非另有说明，本申请中所用的下列术语具有下列含义。一个特定的术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照本领域普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
术语“被取代”是指特定原子上的任意一个或多个氢原子被取代基取代，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧代(即＝O)时，意味着两个氢原子被取代，氧代不会发生在芳香基上。
术语“任选”或“任选地”是指随后描述的事件或情况可以发生或不发生，该描述包括发生所述事件 或情况和不发生所述事件或情况。术语“任选被取代的”表示被取代或未被取代的，例如，乙基“任选”被卤素取代，指乙基可以是未被取代的(CH 2CH 3)、单取代的(如CH 2CH 2F)、多取代的(如CHFCH 2F、CH 2CHF 2等)或完全被取代的(CF 2CF 3)。本领域技术人员可理解，对于包含一个或多个取代基的任何基团，不会引入任何在空间上不可能存在和/或不能合成的取代或取代模式。
本文中的C m-n，是该部分具有给定范围中的整数或小数个碳原子。例如“C 1-6”是指该基团可具有1个碳原子、2个碳原子、3个碳原子、4个碳原子、5个碳原子或6个碳原子。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被2个R所取代，则每个R都有独立的选项。
当一个连接基团的数量为0时，比如-(CH 2) 0-，表示该连接基团为共价键。
当其中一个变量选自共价键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表共价键时表示该结构实际上是A-Z。
术语“卤”或“卤素”是指氟、氯、溴和碘。
术语“羟基”指-OH基团。
术语“氰基”指-CN基团。
术语“巯基”指-SH基团。
术语“氨基”指-NH 2基团。
术语“硝基”指-NO 2基团。
术语“烷基”是指通式为C nH 2n+1的烃基。该烷基可以是直链或支链的。例如，术语“C 1- 6烷基”指含有1至6个碳原子的烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、1-甲基丁基、2-甲基丁基、3-甲基丁基、新戊基、己基、2-甲基戊基等)。类似地，烷氧基、烷基氨基、二烷基氨基、烷基磺酰基和烷硫基的烷基部分(即烷基)具有上述相同定义。
术语“烷氧基”指-O-烷基。
术语“烷基氨基”指-NH-烷基。
术语“二烷基氨基”指-N(烷基) 2。
术语“烷基磺酰基”指-SO 2-烷基。
术语“烷硫基”指-S-烷基。
术语“烯基”是指由碳原子和氢原子组成的直链或支链的具有至少一个双键的不饱和脂肪族烃基。烯基的非限制性实例包括但不限于乙烯基、1-丙烯基、2-丙烯基、1-丁烯基、异丁烯基、1,3-丁二烯基等。
术语“炔基”是指由碳原子和氢原子组成的直链或支链的具有至少一个三键的不饱和脂肪族烃基。炔基的非限制性实例包括但不限于乙炔基(-C≡CH)、1-丙炔基(-C≡C-CH 3)、2-丙炔基(-CH 2-C≡CH)、1,3-丁二炔基(-C≡C-C≡CH)等。
术语“环烷基”指完全饱和的并且可以以单环、桥环或螺环存在的碳环。除非另有指示，该碳环通常为3至10元环。环烷基非限制性实例包括但不限于环丙基、环丁基、环戊基、环己基、降冰片基(双环[2.2.1]庚基)、双环[2.2.2]辛基、金刚烷基等。
术语“环烯基”是指不完全饱和的并且可以以单环、桥环或螺环存在的非芳族碳环。除非另有指示，该碳环通常为5至8元环。环烯基的非限制性实例包括但不限于环戊烯基、环戊二烯基、环己烯基、环己二烯基、环庚烯基、环庚二烯基等。
术语“杂环基”是指完全饱和的或部分不饱和的(但不是完全不饱和的杂芳族)并且可以以单环、桥环或螺环存在的非芳族环。除非另有指示，该杂环通常为含有1至3个独立地选自硫、氧和/或氮的杂原子(优选1或2个杂原子)的3至7元环。杂环基的非限制性实例包括但不限于环氧乙烷基、四氢呋喃基、二氢呋喃基、吡咯烷基、N-甲基吡咯烷基、二氢吡咯基、哌啶基、哌嗪基、吡唑烷基、4H-吡喃基、吗啉基、硫代吗啉基、四氢噻吩基等。
术语“杂环烷基”是指完全饱和的并且可以以单环、桥环或螺环存在的环状基团。除非另有指示，该杂环通常为含有1至3个独立地选自硫、氧和/或氮的杂原子(优选1或2个杂原子)的3至7元环。3元杂环烷基的实例包括但不限于环氧乙烷基、环硫乙烷基、环氮乙烷基，4元杂环烷基的非限制性实例包括但不限于吖丁啶基、噁丁环基、噻丁环基，5元杂环烷基的实例包括但不限于四氢呋喃基、四氢噻吩基、 吡咯烷基、异噁唑烷基、噁唑烷基、异噻唑烷基、噻唑烷基、咪唑烷基、四氢吡唑基，6元杂环烷基的实例包括但不限于哌啶基、四氢吡喃基、四氢噻喃基、吗啉基、哌嗪基、1,4-噻噁烷基、1,4-二氧六环基、硫代吗啉基、1,3-二噻烷基、1,4-二噻烷基，7元杂环烷基的实例包括但不限于氮杂环庚烷基、氧杂环庚烷基、硫杂环庚烷基。优选为具有5或6个环原子的单环杂环烷基。
术语“芳基”是指具有共轭的π电子体系的全碳单环或稠合多环的芳香环基团。例如，芳基可以具有6-20个碳原子、6-14个碳原子或6-12个碳原子。芳基的非限制性实例包括但不限于苯基、萘基、蒽基和1,2,3,4-四氢化萘等。
术语“杂芳基”是指单环或稠合多环体系，其中含有至少一个选自N、O或S的环原子，其余环原子为C，并且具有至少一个芳香环。优选的杂芳基具有单个4至8元环，尤其是5至8元环，或包含6至14个，尤其是6至10个环原子的多个稠合环。杂芳基的非限制性实例包括但不限于吡咯基、呋喃基、噻吩基、咪唑基、噁唑基、吡唑基、吡啶基、嘧啶基、吡嗪基、喹啉基、异喹啉基、四唑基、三唑基、三嗪基、苯并呋喃基、苯并噻吩基、吲哚基、异吲哚基等。
“衍生物”：母体化合物分子中的原子或原子团被其它原子或原子团取代形成的化合物称为母体化合物的衍生物。
本申请的化合物和中间体还可以以不同的互变异构体形式存在，并且所有这样的形式包含于本申请的范围内。术语“互变异构体”或“互变异构体形式”是指可经由低能垒互变的不同能量的结构异构体。例如，质子互变异构体(也称为质子转移互变异构体)包括经由质子迁移的互变，如酮-烯醇及亚胺-烯胺异构化。质子互变异构体的具体实例是咪唑部分，其中质子可在两个环氮间迁移。价互变异构体包括通过一些成键电子的重组的互变。
本申请化合物可以是不对称的，例如，具有一个或多个立体异构体。除非另有说明，所有立体异构体都包括，如对映异构体和非对映异构体。本申请的含有不对称碳原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来。光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。
本发明中所标记合成的化合物的任何原子若没有特别指定，可代表该原子的任何一种稳定的同位素。除非特别说明，当结构中某一位置被定义为H即氢(H-1)时，该位置仅含天然存在的同位素。同样，除非特别说明，当结构中某一位置被定义为D即氘(H-2)时，该位置含同位素量至少比天然存在的同位素量(0.015％)大3340倍(即至少含50.1％氘同位素)，当所标记合成的化合物的结构中某一个或多个位置被定义为D即氘(H-2)时，该结构所示的化合物的含量可至少为52.5％、至少为60％、至少为67.5％、至少为75％、至少为82.5％、至少为90％、至少为95％、至少为97％、至少为98.5％、至少为99％、至少为99.5％。本发明中所标记合成的化合物的氘代率是指标记合成的同位素含量与天然存在的同位素量的比值。本发明中所标记合成的化合物的每个指定氘原子的氘代率可至少为3500倍(52.5％)、至少为4000倍(60％)、至少为4500倍(67.5％)、至少为5000倍(75％)、至少为5500倍(82.5％)、至少为6000倍(90％)、至少为6333.3倍(95％)、至少为6466.7倍(97％)、至少为6566.7倍(98.5％)、至少为6600倍(99％)、至少为6633.3倍(99.5％)。本发明中的同位素体(isotopologues)是指在化学结构方面仅有同位素组成上不同的化合物。本发明中所标记合成的化合物具有相同的化学结构、仅在其分子的原子组成中同位素的变化。因此，本发明中所标记合成的在特定位置含氘化合物也同样会含非常少的该位置的氢同位素体，本发明中所标记合成的化合物中的某位置的氢同位素体的量取决许多因素，其中包括氘代试剂(D 2O、D 2、NaBD 4、LiAlD 4等)的氘同位素纯度以及引入氘同位素合成方法的有效性。然而，如前所述这种某位置的氢同位素体的量总数将少于49.9％。本发明中所标记合成的化合物中的某位置的氢同位素体的量总数将少于47.5％、40％、32.5％、25％、17.5％、10％、5％、3％、1％或0.5％。
本发明中，任何未指定为氘的各原子以其天然同位素丰度存在。
术语“旁观者效应”(Bystander effect)，如本文所用，指代这样的效应，其中通过可断裂或不可断裂接头偶联至抗体或其抗原结合片段的细胞毒药物在从抗体或其抗原结合片段释放后有能力扩散穿过细胞膜，并从而引起对相邻细胞的杀伤。扩散穿过细胞膜的能力与细胞毒药物或细胞毒药物和接头的组合的疏水性有关。此类细胞毒药物可以例如是艾日布林或MMAE。尤其是在具有异质靶表达的肿瘤和其中抗体穿透可能受限的实体瘤中，旁观者效应可能是期望的。
术语“治疗”意为将本申请所述化合物或药物组合物进行给药以预防、改善或消除疾病或与所述疾病相关的一个或多个症状，且包括但不限于：
(i)预防疾病或疾病状态在哺乳动物中出现，特别是当这类哺乳动物易患有该疾病状态，但尚未被诊断为已患有该疾病状态时；
(ii)抑制疾病或疾病状态，即遏制其发展；
(iii)缓解疾病或疾病状态，即使该疾病或疾病状态消退；
(iv)降低疾病或疾病状态的任何直接或间接病理学后果。
术语“治疗有效量”意指(i)治疗或预防特定疾病、病况或障碍，(ii)减轻、改善或消除特定疾病、病况或障碍的一种或多种症状，或(iii)预防或延迟本文中所述的特定疾病、病况或障碍的一种或多种症状发作的本申请化合物的用量。构成“治疗有效量”的本申请化合物或药物组合物的量可根据一些因素而变化，例如取决于该化合物或药物组合物及其在个体中引发所需应答的能力，疾病状态及其严重性，给药方式以及待被治疗的哺乳动物的年龄、性别和体重。有效量也可例行性地由本领域技术人员根据其自身的知识及本发明内容而确定。
术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
作为药学上可接受的盐，例如，可以提及金属盐、铵盐、与有机碱形成的盐、与无机酸形成的盐、与有机酸形成的盐、与碱性或者酸性氨基酸形成的盐等。
术语“溶剂化物”是指化合物与溶剂分子缔合形成的物质。
术语“抗体”以其最广义使用，特别涵盖完整的单克隆抗体、多克隆抗体、至少由两种完整抗体所形成的多特异性抗体(例如双特异性抗体)、多功能抗体以及抗体片段，只要它们具有所需的生物学活性。
术语“人源化抗体”是指一种抗体，包含来源于非人源抗体的CDR区，并且该抗体分子的其它部分来源于一种或多种人抗体。
术语“突变体”用于指包含如下从肽的氨基酸序列衍生出的氨基酸序列的肽：用不同于原始肽的氨基酸置换一个或两个或更多个氨基酸，缺失一个或两个或更多个野生型氨基酸，插入在野生型中不存在的一个或两个或更多个氨基酸，和/或将在野生型中不存在的氨基酸添加至野生型的氨基端(N端)和/或羧基端(C端)(被统称为“突变”)。在本发明中，“插入”也可以被包括在“添加”中。
术语“CDR”(互补决定区)，也称为“高变区”，是指在序列上高度可变和/或形成结构限定的环的抗体可变结构域的每个区域。天然四链抗体通常包含六个CDR，三个在重链可变区中，三个在轻链可变区中。
术语“可变区”是指抗体的轻链或重链的N端结构域限定的约100至110个或更多个氨基酸的主要负责抗原识别的结构域。术语轻链可变区(VL)和重链可变区(VH)分别指这些轻链和重链结构域。
术语“Fab”是指含有轻链的恒定结构域(CL)和重链的第一恒定结构域(CH1)连同分别在轻链和重链上的可变结构域VL(轻链可变区)和VH(重链可变区)。可变结构域包含参与抗原结合的互补决定区(CDR)。
术语“scFv”包括抗体的VH和VL结构域，其中这些结构域存在于单一多肽链中。在一些实施方案中，scFv还包含介于VH和VL结构域之间的多肽接头，其使得scFv能够形成抗原结合所需的结构。
术语“ECD”为胞外结构域。HER2的胞外结构域包括四个结构域，分别为ECD1、ECD2、ECD3和ECD4。
术语“抗体部分”是指抗体药物偶联物中的抗体部分。在某些特定的方案中，其与中间接头部分通过特定官能团连接，该抗体部分可以与抗原特异性结合。
术语“接头部分”是指抗体药物偶联物中将抗体部分与细胞毒药物部分相连接的部分，可以是可切割的或者不可切割的，可切割接头可以在靶细胞内断裂，从而释放细胞毒药物。
术语“细胞毒药物部分”是指抗体药物偶联物中的细胞毒药物部分。在某些特定的方案中，其与中间接头部分通过官能团连接，在肿瘤细胞内，会游离出细胞毒药物分子，从而发挥抗肿瘤效果。
术语“曲妥珠单抗”通用名Trastuzumab，是一种重组的人源化单克隆抗体，选择性地作用于人表皮生长因子受体-2(HER2)的ECD4，可以用于治疗HER2阳性的癌症，其一个实例是以商品名
Figure PCTCN2022119215-appb-000014
上市的治疗性单克隆抗体产品。
术语“HER2”是EGFR家族的第二个成员，具有酪氨酸激酶活性，其中HER2表达水平可以按照免疫组织化学检测法进行检测，HER2阳性指IHC3+，HER2阴性指IHC1+/0，对于IHC2+的情况应该再进行ISH检测以进一步明确。
术语“三阴性乳腺癌”是指雌激素受体、孕激素受体和人表皮生长因子受体2表达都为阴性的乳腺癌。
术语“EC 50”是指有效浓度，该浓度引起抗原结合构建体的50％最大应答。EC 50可以通过ELISA或FACS分析或本领域已知的任何其它方法进行测量。
术语“同一性”，也称一致性。氨基酸序列的“同一性百分数(％)”是指将待比对序列与本文中所示的具体氨基酸序列进行比对并且如有必要的话为达到最大序列同一性百分数而引入空位后，并且不考虑任何保守置换作为序列同一性的一部分时，待比对序列中与本文中所示的具体氨基酸序列的氨基酸残基相同的氨基酸残基百分数。同一性的氨基酸序列比对可以采用本领域范围内的多种方式进行，例如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可决定用于比对序列的适宜参数，包括在比较序列的全长里获得最大比对需要的任何算法。
在本文中，术语“受试者”、“患者”或“主体”可互换使用。在一些实施方案中，术语“受试者”、“患者”或“主体”是哺乳动物。在部分实施方案中，所述受试者、患者或主体是小鼠。在部分实施方案中，所述受试者、患者或主体是人。
如本文所用，“约”表示在本领域普通技术人员判定的对特定值可以接受的误差范围内，其部分取决于如何测量或测定该值，即测量系统的限制。例如，“约”按照本领域实践可表示1倍或超过1倍标准偏差以内。或者，“约”可以表示多至±5％的范围，例如在所给定的具体数值范围±2％范围内、±1％范围内或±0.5％范围内波动。当本申请或发明申请专利范围中给出特定值时，除非另有说明，“约”的含义应认为是在该特定值的可接受的误差范围内。在本文中，除非另有说明，步骤参数或条件的值默认均由“约”修饰。
具体实施方式
本发明还提供了以下一些具体的实施方案，但本发明的保护范围不限于此：
实施方案1.一种具有通式Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中Ab表示抗体部分，L表示接头部分，U表示细胞毒药物部分，n为选自1至10的整数或小数，其中，所述抗体药物偶联物包含以下式IIa所示的结构：
Figure PCTCN2022119215-appb-000015
其中，
R a选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
R b选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
或者，
R a与R b与其相连接的原子一起形成任选被取代的5至8元的杂环基。
实施方案2.根据实施方案1所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述 抗体药物偶联物包含以下式IIIa所示的结构：
Figure PCTCN2022119215-appb-000016
实施方案3.根据实施方案1或2所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗体药物偶联物为以下式IV所示结构：
Figure PCTCN2022119215-appb-000017
其中，
Ab表示抗体部分，
n为选自1-10的整数或小数。
实施方案4.根据实施方案1-3中任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，R a与R b各自独立地选自氢原子、甲基、乙基、丙基或异丙基。
实施方案5.根据实施方案1或2所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗体药物偶联物包含以下式IIIa-1所示的结构：
Figure PCTCN2022119215-appb-000018
实施方案6.根据实施方案3所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗体药物偶联物为以下式IV-1所示结构：
Figure PCTCN2022119215-appb-000019
实施方案7.根据实施方案1-6中任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述n为2-4.5、2.2-4.5、2.2-2.7、2.5-4.5、2.7-4.5、3.3-4.5、3.4-4.5、3.5-4.5、3.9-4.5、3.3-4、3.3-3.9、3.4-3.9、7-8、7-7.5、7-7.8、7.1-7.5、7.1-7.8、7.5-7.8、或7.5-8。
实施方案8.根据实施方案7所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述n为约2.2、约2.7、约3.3、约3.4、约3.9、约4、约7、约7.1、约7.5、约7.8或约8。
实施方案9.根据实施方案1-8中任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述Ab为抗HER2抗体或其抗原结合片段。
实施方案10.根据实施方案9所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗HER2抗体或其抗原结合片段包含SEQ ID NO:1所示氨基酸序列的HCDR1，包含SEQ ID NO:2所示氨基酸序列的HCDR2，包含SEQ ID NO:3所示氨基酸序列的HCDR3，包含SEQ ID NO:4所示氨基酸序列的LCDR1，包含SEQ ID NO:5所示氨基酸序列的LCDR2和包含SEQ ID NO:6所示氨基酸序列的LCDR3。
实施方案11.根据实施方案10所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗HER2抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含与SEQ ID NO:7所示氨基酸序列具有至少80％同一性的氨基酸序列，所述轻链可变区包含与SEQ ID NO:8所示氨基酸序列具有至少80％同一性的氨基酸序列；或所述重链可变区包含SEQ ID NO:7所示氨基酸序列，所述轻链可变区包含SEQ ID NO:8所示氨基酸序列。
实施方案12.根据实施方案10或11所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗HER2抗体或其抗原结合片段包含重链和轻链，所述重链包含与SEQ ID NO:9所示氨基酸序列具有至少80％同一性的氨基酸序列，所述轻链包含与SEQ ID NO:10所示氨基酸序列具有至少80％同一性的氨基酸序列。
实施方案13.根据实施方案9所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗HER2抗体为曲妥珠单抗。
实施方案14.根据实施方案9-13中任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗体药物偶联物或其药学上可接受的盐或溶剂化物展现出下述性质中的一种或多种的组合：
(a)结合HER2；
(b)阻断HER2与配体的结合；
(c)在表达HER2的细胞中显示内吞作用；
(d)对表达HER2的肿瘤细胞具有杀伤活性；
(e)具有旁观者效应。
实施方案15.一种药物组合物，其包含实施方案1-14中任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物；任选地，所述药物组合物还包括可药用载体。
实施方案16.实施方案1-14任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物或者实施方案15所述的药物组合物在制备用于治疗癌症的药物中的用途；优选地，所述癌症为HER2阳性癌症；优选地，所述癌症为胆道癌、癌肉瘤、食管癌、胃食管结合部癌、乳腺癌、胃癌、胰腺癌、头颈 癌、结直肠癌、肾癌、宫颈癌、卵巢癌、子宫内膜癌、子宫癌、黑色素瘤、咽癌、口腔癌、皮肤癌、肺癌、尿道癌、前列腺癌、膀胱癌、胃肠道间质瘤、鳞状细胞癌、腹膜癌、肝癌、子宫癌、唾液腺癌、外阴癌、甲状腺癌、阴茎癌、白血病、恶性淋巴瘤、浆细胞瘤或骨髓瘤。
实施方案17.一种治疗癌症的方法，其包括向有需要的患者施用治疗有效量的实施方案1-14任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物或者实施方案15所述的药物组合物；优选地，所述癌症为HER2阳性癌症。
实施方案18.根据实施方案17所述的方法，所述方法包括使肿瘤细胞与所述抗体药物偶联物或其药学上可接受的盐或溶剂化物、或者所述药物组合物接触，从而杀伤肿瘤细胞或抑制肿瘤细胞生长。
实施方案19.根据实施方案17或18所述的方法，其中所述癌症为胆道癌、癌肉瘤、食管癌、胃食管结合部癌、乳腺癌、胃癌、胰腺癌、头颈癌、结直肠癌、肾癌、宫颈癌、卵巢癌、子宫内膜癌、子宫癌、黑色素瘤、咽癌、口腔癌、皮肤癌、肺癌、尿道癌、前列腺癌、膀胱癌、胃肠道间质瘤、鳞状细胞癌、腹膜癌、肝癌、子宫癌、唾液腺癌、外阴癌、甲状腺癌、阴茎癌、白血病、恶性淋巴瘤、浆细胞瘤或骨髓瘤。
实施方案20.一种具有式III所示结构的接头-药物中间体化合物：
Figure PCTCN2022119215-appb-000020
其中，
R a选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
R b选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
或者R a与R b与其相连接的原子一起形成任选被取代的5至8元的杂环基。
实施方案21.根据实施方案20所述的接头-药物中间体化合物，其中，R a与R b各自独立地选自氢原子、甲基、乙基、丙基或异丙基。
为清楚起见，进一步用实施例来阐述本发明，但是实施例并非限制本申请的范围。本申请所使用的试剂通常是市售的，无需进一步纯化即可使用。
本申请实施例中采用的曲妥珠单抗(Trastuzumab)按照抗体的常规方法进行制备，先进行表达载体(包括例如CN107001463A披露的pcDNA3.1载体、CN109422811A披露的pCHO1.0载体等)构建，转染CHO宿主细胞之后进行表达，并采用多步层析的方法进行纯化；曲妥珠单抗重链和轻链的氨基酸序列分别如SEQ ID NO:9和10所示。DS-8201a为第一三共株式会社市售ADC药物Enhertu的活性成分，其结构和合成参见专利公开WO2022033578A1中实施例15的Trastuzumab-DXD；T-DM1为曲妥珠单抗-美坦新偶联物，商品名
Figure PCTCN2022119215-appb-000021
本申请实施例中涉及的细胞及其来源如下表所示：
细胞 来源
NCI-N87 中国科学院细胞库
BT474 中科院
SKBR3 中美冠科
SK-OV3 中国科学院细胞库
JIMT-1 ATCC
Capan1 北纳生物
MCF-7 中国科学院细胞库
KYSE410 雅吉生物
MDA-MB-468 北纳生物
。
实施例1化合物III-1的制备
Figure PCTCN2022119215-appb-000022
称取100mg化合物A(0.12mmol)和75mg化合物B(0.145mmol)加入到20mL反应管中(化合物A的CAS号为2413428-36-9，化合物B的CAS号为441045-17-6)，加入2mL N,N-二甲基甲酰胺，降温到0℃，加入70mg 2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(0.18mmol)与49mg N,N-二异丙基乙胺(0.36mmol)，0℃下反应1h，经制备液相纯化得到化合物III-1(MC-GGFG-Eribulin，或称为MC-GGFG-艾日布林)约70mg。经ESI-MS分析，化合物III-1的m/z＝1241.72[M+H] +。化合物III-1的氢谱如下：
1H NMR(500MHz,DMSO)δ8.22(t,J＝5.3Hz,1H),8.12(d,J＝8.0Hz,1H),8.06(t,J＝5.4Hz,1H),7.99(t,J＝5.3Hz,1H),7.65(d,J＝5.2Hz,1H),7.30-7.21(m,4H),7.18(d,J＝6.4Hz,1H),6.99(s,2H),5.02(d,J＝26.0Hz,2H),4.79(d,J＝38.9Hz,2H),4.65-4.60(m,2H),4.56(d,J＝3.7Hz,1H),4.50-4.42(m,1H),4.26(d,J＝10.1Hz,1H),4.21-4.14(m,1H),4.10(s,3H),4.05-3.98(m,1H),3.86-3.64(m,8H),3.60-3.45(m,4H),3.37-3.30(m,2H),3.26(s,4H),3.17-3.09(m,1H),3.08-2.99(m,2H),2.84(d,J＝10.4Hz,1H),2.86-2.66(m,3H),2.56-2.50m,1H),2.37-2.18(m,5H),2.15-2.05(m,3H),2.05-1.96(m,2H),1.95-1.84(m,4H),1.75-1.57(m,6H),1.55-1.38(m,6H),1.35-1.25(m,3H),1.23-1.12(m,3H),1.03(d,J＝6.0Hz,3H),1.00-0.92(m,1H).
实施例2抗体药物偶联物的制备
试剂：
溶液A：pH 7.4 PBS缓冲液
溶液B：10mM TCEP(三(2-羧基乙基)膦盐酸盐)水溶液
溶液C：DMSO
溶液D：组氨酸缓冲液(含L-组氨酸0.89mg/mL和盐酸L-组氨酸一水合物4.04mg/mL)
溶液E：700mg/mL蔗糖溶液(用溶液D配制)
溶液F：20mg/mL吐温80(用溶液D配制)
实验流程：
1、抗体置换
a、将30KD的超滤离心管用溶液A充分润湿
b、将抗体置换到溶液A中
c、加入适量溶液A调节抗体浓度
2、抗体还原
a、计算抗体摩尔量，记为N1
b、向抗体溶液中加入适量溶液B，使反应体系TCEP摩尔量为N2
c、铝箔包扎，置于旋转培养仪上低速(20rpm)震摇，37℃避光反应1h
3、偶联
a、取适量linker-payload，用DMSO溶解，终浓度为10mg/mL
b、向抗体溶液中加入DMSO，使浓度为5％，再加入适量linker-payload溶液，使摩尔量为N3
c、铝箔包扎，置于旋转培养仪上低速(20rpm)震摇，22℃避光反应1.5h
4、偶联终止
a、用溶液D将超滤离心管润湿
b、将抗体置换至溶液D中，加入适量溶液E、F，-80℃冻存
抗体药物偶联物的DAR值(每一分子抗体的药物平均连接数)测定
采用LC-MS方法测定DAR值。取50μg ADC样品，加入1μL糖苷酶PNGase F(瑞安生物，中国)，在37℃孵育20小时。实验中的质谱仪为高分辨的Xevo G2-XS(Waters，美国)。将样品浓度调至5μM，采用直接进样法，采集正离子模式下的质谱数据。采集的非变性质谱数据采用软件UNIFI 1.8.2.169(Waters，美国)进行分析处理。
抗体药物偶联物的蛋白浓度测定
采用lowry法检测蛋白浓度。用酶标仪测定样品在OD 650波长的吸光度值，拟合标准曲线，将样品吸光度值带入标准曲线，计算蛋白浓度。
通过以上方法制备式IV-1的抗体药物偶联物(包括IV-1(Trastuzumab))以及式IV系列的其他抗体药物偶联物：
Figure PCTCN2022119215-appb-000023
分别制备得到样品1：DAR＝3.3、蛋白浓度＝3.94mg/mL和样品2：DAR＝7.0、蛋白浓度＝3.91mg/mL。
实施例3小分子细胞毒化合物以及抗体药物偶联物的细胞活性
将小分子细胞毒化合物使用培养基稀释至35000ng/mL-0.0896ng/mL，共9个浓度。取对数生长期肿瘤细胞，调整密度至1×10 5cells/mL进行铺板，每孔加入100μL，并设置无细胞空白孔作为对照。加入上述梯度稀释的样品，每孔50μL。于37℃、5％CO 2二氧化碳培养箱中培养5天。弃掉培养液，加入CCK-8(日本同仁化学，货号：CK04)工作液，每孔100μL，孵育显色4-5小时后置于酶标仪中(厂家Thermo，型号：VarioskanFlash)，以630nm为参比波长，读取并记录波长450nm下孔板的吸光度值。计算肿瘤细胞增殖抑制率。
将抗体药物偶联物使用培养基稀释至5000ng/mL-0.0128ng/mL，共9个浓度。取对数生长期肿瘤细胞，调整密度至2×10 4cells/mL进行铺板，每孔加入100μL，并设置无细胞空白孔作为对照。加入上述梯度稀释的样品，每孔50μL。于37℃、5％CO 2二氧化碳培养箱中培养。弃掉培养液，加入CTG检测液(Promega，货号：G7572)，每孔100μL，孵育显色10min后置于多功能读板(厂家Thermo，型号：VarioskanFlash)，读取化学发光值。计算肿瘤细胞增殖抑制率。
IV-1(Trastuzumab)细胞活性：
Figure PCTCN2022119215-appb-000024
实施例4抗体药物偶联物的制备
试剂：
溶液G：组氨酸/盐酸组氨酸缓冲液(L-组氨酸1.43mg/mL、盐酸L-组氨酸一水合物2.27mg/mL)；
溶液H：10mM TCEP(三(2-羧基乙基)膦盐酸盐)水溶液；
溶液I：DMSO(二甲基亚砜)；
溶液J：500mg/mL蔗糖溶液(用溶液G配制)；
溶液K：30mg/mL吐温80(用溶液G配制)；
溶液L：10％DMSO的溶液G；
溶液M：0.3M Na 2HPO 4；
抗体：曲妥珠单抗；
linker-payload(接头-药物中间体化合物)：实施例1中式III-1所示结构的化合物MC-GGFG-艾日布林。
(1)按照如下实验流程制备DAR为2.2-2.7或7.1-7.5的曲妥珠单抗-艾日布林偶联物，分别命名为曲妥珠单抗-艾日布林-D2和曲妥珠单抗-艾日布林-D8：
实验流程：
1、抗体置换：
将30KD的超滤离心管用溶液G充分润湿，将抗体置换到溶液G中，加入适量溶液G调节抗体浓度为10mg/mL；之后加入适量溶液M调节抗体溶液的pH约为7.0。
2、抗体还原：
计算抗体摩尔量，记为N1；向抗体溶液中加入适量溶液H，使反应体系TCEP摩尔量为N2；将获得的混合物于37℃避光振荡1h，从而抗体二硫键被还原，获得反应溶液1。
3、抗体与接头-药物中间体化合物的偶联：
取适量linker-payload，用50％丙酮水溶液溶解，终浓度为10mg/mL；向反应溶液1中加入溶液I(溶液I:反应溶液1(v/v)＝1:10)，混匀，再加入适量的上述用丙酮水溶液溶解的linker-payload溶液，使linker-payload的摩尔量为N3；反应混合物于22℃避光振荡1h，获得反应溶液2。
4、偶联反应的终止：
用溶液L将超滤离心管润湿；先后用20倍体积的溶液L和20倍体积的溶液G来超滤反应溶液2，加 入适量溶液J、K，-80℃冻存。
其中，实验条件及组别如下表1-1所示。
表1-1实验条件及组别
ADC 抗体 linker-payload N1:N2 N1:N3
曲妥珠单抗-艾日布林-D2 曲妥珠单抗 MC-GGFG-艾日布林 1:2 1:3
曲妥珠单抗-艾日布林-D8 曲妥珠单抗 MC-GGFG-艾日布林 1:8.5 1:10.5
(2)按照如下实验流程制备DAR为3.4-3.9的曲妥珠单抗-艾日布林偶联物，命名为曲妥珠单抗-艾日布林-D4：
实验流程：
1、抗体置换：
将30KD的超滤离心管用溶液G充分润湿，将抗体置换到溶液G中，加入适量溶液G调节抗体浓度为10mg/mL；之后加入适量溶液M调节抗体溶液的pH约为7.0。
2、抗体还原：
计算抗体摩尔量，记为N1；向抗体溶液中加入适量溶液H，使反应体系TCEP摩尔量为N2；将获得的混合物于5-10℃避光反应6h，从而抗体二硫键被还原，获得反应溶液3。
3、抗体与接头-药物中间体化合物的偶联：
取适量linker-payload，用50％丙酮水溶液溶解，终浓度为10mg/mL；向反应溶液3中加入适量的上述用丙酮水溶液溶解的linker-payload溶液，使linker-payload的摩尔量为N3；反应混合物于5-10℃避光反应40min，获得反应溶液4。
4、偶联反应的终止：
用溶液L将超滤离心管润湿；先后用20倍体积的溶液L和20倍体积的溶液G来超滤反应溶液4，加入适量溶液J、K，-80℃冻存。
其中，实验条件及组别如下表1-2所示。
表1-2实验条件及组别
ADC 抗体 linker-payload N1:N2 N1:N3
曲妥珠单抗-艾日布林-D4 曲妥珠单抗 MC-GGFG-艾日布林 1:2.58 1:5.1
实施例5抗体药物偶联物的DAR值测定
采用键合丁基的无孔聚苯乙烯/二乙烯苯(PS/DVB)填料对上述实施例4制备的曲妥珠单抗-艾日布林偶联物各组分进行分离，利用中性高盐流动相提高蛋白分子的疏水性质，从而和色谱柱中疏水的键合相结合，而后通过逐步降低盐浓度，并逐步增大异丙醇的比例洗脱物质，疏水性小的组分先洗脱，疏水性大的组分后洗脱。
色谱柱规格为Sepax HIC-Butyl、4.6×100mm、5μm，柱温25℃。流动相A为10mM磷酸盐缓冲液-1.5M硫酸铵，pH 7.0(称取无水磷酸氢二钠1.42g、硫酸铵198.21g，加入约800mL超纯水，搅拌至充分溶解后，用磷酸调节至pH 7.0±0.1，定容至1L，混匀后经0.22μm滤膜过滤)。流动相B为10mM磷酸盐缓冲液，pH 7.0(称取无水磷酸氢二钠1.42g，加入约800mL超纯水，搅拌至充分溶解后，用磷酸调节至pH 7.0±0.1，定容至1L，混匀后经0.22μm滤膜过滤)。流动相C为100％异丙醇。流速为0.5mL/min，梯度洗脱30min，流动相参数为：0-15min由75％流动相A加25％流动相B升至75％流动相B加25％流动相C，15-20min为75％流动相B加25％流动相C，20-30min为75％流动相A加25％流动相B)。用第0min初始流动相将曲妥珠单抗-艾日布林偶联物稀释1倍作为供试品溶液，根据曲妥珠单抗-艾日布林偶联物浓度调整进样体积进样50μg蛋白，在280nm波长处检测吸光度值。
数据处理，采用面积归一化法对结果进行定量分析。分别计算含有0、1、2、3、4、5、6、7和8个细胞毒药物的ADC的峰面积百分比，并计算DAR值结果。计算公式为：DAR值＝(含有0个细胞毒药物的ADC峰面积百分比×0+含有1个细胞毒药物的ADC峰面积百分比×1+含有2个细胞毒药物的ADC峰面积百分比×2+含有3个细胞毒药物的ADC峰面积百分比×3+含有4个细胞毒药物的ADC峰面积百分比×4+含有5个细胞毒药物的ADC峰面积百分比×5+含有6个细胞毒药物的ADC峰面积百分比×6+含有7个细胞 毒药物的ADC峰面积百分比×7+含有8个细胞毒药物的ADC峰面积百分比×8)/100％。
通过实施例4和实施例5的方法制备并测定曲妥珠单抗-艾日布林偶联物，其具有如下结构：
Figure PCTCN2022119215-appb-000025
其中，曲妥珠单抗-艾日布林-D2，其测得的DAR值为2.2-2.7；曲妥珠单抗-艾日布林-D4，其测得的DAR值为3.4-3.9；曲妥珠单抗-艾日布林-D8，其测得的DAR值为7.1-7.5。
实施例6抗体药物偶联物的聚集体验证
采用凝胶色谱柱对上述实施例4制备的曲妥珠单抗-艾日布林偶联物各组分进行分离。利用添加10％异丙醇的中性pH缓冲液作为流动相进行洗脱，各组分按照分子量由大到小的顺序依次被洗出。色谱柱为ACQUITY UPLC Protein BEH SEC Column
Figure PCTCN2022119215-appb-000026
1.7μm、4.6×300mm规格的凝胶色谱柱，柱温25℃。流动相为50mM磷酸盐缓冲液-200mM氯化钠-10％异丙醇，pH 7.0(称取十二水合磷酸氢二钠12.53g、二水合磷酸二氢钠2.33g、氯化钠11.69g，加入约800mL超纯水，搅拌至充分溶解，加超纯水至1000mL备用，取异丙醇100mL加上述溶液至1000mL，混匀后经0.22μm滤膜过滤)。精密取20μg曲妥珠单抗-艾日布林偶联物注入液相色谱仪，在280nm波长处检测。流速为0.3mL/min，等度洗脱15min。
数据处理，采用面积归一化法对结果进行定量分析。分别计算聚集体、免疫球蛋白单体和低分子量杂质的峰面积百分比，其中主峰前为聚集体，主峰为免疫球蛋白单体，主峰后为低分子量杂质。曲妥珠单抗-艾日布林偶联物的单体、聚集体及低分子量杂质含量比例见表2。
表2曲妥珠单抗-艾日布林偶联物的单体、聚集体及低分子量杂质含量
Figure PCTCN2022119215-appb-000027
实施例7抗体药物偶联物的细胞结合活性
基于FACS的方法，以DS-8201a和曲妥珠单抗作为对照，分析上述实施例4制备的曲妥珠单抗-艾日布林偶联物与不同HER2表达水平细胞的结合活性，包括HER2高表达水平的NCI-N87、BT474细胞，HER2中表达水平的JIMT-1、Capan1细胞，HER2低表达水平的MCF-7细胞以及HER2阴性的MDA-MB-468细胞。
将1×10 5个细胞加入到96孔细胞培养板的各孔中，曲妥珠单抗-艾日布林偶联物以135.14nM起始浓度，5倍稀释、9个浓度梯度，稀释于FACS buffer(Miltenyi Biotec，货号：130-091-221)中，于4℃孵育60min后，1000rpm离心5min，弃上清，用预冷PBS(pH 7.4)洗涤3次，加入按照1:200(v/v)稀释的羊抗人IgG Fcγ-PE二抗(Jackson immunoresearch，货号：109-116-170)，100μL/孔，4℃孵育30min。用预冷PBS(pH 7.4)洗涤3次，用100μL PBS(pH 7.4)重悬，然后使用流式细胞仪(Sartorius，iQUE)分析检测荧光信号。通过染色的平均荧光强度(MFI)来测量曲妥珠单抗-艾日布林偶联物和对照与上述各细胞的结合活性。使用GraphPad Prism5分析数据，结果如图1A-1F所示，计算的EC 50在下表3中示出。结果表明，各DAR值的曲妥珠单抗-艾日布林偶联物与HER2高、中、低表达水平细胞的结合活性均与曲妥珠单抗的相当，与HER2阴性的MDA-MB-468细胞无结合。
表3曲妥珠单抗-艾日布林偶联物与不同HER2表达水平细胞的结合活性
Figure PCTCN2022119215-appb-000028
实施例8抗体药物偶联物的内吞实验
基于FACS的方法，以DS-8201a和曲妥珠单抗作为对照，分析上述实施例4制备的曲妥珠单抗-艾日布林偶联物在不同HER2表达水平细胞中的内吞情况，包括HER2高表达水平的SKBR3、BT474细胞，HER2中表达水平的JIMT-1细胞以及HER2低表达水平的MCF-7细胞。
调整细胞密度为1×10 6个/mL，50μL/孔加入96孔细胞培养板。样品准备：将曲妥珠单抗-艾日布林偶联物预稀释至浓度为20μg/mL，标记为S1，其后按3倍浓度梯度稀释，获得9个样品S1-S9；把梯度稀释的样品溶液加到细胞培养板中，每孔加50μL，4℃孵育30min。孵育结束后取出96孔细胞培养板，置于4℃，400g离心4min，弃上清。按照1:200(v/v)稀释pHrodo TM Green Maleimide(绿色马来酰亚胺；Invitrogen，货号：P35370)标记的AffiniPure Goat Anti-Human IgG,Fcγfragment specific(羊抗人IgG，Fc片段特异性抗体；Jackson Immuno；货号：109-005-190)，每孔加50μL后于4℃孵育。30min后洗涤，每孔加入50μL细胞培养基，混匀后置于37℃内化2h，置于流式细胞仪(Sartorius，iQUE)，测定BL1通道的荧光读值。使用GraphPad Prism5分析数据，结果如图2A-2D所示，计算的EC 50在下表4中示出。结果表明，各DAR值的曲妥珠单抗-艾日布林偶联物在不同HER2表达水平细胞中的内吞作用与曲妥珠单抗的相当。
表4曲妥珠单抗-艾日布林偶联物在不同HER2表达水平细胞中的内吞作用
Figure PCTCN2022119215-appb-000029
实施例9抗体药物偶联物的细胞杀伤活性
为检测上述实施例4制备的曲妥珠单抗-艾日布林偶联物对肿瘤细胞的增殖抑制作用，以DS-8201a作为对照，分别用不同HER2表达水平的细胞进行杀伤活性检测，包括HER2高表达水平的NCI-N87、BT474、SK-OV3细胞，HER2中表达水平的JIMT-1、Capan1细胞，以及HER2低表达水平的MCF-7、KYSE410细胞。
取对数生长期的细胞加入96孔板，100μL/孔，细胞密度为1×10 4个/mL或2×10 4个/mL。37℃、5％CO 2贴壁过夜培养。样品准备：将曲妥珠单抗-艾日布林偶联物用含10％FBS的基础培养基配制成待测样品(5μg/mL起始，5倍梯度稀释、九个梯度)。取出过夜贴壁培养的细胞，实验组加入50μL/孔稀释好的待测样品，对照组加入50μL/孔含10％FBS的基础培养基，继续培养96h、120h或144h后采用CellTiter-Glo Luminescent Cell试剂盒(Promega，货号：G7572)检测，取出96孔板，在每孔中加入75μL CTG检测液(Promega，货号：G7572)，震荡混匀，室温避光孵育10min后每孔各吸取180μL转移至不透明白板，去除气泡，读取化学发光值，并计算杀伤率。
杀伤率(％)＝(1-实验组发光值/对照组发光值)×100％。
用Graphpad Prism5分析处理数据，结果如图3A-3G所示，计算的EC 50在下表5中示出。结果表明，DAR值越大，曲妥珠单抗-艾日布林偶联物对HER2高、中、低表达水平细胞的杀伤活性越强，且各DAR值的曲妥珠单抗-艾日布林偶联物的杀伤活性均优于DS-8201a。
表5曲妥珠单抗-艾日布林偶联物对不同HER2表达水平细胞的杀伤活性
Figure PCTCN2022119215-appb-000030
实施例10抗体药物偶联物的旁观者效应
为检测抗体药物偶联物的旁观者效应，以T-DM1、DS-8201a作为对照，并设置空白对照组，使用NCI-N87作为阳性细胞、MDA-MB-468作为阴性细胞，基于FACS的方法进行了检测。取对数生长期的NCI-N87和MDA-MB-468细胞，调整两种细胞的活细胞密度分别为2×10 5个/mL，每孔加入两种细胞各1mL至6孔细胞培养板，置于37℃、5％CO 2细胞培养箱中过夜培养。过夜后将上述实施例4制备的曲妥珠单抗-艾日布林偶联物预稀释至0.5μg/mL(终浓度)，按孔板顺序加入细胞板中。孵育5d后，取6孔细胞培养板细胞，胰酶消化计数，收集不同加样孔细胞，用Running buffer(Miltenyi Biotec，货号：130-091-221)调整细胞密度至1×10 6个/mL，100μL/孔加入96孔V底板。在96孔V底板中加入直标抗体PE anti-human CD340 antibody(Biolegend，货号：324406)，4℃孵育60min。孵育后洗涤细胞两次，重悬于Running buffer溶液中，流式细胞仪(
Figure PCTCN2022119215-appb-000031
NxT)检测信号。使用GraphPad Prism5分析数据，结果如图4所示。结果表明，各DAR值的曲妥珠单抗-艾日布林偶联物均具有旁观者效应，且其DAR值越大，对阴性细胞的杀伤活性越强。
实施例11抗体药物偶联物在JIMT-1人乳腺癌细胞裸小鼠皮下移植瘤模型中的药效学评价
通过曲妥珠单抗耐药细胞株JIMT-1人乳腺癌细胞裸小鼠皮下移植瘤模型评价上述实施例4制备的曲妥珠单抗-艾日布林偶联物的体内药效。
在SPF级雌性裸小鼠(来源：常州卡文斯实验动物有限公司)右侧腋窝皮下接种JIMT-1细胞，2×10 6个/只。待肿瘤平均体积达100-300mm 3时，将动物分成8组，每组6只，具体分组和给药方案在表6中示出。
表6分组和给药方案
组别 药物 剂量(mg/kg) 给药途径 给药频次
1 溶媒对照 N/A 尾静脉注射 单次
2 曲妥珠单抗-艾日布林-D2 1 尾静脉注射 单次
3 曲妥珠单抗-艾日布林-D2 3 尾静脉注射 单次
4 曲妥珠单抗-艾日布林-D4 1 尾静脉注射 单次
5 曲妥珠单抗-艾日布林-D4 3 尾静脉注射 单次
6 曲妥珠单抗-艾日布林-D8 1 尾静脉注射 单次
7 曲妥珠单抗-艾日布林-D8 3 尾静脉注射 单次
分组当天为d0天，分组后d1天尾静脉给药。每周测2-3次瘤体积，同时给小鼠称重，记录数据；每日观察与记录小鼠一般表现。实验结束后剥取肿瘤并称重、拍照。
检测指标包括：
肿瘤体积TV(mm 3)＝1/2×(a×b 2)；其中，a为长径，b为短径。
相对肿瘤体积RTV＝TV t/TV 0；其中，TV 0为d0天肿瘤体积，TV t为每一次测量时的肿瘤体积。
相对肿瘤增殖率T/C(％)＝T RTV/C RTV×100％；其中，T RTV为治疗组RTV，C RTV为对照组RTV。
肿瘤生长抑制率：1-T/C。
抑瘤率TGI(％)＝(1-TW/TW 0)×100％；其中，TW为治疗组瘤重，TW 0为对照组瘤重。
体重变化率，WCR(％)＝(Wt t-Wt 0)/Wt 0×100％；其中，Wt 0为d0天动物体重，Wt t为每一次测量时的动物体重。
各药物对肿瘤体积、瘤重、及小鼠体重的影响见图5-7，检测指标结果在下表7中示出。到d21天实验结束，无动物死亡，各治疗组的小鼠体重增长与模型组相当或高于模型组，药物无明显毒性作用，安全 性好。各DAR值的曲妥珠单抗-艾日布林偶联物均表现出较好的体内肿瘤增殖抑制活性，且存在剂量依赖性；在低剂量组中，曲妥珠单抗-艾日布林偶联物的DAR值越大，体内肿瘤增殖抑制活性越强。
表7曲妥珠单抗-艾日布林偶联物在JIMT-1人乳腺癌细胞裸小鼠移植瘤模型中的肿瘤抑制作用
组别 肿瘤生长抑制率1-T/C 抑瘤率TGI
2 50.0％ 48.0％
3 96.9％ 96.1％
4 59.4％ 57.6％
5 93.7％ 93.9％
6 65.6％ 67.2％
7 93.7％ 93.4％
根据本发明所公开的内容，虽然根据优选实施方案对本发明的方法进行了描述，但对本领域技术人员而言，在不背离本发明的概念、精神和范围的情况下，可对在此所述的方法以及所述方法的步骤或步骤的顺序进行改变。
本文所引用的所有文献的公开内容通过引用结合于此，引用程度为，他们提供示例性的，程序上和其他的细节补充本文所述内容。

Claims (20)

  1. 一种具有通式Ab-(L-U) n的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中Ab表示抗体部分，L表示接头部分，U表示细胞毒药物部分，n为选自1至10的整数或小数，其特征在于，所述抗体药物偶联物包含以下式IIa所示的结构：
    Figure PCTCN2022119215-appb-100001
    其中，
    R a选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
    R b选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
    或者，
    R a与R b与其相连接的原子一起形成任选被取代的5至8元的杂环基。
  2. 根据权利要求1所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗体药物偶联物包含以下式IIIa所示的结构：
    Figure PCTCN2022119215-appb-100002
  3. 根据权利要求1或2所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗体药物偶联物为以下式IV所示结构：
    Figure PCTCN2022119215-appb-100003
    其中，
    Ab表示抗体部分，
    n为选自1-10的整数或小数。
  4. 根据权利要求1-3中任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，R a与R b各自独立地选自氢原子、甲基、乙基、丙基或异丙基。
  5. 根据权利要求1或2所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗体药物偶联物包含以下式IIIa-1所示的结构：
    Figure PCTCN2022119215-appb-100004
  6. 根据权利要求3所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗体药物偶联物为以下式IV-1所示结构：
    Figure PCTCN2022119215-appb-100005
  7. 根据权利要求1-6中任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述n为2-4.5、2.2-4.5、2.2-2.7、2.5-4.5、2.7-4.5、3.3-4.5、3.4-4.5、3.5-4.5、3.9-4.5、3.3-4、3.3-3.9、3.4-3.9、7-8、7-7.5、7-7.8、7.1-7.5、7.1-7.8、7.5-7.8、或7.5-8。
  8. 根据权利要求7所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述n为约2.2、约2.7、约3.3、约3.4、约3.9、约4、约7、约7.1、约7.5、约7.8或约8。
  9. 根据权利要求1-8中任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述Ab为抗HER2抗体或其抗原结合片段。
  10. 根据权利要求9所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗HER2抗体或其抗原结合片段包含SEQ ID NO:1所示氨基酸序列的HCDR1，包含SEQ ID NO:2所示氨基酸序列的HCDR2，包含SEQ ID NO:3所示氨基酸序列的HCDR3，包含SEQ ID NO:4所示氨基酸序列的LCDR1，包含SEQ ID NO:5所示氨基酸序列的LCDR2和包含SEQ ID NO:6所示氨基酸序列的LCDR3。
  11. 根据权利要求10所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗HER2抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含与SEQ ID NO:7所示氨基酸序列具有至少80％同一性的氨基酸序列，所述轻链可变区包含与SEQ ID NO:8所示氨基酸序列具有至少80％同一性的氨基酸序列；或所述重链可变区包含SEQ ID NO:7所示氨基酸序列，所述轻链可变区包含SEQ ID NO:8所示氨基酸序列。
  12. 根据权利要求10或11所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗HER2抗体或其抗原结合片段包含重链和轻链，所述重链包含与SEQ ID NO:9所示氨基酸序列具有至少80％同一性的氨基酸序列，所述轻链包含与SEQ ID NO:10所示氨基酸序列具有至少80％同一性的氨基酸序列。
  13. 根据权利要求9所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗HER2抗体为曲妥珠单抗。
  14. 根据权利要求9-13中任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其中，所述抗体药物偶联物或其药学上可接受的盐或溶剂化物展现出下述性质中的一种或几种的组合：
    (a)结合HER2；
    (b)阻断HER2与配体的结合；
    (c)在表达HER2的细胞中显示内吞作用；
    (d)对表达HER2的肿瘤细胞具有杀伤活性；
    (e)具有旁观者效应。
  15. 一种药物组合物，其包含权利要求1-14中任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物；任选地，所述药物组合物还包括可药用载体。
  16. 权利要求1-14任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物或者权利要求15所述的药物组合物在制备用于治疗癌症的药物中的用途；优选地，所述癌症为HER2阳性癌症；优选地，所述癌症为胆道癌、癌肉瘤、食管癌、胃食管结合部癌、乳腺癌、胃癌、胰腺癌、头颈癌、结直肠癌、肾癌、宫颈癌、卵巢癌、子宫内膜癌、子宫癌、黑色素瘤、咽癌、口腔癌、皮肤癌、肺癌、尿道癌、前列腺癌、膀胱癌、胃肠道间质瘤、鳞状细胞癌、腹膜癌、肝癌、子宫癌、唾液腺癌、外阴癌、甲状腺癌、阴茎癌、白血病、恶性淋巴瘤、浆细胞瘤或骨髓瘤。
  17. 一种治疗癌症的方法，其包括向有需要的患者施用治疗有效量的权利要求1-14任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化物或者权利要求15所述的药物组合物；优选地，所述癌症为HER2阳性癌症；优选地，所述癌症为胆道癌、癌肉瘤、食管癌、胃食管结合部癌、乳腺癌、胃癌、胰腺癌、头颈癌、结直肠癌、肾癌、宫颈癌、卵巢癌、子宫内膜癌、子宫癌、黑色素瘤、咽癌、口腔癌、皮肤癌、肺癌、尿道癌、前列腺癌、膀胱癌、胃肠道间质瘤、鳞状细胞癌、腹膜癌、肝癌、子宫癌、唾液腺癌、外阴癌、甲状腺癌、阴茎癌、白血病、恶性淋巴瘤、浆细胞瘤或骨髓瘤。
  18. 根据权利要求17所述的方法，所述方法包括使肿瘤细胞与所述抗体药物偶联物或其药学上可接受的盐或溶剂化物、或者所述药物组合物接触，从而杀伤肿瘤细胞或抑制肿瘤细胞生长。
  19. 一种具有式III所示结构的接头-药物中间体化合物：
    Figure PCTCN2022119215-appb-100006
    其中，
    R a选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
    R b选自氢原子、氘原子、任选被取代的C 1-6烷基、任选被取代的C 3-7环烷基、任选被取代的C 3-7杂环基、任选被取代的C 6-10芳基、任选被取代的C 5-12杂芳基；
    或者R a与R b与其相连接的原子一起形成任选被取代的5至8元的杂环基。
  20. 根据权利要求19所述的接头-药物中间体化合物，其特征在于，R a与R b各自独立地选自氢原子、甲基、乙基、丙基或异丙基。
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108883198A (zh) * 2016-03-02 2018-11-23 卫材研究发展管理有限公司 基于艾日布林的抗体-药物偶联物和使用方法
WO2019200322A1 (en) * 2018-04-13 2019-10-17 Genentech, Inc. Stable anti-cd79b immunoconjugate formulations
CN110997010A (zh) * 2019-08-07 2020-04-10 烟台迈百瑞国际生物医药有限公司 一种抗体药物偶联物及其应用
CN111116745A (zh) * 2018-11-01 2020-05-08 上海新理念生物医药科技有限公司 抗CD79b抗体、其药物偶联物及其应用
WO2021132166A1 (ja) * 2019-12-23 2021-07-01 エーザイ・アール・アンド・ディー・マネジメント株式会社 エリブリンをベースとする抗体－薬物コンジュゲートの製造方法
WO2021148003A1 (zh) * 2020-01-22 2021-07-29 上海森辉医药有限公司 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
WO2022022508A1 (zh) * 2020-07-27 2022-02-03 上海拓界生物医药科技有限公司 抗cd79b抗体药物偶联物、其制备方法及其医药用途
WO2022105878A1 (en) * 2020-11-20 2022-05-27 Bliss Biopharmaceutical (Hangzhou) Co., Ltd. Modified egfr antibody with reduced affinity, drug conjugate, and use thereof
WO2022104697A1 (en) * 2020-11-20 2022-05-27 Bliss Biopharmaceutical (Hangzhou) Co., Ltd. Modified egfr antibody with reduced affinity

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ728749A (en) 2014-08-05 2023-06-30 Cb Therapeutics Inc Anti-pd-l1 antibodies
CN109422811A (zh) 2017-08-29 2019-03-05 信达生物制药(苏州)有限公司 抗cd47抗体及其用途
CN115702008A (zh) 2020-08-13 2023-02-14 正大天晴药业集团股份有限公司 抗体药物偶联物

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108883198A (zh) * 2016-03-02 2018-11-23 卫材研究发展管理有限公司 基于艾日布林的抗体-药物偶联物和使用方法
WO2019200322A1 (en) * 2018-04-13 2019-10-17 Genentech, Inc. Stable anti-cd79b immunoconjugate formulations
CN111116745A (zh) * 2018-11-01 2020-05-08 上海新理念生物医药科技有限公司 抗CD79b抗体、其药物偶联物及其应用
CN110997010A (zh) * 2019-08-07 2020-04-10 烟台迈百瑞国际生物医药有限公司 一种抗体药物偶联物及其应用
WO2021132166A1 (ja) * 2019-12-23 2021-07-01 エーザイ・アール・アンド・ディー・マネジメント株式会社 エリブリンをベースとする抗体－薬物コンジュゲートの製造方法
WO2021148003A1 (zh) * 2020-01-22 2021-07-29 上海森辉医药有限公司 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
WO2022022508A1 (zh) * 2020-07-27 2022-02-03 上海拓界生物医药科技有限公司 抗cd79b抗体药物偶联物、其制备方法及其医药用途
WO2022105878A1 (en) * 2020-11-20 2022-05-27 Bliss Biopharmaceutical (Hangzhou) Co., Ltd. Modified egfr antibody with reduced affinity, drug conjugate, and use thereof
WO2022104697A1 (en) * 2020-11-20 2022-05-27 Bliss Biopharmaceutical (Hangzhou) Co., Ltd. Modified egfr antibody with reduced affinity

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NAKADA, T. ET AL.: "Novel antibody drug conjugates containing exatecan derivative-based cytotoxic payloads", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 26, no. 6, 8 February 2016 (2016-02-08), XP029436554, DOI: 10.1016/j.bmcl.2016.02.020 *

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