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WO2024160176A1 - 一类连接子药物及其抗体-药物偶联物的制备方法和应用 - Google Patents

一类连接子药物及其抗体-药物偶联物的制备方法和应用 Download PDF

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WO2024160176A1
WO2024160176A1 PCT/CN2024/074508 CN2024074508W WO2024160176A1 WO 2024160176 A1 WO2024160176 A1 WO 2024160176A1 CN 2024074508 W CN2024074508 W CN 2024074508W WO 2024160176 A1 WO2024160176 A1 WO 2024160176A1
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ala
antibody
solvate
gln
drug conjugate
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PCT/CN2024/074508
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English (en)
French (fr)
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李鸿峰
刘伟
马田
刘文超
吴香玉
王延春
徐振一
胡朝红
秦民民
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上海美雅珂生物技术有限责任公司
乐普生物科技股份有限公司
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Publication of WO2024160176A1 publication Critical patent/WO2024160176A1/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
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C33/00—Unsaturated compounds having hydroxy or O-metal groups bound to acyclic carbon atoms
    • C07C33/05—Alcohols containing rings other than six-membered aromatic rings
    • C07C33/16—Alcohols containing rings other than six-membered aromatic rings containing rings with more than six ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
    • C07D207/444—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5
    • C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
    • C07D207/452—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains four or more hetero rings

Definitions

  • the present invention relates to the field of medicine, and in particular, to a linker-drug conjugate, an antibody-drug conjugate, and a preparation method and application thereof.
  • ADC antibody-drug conjugate
  • ADCs have developed rapidly. Currently, 15 ADC drugs have been approved for marketing and more than 200 ADCs have entered clinical trials. ADCs are playing an increasingly important role in the field of tumor targeted therapy.
  • Antibody-drug conjugates consist of three parts: antibody (Ab), linker, and small molecule cytotoxic drug (payload/warhead).
  • the antibody part of ADC should have tumor specificity, good endocytosis efficiency, good antigen affinity, and no or low immunogenicity. IgG1 is the most commonly used antibody part.
  • the warhead part is generally a cytotoxic drug that acts on microtubules, DNA or RNA. Drugs such as camptothecin, maytansine, and auristatin need to be sufficiently toxic, sufficiently water-soluble, and have their targets located inside cells.
  • the linkers that connect antibodies and small molecule cytotoxic drugs are required to be stable in the blood circulation, quickly disintegrate inside cells, and release toxins efficiently.
  • the inventors of the present application creatively designed a new linker structure, thereby providing the present invention.
  • the first aspect of the present invention relates to a linker-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the linker-drug conjugate has a structure as shown in Formula I, MXD Formula I
  • M is a chemical structure comprising a maleimide (m) fragment or a cyclooctyne and its derivative fragments (such as BCN, DIBO, DIBAC, BARAC, etc.);
  • X is a connecting fragment consisting of 1 to 5 amino acids or derivatives thereof, preferably selected from Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala -Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-(3-cyano-alanine), Ala-(3-cyano-alanine), Ala-(3-cyano-alanine) , 3-cyano-alanine, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pro, Ala -Ala-Ala-Gln ⁇ Ala-Ala-Gln ⁇ Ala-Gln ⁇ Ala-G
  • D is a cytotoxin
  • the linker is composed of a chemical structure comprising a maleimide (m) fragment or a cyclooctyne and its derivative fragment (such as BCN, DIBO, DIBAC, BARAC, etc.) and a linker fragment composed of 1 to 5 amino acids or their derivatives, and has the following characteristics: it is stable in blood circulation, can be rapidly disintegrated in cells or tumor microenvironments, and can efficiently release toxins.
  • m maleimide
  • DIBAC DIBAC
  • BARAC a linker fragment composed of 1 to 5 amino acids or their derivatives
  • the chemical structure comprising the maleimide (m) fragment may be selected from 4-(N-maleimidomethyl) yl)-cyclohexane-1-carboxyl (MCC), 6-maleimidocaproyl (MC), M-(PEG) n , MC-(PEG) n , MCC-(PEG) n , etc., each structure is shown below:
  • n represents the degree of polymerization of PEG, and is preferably an integer between 2-12.
  • the chemical structure comprising the maleimide (m) fragment is 6-maleimidocaproyl (MC).
  • the chemical structure comprising the maleimide (m) fragment is MC-(PEG) n, wherein n is selected from 4, 8, and 12.
  • n is selected from 4, 8, and 12.
  • the inventors have found that a lower degree of polymerization of PEG is beneficial to reduce the polymer content (HMW%) in the ADC prepared by the linker drug conjugate to a certain extent, and therefore, the degree of polymerization n of PEG is preferably 4.
  • the chemical structure containing the cyclooctyne fragment can be selected from BCN, DIBO, DIBAC, BARAC, etc., and each structure is shown below:
  • the PAB structure (p-aminobenzyl-carbamoyl) is a self-releasing fragment in the linker and is an essential structure for effective release of the payload.
  • the hydrophobicity of the PAB structure easily leads to the production of more polymers when preparing ADCs. Therefore, further research and development of new linker structures are needed to overcome the above problems.
  • the inventors of the present application have found through experimental studies that if D is a cytotoxin containing a hydroxyl (-OH), primary amine (-NH2) or secondary amine (-NHR) group, the amino acid fragment contained in the linker-drug conjugate of the present application is connected to the Payload containing a hydroxyl (-OH), primary amine ( -NH2 ) or secondary amine (-NHR) structure, and the Payload can be released by enzymatic reaction when the hydrophobic structure PAB is removed, which can not only reduce the hydrophobicity of the Linker-Drug and reduce the polymer content generated when preparing ADC, but also show corresponding cell killing activity.
  • D is a cytotoxin containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group.
  • the cytotoxin containing a hydroxyl (-OH), primary amine (-NH 2 ) or secondary amine (-NHR) group can be selected from topoisomerase I inhibitors such as camptothecin compounds and their derivatives (such as exitecan, 14-aminocamptothecin, 9-aminocamptothecin, belotecan, etc.), topoisomerase II inhibitors such as doxorubicin compounds and their derivatives (such as Daunorubicin), DNA alkylating agents such as maytansine compounds and their derivatives, calicheamicin compounds and their derivatives, multicarmycin compounds and their derivatives (such as Duocarmycin derivatives) and pyrrolobenzodiazepine compounds and their derivatives (such as PBD), calendula compounds and their derivatives (such as MMAE and its derivatives or MMAF and its derivatives, etc.) microtubule inhibitors, Protac compounds and molecular glue compounds (such as len
  • the cytotoxin containing a hydroxyl (-OH), primary amine (-NH 2 ) or secondary amine (-NHR) group is selected from Exatecan, belotecan, and rapamycin. In certain embodiments, the cytotoxin containing a hydroxyl (-OH) group is rapamycin. In certain embodiments, the cytotoxin containing a secondary amine (-NHR) group is belotecan. In certain embodiments, the cytotoxin containing a primary amine (-NH 2 ) group is Exatecan.
  • the linker fragment composed of 1 to 5 amino acids or their derivatives in Formula I is selected from Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-(3-cyano-alanine), Ala-(3-cyano-alanine), Ala-(3-cyano-alanine), 3-cyano-alanine, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pr o ⁇ Ala-Ala-Ala-Ala-Gln ⁇ Ala-Ala-Ala-Gln ⁇ Ala-Ala-Gln
  • the amino acid monomer or its derivative or the fragment composed of multiple amino acids or their derivatives in formula I can be recognized by enzymes in tumor cell lysosomes and dissociate the ester bond or amide bond (peptide bond) formed by the carboxyl group in such compounds and the compound containing hydroxyl (-OH), primary amine (-NH 2 ) or secondary amine (-NHR), without the need for additional self-cleaving fragments (self-immolative moiety).
  • the linker fragment composed of 1 to 5 amino acids or their derivatives in Formula I is selected from Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-L-alanine), Ala-Ala-Ala-(3-cyano-L-alanine), Ala-Ala-(3-cyano-L-alanine), Ala-(3-cyano-L-alanine), Ala-(3-cyano-L-alanine), 3-cyano-L-alanine, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pro ,Ala-Ala-Ala-Gln,Ala-Ala-Gln,Ala-Ala
  • the linker is selected from a linker with asparagine (Asn), such as Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Pro-Asn, Lys-Asn.
  • the linker is Ala-Ala-Asn (AAN).
  • the linker drug conjugate of the present invention is MCC-AAQ-Exatecan, which has the structure shown below:
  • the second aspect of the present invention relates to an antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the antibody-drug conjugate is formed by coupling the linker-drug conjugate described in the first aspect of the present invention to an antibody.
  • the antibody drug conjugate has the structure shown in Formula II, Ab-(LD) p Formula II
  • L is a linker, and its structure is M'-X, wherein M' is connected to Ab, and X is connected to D;
  • M' is a chemical structure formed by connecting M as defined above to Ab via a maleimide group
  • X is a linker fragment consisting of 1 to 5 amino acids or their derivatives as defined above;
  • D is a cytotoxin as defined above
  • p is any value between 2 and 8 (e.g., 2, 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2 , 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, or such as 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5 or 7.5-8).
  • LD indicates that the linker and the cytotoxin are connected by a covalent bond to form an LD molecule
  • Ab-(LD) p indicates that p LD molecules are coupled to Ab by a covalent bond.
  • the drug-antibody ratio refers to the number of drug molecules coupled to the antibody (e.g., p in Formula II).
  • the number of drug molecules contained in the antibody-drug conjugate described herein can be an integer or a decimal. Whether it is an integer or a decimal, it refers to the average number of drug molecules coupled to each antibody.
  • the DAR values of antibody drug conjugates prepared in different batches may not be exactly the same, for example, they may fluctuate within a range of no more than 0.5.
  • the drug-antibody ratio (DAR) can be verified by conventional analytical means, such as mass spectrometry, ELISA assays, HIC and HPLC.
  • the quantitative distribution of ADC in terms of p can also be determined.
  • separation, purification and verification of homogenous ADC with a certain p value from ADC with other drug loads can be achieved by means such as reverse phase HPLC or electrophoresis.
  • one method is to couple the maleimide with the thiol group in the antibody to form an -S-succinimide structure.
  • MCC-AAQ-Exatecan is coupled to the Ab via a covalent bond
  • the structural formula of the formed ADC is as follows:
  • -S- can be either an external thiol group introduced by engineering modification or a thiol group contained in the antibody itself after the antibody Ab is reduced and the disulfide bonds are opened.
  • Another way is to couple the cyclooctyne with the azide group (-N 3 ) introduced in the modified antibody to form a triazole structure.
  • the structure of the formed ADC is as follows:
  • the antibodies that can be used in the present invention are not particularly limited and can be selected from mouse antibodies, rabbit antibodies, phage display antibodies, yeast display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies and multispecific antibodies.
  • the antibody is a monoclonal antibody selected from, but not limited to, HER2 antibody and EGFR antibody.
  • the antibody is a HER2 antibody.
  • the antibody is composed of a light chain and a heavy chain, the light chain comprises CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences thereof are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.
  • the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences thereof are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.
  • the light chain comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 7. In certain embodiments, the light chain further comprises a light chain constant region having an amino acid sequence of SEQ ID NO: 8. In certain embodiments, the amino acid sequence of the light chain is SEQ ID NO: 9.
  • the heavy chain comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 10. In certain embodiments, the heavy chain further comprises a heavy chain constant region having an amino acid sequence of SEQ ID NO: 11. In certain embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 12.
  • the antibody is an EGFR antibody.
  • the antibody is composed of a light chain and a heavy chain, the light chain comprises CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences thereof are shown in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively.
  • the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences thereof are shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.
  • the light chain comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 19. In certain embodiments, the light chain further comprises an amino acid sequence of SEQ ID NO: 20. In certain embodiments, the amino acid sequence of the light chain is SEQ ID NO: 21.
  • the heavy chain comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 22. In certain embodiments, the heavy chain further comprises a heavy chain constant region having an amino acid sequence of SEQ ID NO: 23. In certain embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 24.
  • compositions and pharmaceutical uses are provided.
  • the third aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned linker-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof.
  • the pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immunotherapeutic drug, and an immunosuppressant for treating tumors.
  • the chemotherapy drug is, for example, doxorubicin (Adriamycin), cyclophosphamide, taxanes (such as paclitaxel (Taxol), docetaxel (Taxotere)), capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (Arimidex, Furlong, Aromasin), 5-FU plus folinic acid, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, etc., or a combination thereof.
  • doxorubicin Adriamycin
  • cyclophosphamide taxanes (such as paclitaxel (Taxol),
  • the immunotherapy drug is, for example, PD-1 antibody, PD-L1 antibody, EGFR monoclonal antibody, CD20 monoclonal antibody, HER2 monoclonal antibody (such as Trastuzumab, Trastuzumab biosimilar, Trastuzumab-dkst), etc., or a combination thereof.
  • the immunosuppressant is selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and tacrolimus; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3; (5) alkylating agents, such as cyclophosphamide.
  • glucocorticoids such as cortisone and prednisone
  • microbial metabolites such as cyclosporine and tacrolimus
  • antimetabolites such as azathioprine and 6-mercaptopurine
  • polyclonal and monoclonal anti-lymphocyte antibodies such as anti-lymphocyte globulin and OKT3
  • alkylating agents such as cyclophosphamide.
  • the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, prograf, zenapax, sulelac, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod, etc.
  • the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.
  • the fourth aspect of the present invention provides the use of the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the aforementioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof in the preparation of a drug for preventing and/or treating tumors.
  • the fifth aspect of the present invention provides a method for treating and/or preventing tumors, comprising: administering to a subject in need thereof a therapeutically and/or preventively effective amount of the aforementioned linker-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, or the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof.
  • tumor refers to a lesion formed by excessive proliferation of local cells in an organ or tissue, including blood tumors and solid tumors, including benign tumors and malignant tumors.
  • the tumor is selected from tumors with positive or high expression of the following markers: HER2, EGFR.
  • the tumor is selected from the group consisting of breast cancer, melanoma, meningioma, soft tissue sarcoma, salivary gland tumor, primary liver cancer, intraspinal tumor, mediastinal tumor, brain cancer, bone cancer, penile cancer, osteosarcoma, intracranial tumor, tongue cancer, maxillary sinus cancer, thyroid cancer, malignant lymphoma, multiple myeloma, pituitary adenoma, testicular tumor, non-Hodgkin's lymphoma, bladder cancer, leukemia, gastric cancer, nasopharyngeal cancer, laryngeal cancer, oral cancer, esophageal cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), kidney cancer, cervical cancer, choriocarcinoma, vulvar cancer, skin cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, Kaposi's
  • the sixth aspect of the present invention provides a method for preparing the aforementioned linker-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, the method comprising the following steps:
  • MCC-AAQ-Exatecan The above preparation method is further described below by taking MCC-AAQ-Exatecan as an example.
  • the exemplary preparation process of MCC-AAQ-Exatecan is as follows:
  • the seventh aspect of the present invention provides a method for preparing the aforementioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, the method comprising the following steps:
  • the eighth aspect of the present invention relates to a linker-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the linker-drug conjugate has a structure as shown in Formula I, MXD Formula I
  • M is a chemical structure comprising a maleimide (m) fragment or a cyclooctyne and its derivative fragments (such as BCN, DIBO, DIBAC, BARAC, etc.);
  • X is a linker fragment consisting of 1 to 4 amino acids selected from Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pro, Ala-Ala-Ala-Gln, Ala-Ala-Gln, Ala-Gln, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, Gln;
  • D is a cytotoxin containing a primary amine ( -NH2 ) group.
  • the linker is composed of a chemical structure comprising a maleimide (m) fragment or a cyclooctyne and its derivative fragment (such as BCN, DIBO, DIBAC, BARAC, etc.) and a linker fragment consisting of 1 to 4 amino acids, and has the following characteristics: it is stable in blood circulation, can be rapidly disintegrated in cells or tumor microenvironments, and can efficiently release toxins.
  • m maleimide
  • a cyclooctyne and its derivative fragment such as BCN, DIBO, DIBAC, BARAC, etc.
  • the chemical structure containing the maleimide (m) fragment can be selected from 4-(N-maleimidomethyl)-cyclohexane-1-formyl (mcc), 6-maleimidocaproyl (mc), m-(PEG) n , mc-(PEG) n , mcc-(PEG) n , etc., and each structure is shown below:
  • n represents the degree of polymerization of PEG, and is preferably an integer between 2-12.
  • the chemical structure comprising the maleimide (m) fragment is 6-maleimidocaproyl (mc). In certain embodiments, the chemical structure comprising the maleimide (m) fragment is mc-(PEG) n , wherein n is selected from 4, 8, and 12.
  • n is selected from 4, 8, and 12.
  • the inventors have found that a lower degree of polymerization of PEG is beneficial to reduce the polymer content (HMW%) in the ADC prepared by the linker drug conjugate to a certain extent, and therefore, the degree of polymerization n of PEG is preferably 4.
  • the chemical structure containing the cyclooctyne fragment can be selected from BCN, DIBO, DIBAC, BARAC, etc., and each structure is shown below:
  • the linker fragment composed of 1 to 4 amino acids in Formula I is selected from Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pro, Ala-Ala-Ala-Gln, Ala-Ala-Gln, Ala-Gln, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gln.
  • the above amino acid monomers or fragments composed of multiple amino acids can be recognized by specific enzymes in tumor cell lysosomes and dissociate the amide bond (peptide bond) formed by the carboxyl group in such compounds and the compound containing primary amines, without the need for additional self-cleavage fragments (self-immolative moiety).
  • the linker fragment is selected from a linker fragment with asparagine (Asn), such as Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Pro-Asn, and Lys-Asn.
  • Asparagine (Asn) can be recognized by legumain, which exists not only in the lysosomes of tumor cells, but also in large quantities outside tumor cells. Therefore, it can release small molecules extracellularly, exert a by-stander effect, and has a good killing effect on low endocytic target cells.
  • the linker fragment is Ala-Ala-Asn (AAN).
  • the cytotoxin containing a primary amine ( -NH2 ) group can be selected from topoisomerase I inhibitors such as camptothecin compounds and their derivatives (such as exitecan, 14-aminocamptothecin, 9-aminocamptothecin, etc.), topoisomerase II inhibitors such as doxorubicin compounds and their derivatives (such as daunorubicin), maytansine compounds and their derivatives, calicheamicin compounds and their derivatives, multicarmycin compounds and their derivatives (such as duocarmycin derivatives) and pyrrolobenzodiazepine compounds and their derivatives (such as PBD) and other DNA alkylating agents, calendula compounds and their derivatives (such as deoxycholate), and pyrrolobenzodiazepine compounds and their derivatives (such as dapoxetine).
  • topoisomerase I inhibitors such as camptothecin compounds and their derivatives (such as exitecan, 14-aminocamp
  • cytotoxin containing a primary amine (-NH 2 ) group is Exatecan.
  • the cytotoxin containing a primary amine (—NH 2 ) group is selected from Exatecan, 14-aminocamptothecin (14-AC), 9-aminocamptothecin (9-AC), demethyl MMAE, demethyl MMAF, Daunorubicin, and Lenalidomide.
  • the linker drug conjugate of the present invention is mc-AAN-Exatecan, which has the structure shown below:
  • the ninth aspect of the present invention relates to an antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the antibody-drug conjugate is formed by coupling the linker-drug conjugate according to the eighth aspect of the present invention to an antibody.
  • the antibody drug conjugate has the structure shown in Formula II, Ab-(LD) p Formula II
  • L is a linker, and its structure is M'-X, wherein M' is connected to Ab, and X is connected to D;
  • M' is a chemical structure formed by connecting M as defined above to Ab via a maleimide group
  • X is a linker fragment consisting of 1 to 4 amino acids as defined above;
  • D is a cytotoxin containing a primary amine (-NH 2 ) group as defined above;
  • p is any value between 2 and 8 (e.g., 2, 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5 or 7.5-8).
  • LD indicates that the linker and the cytotoxin are connected by a covalent bond to form an LD molecule
  • Ab-(LD) p indicates that p LD molecules are coupled to Ab by a covalent bond.
  • the drug-antibody ratio refers to the number of drug molecules coupled to the antibody (e.g., p in Formula I).
  • the number of drug molecules contained in the antibody-drug conjugates described herein may be an integer or a decimal. Whether it is an integer or a decimal, it refers to the average number of drug molecules coupled to each antibody.
  • "p is any value between 2-8", which means that p can be any integer selected from 2-8 (including endpoints 2 and 8), or any decimal selected from 2-8, such as 2.3, 3.9, 4.0, 4.2.
  • the DAR values of antibody-drug conjugates prepared in different batches may not be exactly the same, for example, they may float within a range of no more than 0.5.
  • the drug-antibody ratio (DAR) can be verified by conventional analytical means, such as mass spectrometry, ELISA assays, HIC and HPLC.
  • the quantitative distribution of ADC in terms of p can also be determined.
  • separation, purification and verification of homogenous ADC with a certain p value from ADC with other drug loads can be achieved by means such as reverse phase HPLC or electrophoresis.
  • one method is to couple the maleimide with the thiol group in the antibody to form an -S-succinimide structure.
  • the structural formula of the formed ADC is as follows:
  • -S- can be either an external thiol group introduced by engineering modification or a thiol group contained in the antibody itself after the antibody Ab is reduced and the disulfide bonds are opened.
  • Another way is to link the antibody through the chemical structure of cyclooctyne and its derivative fragments (such as BCN, DIBO, DIBAC, BARAC, etc.), in which cyclooctyne is coupled with the azide group (-N 3 ) introduced in the modified antibody to form a triazole structure.
  • cyclooctyne and its derivative fragments such as BCN, DIBO, DIBAC, BARAC, etc.
  • the antibodies that can be used in the present invention are not particularly limited and can be selected from mouse antibodies, rabbit antibodies, phage display antibodies, yeast display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies and multispecific antibodies.
  • the antibody is a monoclonal antibody selected from, but not limited to, HER2 antibody and EGFR antibody.
  • the antibody is a HER2 antibody.
  • the antibody is composed of a light chain and a heavy chain, the light chain comprises CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences thereof are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.
  • the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences thereof are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.
  • the light chain comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 7. In certain embodiments, the light chain further comprises a light chain constant region having an amino acid sequence of SEQ ID NO: 8. In certain embodiments, the amino acid sequence of the light chain is SEQ ID NO: 9.
  • the heavy chain comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 10. In certain embodiments, the heavy chain further comprises a heavy chain constant region having an amino acid sequence of SEQ ID NO: 11. In certain embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 12.
  • the antibody is an EGFR antibody.
  • the antibody consists of a light chain and a heavy chain, the light chain comprises CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences thereof are shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively.
  • the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.
  • the light chain comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 19. In certain embodiments, the light chain further comprises a light chain constant region having an amino acid sequence of SEQ ID NO: 20. In certain embodiments, the amino acid sequence of the light chain is SEQ ID NO: 21.
  • the heavy chain comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 22. In certain embodiments, the heavy chain further comprises a heavy chain constant region having an amino acid sequence of SEQ ID NO: 23. In certain embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 24.
  • compositions and pharmaceutical uses are provided.
  • the tenth aspect of the present invention provides a pharmaceutical composition comprising the aforementioned linker-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, or the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof.
  • the pharmaceutical composition further comprises at least one of a chemotherapeutic drug, an immunotherapeutic drug, and an immunosuppressant for treating tumors.
  • the chemotherapy drug is, for example, doxorubicin (Adriamycin), cyclophosphamide, taxanes (such as paclitaxel (Taxol), docetaxel (Taxotere)), capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (Arimidex, Furlong, Aromasin), 5-FU plus folinic acid, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, etc., or a combination thereof.
  • doxorubicin Adriamycin
  • cyclophosphamide taxanes (such as paclitaxel (Taxol),
  • the immunotherapy drug is, for example, PD-1 antibody, PD-L1 antibody, EGFR monoclonal antibody, CD20 monoclonal antibody, HER2 monoclonal antibody (such as Trastuzumab, Trastuzumab biosimilar, Trastuzumab-dkst), etc., or a combination thereof.
  • the immunosuppressant is selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and tacrolimus; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3; (5) alkylating agents, such as cyclophosphamide.
  • glucocorticoids such as cortisone and prednisone
  • microbial metabolites such as cyclosporine and tacrolimus
  • antimetabolites such as azathioprine and 6-mercaptopurine
  • polyclonal and monoclonal anti-lymphocyte antibodies such as anti-lymphocyte globulin and OKT3
  • alkylating agents such as cyclophosphamide.
  • the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, prograft, zenapax, sulelac, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod, etc.
  • the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.
  • the eleventh aspect of the present invention provides the use of the aforementioned linker-drug conjugate, or a pharmaceutically acceptable salt, solvate or a solvate of the salt thereof, or the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or a solvate of the salt thereof in the preparation of a drug for preventing and/or treating tumors.
  • the twelfth aspect of the present invention provides a method for treating and/or preventing tumors, comprising: administering to a subject in need thereof a therapeutically and/or preventively effective amount of the aforementioned linker-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, or the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof.
  • tumor refers to a lesion formed by excessive proliferation of local cells in an organ or tissue, including blood tumors and solid tumors, including benign tumors and malignant tumors.
  • the tumor is selected from tumors with positive or high expression of the following markers: HER2, EGFR.
  • the tumor is selected from the group consisting of breast cancer, melanoma, meningioma, soft tissue sarcoma, salivary gland tumor, primary liver cancer, intraspinal tumor, mediastinal tumor, brain cancer, bone cancer, penile cancer, osteosarcoma, intracranial tumor, tongue cancer, maxillary sinus cancer, thyroid cancer, malignant lymphoma, multiple myeloma, pituitary adenoma, testicular tumor, non-Hodgkin's lymphoma, bladder cancer, leukemia, gastric cancer, nasopharyngeal cancer, laryngeal cancer, oral cancer, esophageal cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), kidney cancer, cervical cancer, choriocarcinoma, vulvar cancer, skin cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, Kaposi's
  • the thirteenth aspect of the present invention provides a method for preparing the aforementioned linker-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, the method comprising the following steps:
  • mc-AAN-Exatecan The above preparation method is further described below by taking mc-AAN-Exatecan as an example.
  • the exemplary preparation process of mc-AAN-Exatecan is as follows:
  • the intermediate 2 is subjected to a de-Fmoc reaction to obtain the intermediate 3, wherein the intermediate 3 has the following structural formula:
  • the fourteenth aspect of the present invention provides a method for preparing the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, the method comprising the following steps:
  • the linker provided by the present invention can release the payload through an enzymatic reaction after removing the hydrophobic structure PAB, which can not only reduce the hydrophobicity of the linker-drug and reduce the polymer content generated when preparing ADC, but also exhibit corresponding cell killing activity.
  • any numerical range should be understood to include any value or any sub-range within the range.
  • the term "antibody” refers to an immunoglobulin molecule generally composed of two pairs of identical polypeptide chains (each pair having one "light” (L) chain and one "heavy” (H) chain).
  • the light chain of an antibody can be divided into two types: ⁇ and ⁇ .
  • the heavy chain can be divided into five types: ⁇ , ⁇ , ⁇ , ⁇ or ⁇ .
  • antibodies can be divided into five types: IgM, IgD, IgG, IgA and IgE.
  • the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region ( VH ) and a heavy chain constant region ( CH ).
  • the heavy chain constant region consists of three domains ( CH1 , CH2 and CH3 ).
  • Each light chain consists of a light chain variable region ( VL ) and a light chain constant region ( CL ).
  • the light chain constant region consists of one domain, CL .
  • the constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the component C1q of the complement system.
  • the VH and VL regions can also be subdivided into regions of high variability, called complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementary determining regions
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions ( VH and VL ) of each heavy chain/light chain pair form the antibody binding site, respectively.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or the definitions of Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.
  • antibodies do not contain many free and reactive cysteine thiol groups to which drug moieties can be attached; in fact, most cysteine thiol groups in antibodies exist as disulfide bridges.
  • antibodies can be reduced under partially or fully reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine thiol groups.
  • DTT dithiothreitol
  • TCEP tricarbonylethylphosphine
  • the term "pharmaceutically acceptable salt” refers to: (i)
  • the acidic functional groups present may be salts formed with appropriate inorganic or organic cations (bases), and include, but are not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, such as ammonium salts; organic base salts, such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclo
  • Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic substance with a suitable acid to provide a pharmaceutically acceptable anion, or by reacting a sufficient amount of an acidic substance with a suitable base to provide a pharmaceutically acceptable cation.
  • solvate refers to these forms of the antibody drug conjugate of the present invention: the antibody drug conjugate forms a complex in a solid or liquid form by coordination with solvent molecules.
  • Hydrate is a specific form of solvate, which has coordinated water molecules.
  • hydrate is a preferred solvate.
  • the method for preparing the pharmaceutical composition includes incorporating appropriate pharmaceutical excipients, carriers, diluents, etc., which are non-toxic to cells or mammals exposed thereto at the doses and concentrations used.
  • the pharmaceutical excipients refer to excipients and additives used in the production of drugs and the preparation of prescriptions. They refer to substances that have been reasonably evaluated in terms of safety and are included in drug preparations in addition to active ingredients. In addition to shaping, acting as carriers, and improving stability, pharmaceutical excipients also have It has important functions such as solubilization, solubilization, and controlled release, and is an important ingredient that may affect the quality, safety, and effectiveness of drugs. According to its source, it can be divided into natural products, semi-synthetics, and fully synthetics.
  • solvents solvents, propellants, solubilizers, solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc.; according to its route of administration, it can be divided into oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration, and ocular administration, etc.
  • the same pharmaceutical excipient can be used in drug preparations for different administration routes
  • the pharmaceutical composition can be prepared into various suitable dosage forms according to the administration route, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder sprays, sprays, etc.
  • the pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the antibody-drug conjugate of the present invention, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof.
  • treatment generally refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be preventive, in terms of completely or partially preventing a disease or its symptoms; and/or therapeutic, in terms of partially or completely stabilizing or curing a disease and/or side effects resulting from a disease.
  • treatment encompasses any treatment of a patient's disease, including: (a) preventing the disease or symptoms from occurring in a patient who is susceptible to the disease or symptoms but has not yet been diagnosed with the disease; (b) inhibiting the symptoms of a disease, i.e., preventing its development; or (c) alleviating the symptoms of a disease, i.e., causing the disease or symptoms to regress.
  • a vertebrate refers to a mammal.
  • Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats.
  • a mammal refers to a human.
  • an amount refers to an amount effective at the necessary dosage and time to achieve the desired therapeutic or preventive effect.
  • the "therapeutically effective amount” of the substance/molecule of the present invention may vary according to factors such as the disease state, age, sex and weight of the individual and the ability of the substance/molecule to elicit the desired response in the individual.
  • a therapeutically effective amount also encompasses an amount in which the therapeutically beneficial effects of the substance/molecule outweigh any toxic or harmful consequences.
  • a “prophylactically effective amount” refers to an amount effective at the necessary dosage and time to achieve the desired preventive effect.
  • the effective amount for prevention is lower than the effective amount for treatment.
  • the effective amount for treatment can reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow down to a certain extent, preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down to a certain extent, preferably stop) tumor metastasis; inhibit tumor growth to a certain extent; and/or alleviate one or more symptoms associated with cancer to a certain extent.
  • X is Ala-Ala-Gln
  • X is a linking fragment composed of Ala, Ala and Gln connected in sequence to form a peptide bond
  • those skilled in the art can understand that it does not limit the specific stereo configuration of Ala, Ala and Gln, such as L-type or D-type.
  • X is Ala-Ala-(3-cyano-alanine)
  • X is a linking fragment composed of Ala, Ala, (3-cyano-alanine) connected in sequence to form a peptide bond
  • those skilled in the art can also understand that it does not limit the specific stereo configuration of Ala, Ala, (3-cyano-alanine), such as L-type or D-type, or S-type or R-type.
  • Other similar limitations of X in the present invention are understood with reference to the above content.
  • X is selected from Ala-Ala-Gln, Ala-Ala-(3-cyano-alanine) and deuterated products thereof, it means that X may be Ala-Ala-Gln, Ala-Ala-(3-cyano-alanine), a deuterated product of Ala-Ala-Gln, or a deuterated product of Ala-Ala-(3-cyano-alanine).
  • deuterated compound refers to a compound formed by replacing one or more (such as 2, 3, 4 or more) hydrogen atoms (the position of the hydrogen atoms is not specifically limited, and can be hydrogen atoms on an alkyl group or a hydrogen atom on an amino group, etc.) in a certain compound with deuterium atoms.
  • chimeric antibody refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from mouse antibodies and the constant region sequences are derived from human antibodies.
  • Humanized antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulins.
  • humanized antibodies are human immunoglobulins (recipient antibodies) in which residues in the complementary determining regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • CDRs complementary determining regions
  • telomeres in the CDR1, CDR2 and/or CDR3 regions of VH and/or VL can be mutated to improve one or more binding properties (e.g., affinity) of the antibody.
  • PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions.
  • the mutations in the CDRs are usually no more than one or two.
  • Fmoc-Ala-Ala-OH N-[fluorenylmethoxycarbonyl]-L-alanyl-L-alanine
  • HOSu N-hydroxysuccinimide
  • L-Asn L-asparagine
  • the specific steps are: add Fmoc-Ala-Ala-OH (1.0 eq), HOSu (1.3 eq), THF (10 v/w) to the reaction bottle at room temperature, control the temperature at room temperature, slowly add DCC (1.3 eq) under stirring conditions, react at room temperature, detect by HPLC until Fmoc-Ala-Ala-OH residue is less (area normalization method), filter the reaction solution, and rinse the filter cake with THF (2.5 v/w).
  • the intermediate 2 is reacted with 4-(N-maleimidomethyl)cyclohexanecarboxylic acid-N-succinimidyl ester to obtain the intermediate 3.
  • reaction formula is as follows:
  • UV/BCA method to detect the protein concentration of antibody drug conjugates, use HIC for DAR detection, and SEC for purity detection.
  • the method for preparing antibody drug conjugates from other linker drug conjugates of the present invention can refer to the preparation method of the above-mentioned antibody conjugate EGFR antibody-MCC-AAQ-Exatecan.
  • the method for preparing an antibody drug conjugate (such as the antibody drug conjugate HER2 antibody-MCC-AAN-Exatecan) by using the linker drug conjugate of the present invention (such as the linker drug conjugate shown in Formula I) and HER2 antibody can refer to the method for preparing the antibody conjugate EGFR antibody-MCC-AAQ-Exatecan in Example 7 above.
  • the different antibody-drug conjugates of the present invention were subjected to in vitro activity tests to detect their cell killing activities.
  • the test method is as follows:
  • the target cells are plated in a certain number into a 96-well black bottom transparent cell culture plate. After 24 hours of incubation, the samples diluted in concentration gradients are added. After 72 hours or 144 hours of incubation, the color reagent is used to develop the color for 60 minutes, and the plate is read using an ELISA reader. The more live cells there are, the higher the signal value. According to the different signal values obtained at different concentration gradient points, a four-parameter fitting curve is formed on the ELISA reader to obtain the C value, i.e., the IC 50 value.
  • the present invention also provides the following embodiments.
  • Fmoc-Ala-OH (N-fluorenylmethoxycarbonyl-L-alanine, CAS No.: 35661-39-3) was activated by HOSu (N-hydroxysuccinimide, CAS No.: 6066-82-6), and then reacted with L-Ala (L-alanine, CAS No.: 56-41-7) to obtain intermediate 1; specifically, Fmoc-Ala-OH (3 g, 1.0 eq) and HOSu (1.45 g, 1.3 eq) were added to the reaction flask, 21 mL of THF was added, the temperature was controlled at room temperature, DCC (2.59 g, 1.3 eq) was slowly added under stirring, and the reaction was carried out at room temperature and monitored by HPLC.
  • reaction solution was filtered and the filter cake was rinsed with THF (6 mL). Purified water (15 mL) was added to the filtrate, followed by L-Ala (1.12 g, 1.3 eq) and solid sodium bicarbonate (0.81 g, 1.0 eq). The reaction was stirred at room temperature and monitored by HPLC. After the reaction was completed, citric acid (2.02 g, 1.0 eq) was added and stirred. The reaction solution was then extracted with ethyl acetate. The organic phase was concentrated, and DMF (12 mL) was added to dissolve the product. After filtering, the product was passed through pre-HPLC.
  • the intermediate 1 (N-[fluorenylmethoxycarbonyl]-L-alanyl-L-alanine, CAS No.: 87512-31-0) was activated by HOSu (N-hydroxysuccinimide, CAS No.: 6066-82-6), and then reacted with L-Asn (L-asparagine, CAS No.: 70-47-3) to obtain the intermediate 2; specifically, the intermediate 1 (0.87 g, 1.0 eq), HOSu (0.34 g, 1.3 eq), and THF (9 mL) were added to the reaction flask, the temperature was controlled at room temperature, DCC (0.61 g, 1.3 eq) was slowly added under stirring, the reaction was carried out at room temperature, and HPLC was used for monitoring.
  • reaction solution was filtered and the filter cake was rinsed with THF (2 mL). Purified water (10 mL) was added to the filtrate, and L-Asn (0.34 g, 1.1 eq) and solid sodium bicarbonate (0.19 g, 1.0 eq) were added. The reaction was stirred at room temperature and monitored by HPLC. After the reaction was completed, citric acid monohydrate (0.48 g, 1.0 eq) was added and stirred. The reaction solution was concentrated to remove most of the solvent, and a purified residue was prepared. The prepared solution was concentrated until no obvious droplets flowed out, and then extracted with ethyl acetate. The organic phase was concentrated to dryness to obtain 826 mg of the intermediate II. The reaction yield of this step was 73%.
  • the reaction formula is as follows:
  • the intermediate 3 was obtained; specifically, the intermediate 2 (100 mg) and DMF (1.5 mL) were added to the reaction flask, the temperature was controlled at room temperature, DEA (300 ⁇ L) was added dropwise, and the reaction was carried out at room temperature, and HPLC was monitored until there was no residue of the intermediate 2, and the DMF was removed by concentration, DCM (4 mL) and purified water (4 mL) were added, and the mixture was separated after stirring, and the aqueous phase was concentrated to dryness to obtain 92 mg of the intermediate 3.
  • the reaction yield of this step was 166% (part of the solvent was included), and the reaction formula is as follows:
  • Intermediate 4 and isotecan mesylate are subjected to condensation reaction to obtain intermediate 4; specifically, at room temperature, intermediate 4 (26 mg, 1.0 eq) is added to the reaction bottle, DMF (1.5 mL) is added, isotecan mesylate (29.6 mg, 1.0 eq), EEDQ (20.7 mg, 1.5 eq), HATU (31.8 mg, 1.5 eq), DMAP (0.7 mg, 0.1 eq), DIPEA (29.2 ⁇ L, 3.0 eq) are added in sequence, and the reaction is carried out at room temperature. HPLC monitoring, after the reaction is completed, the preparation is purified, and the preparation solution is concentrated to obtain 16.5 mg of the product (NMR is as follows), and the reaction yield of this step is 33.5%.
  • the reaction formula is as follows:
  • HER2 antibody Take 10 mg of HER2 antibody, dilute it with PBS buffer to a concentration of 5 mg/mL, and adjust the pH of the antibody to about 7.5 with Tris-EDTA solution; use Nanodrop to detect the protein concentration, weigh the net weight of the antibody solution, and calculate the total amount of protein. Add 8 times the molar number of TCEP solution to the antibody, place it on a 3D shaker, react for more than 120 minutes at room temperature, and mix continuously.
  • UV/BCA method to detect the protein concentration of antibody drug conjugates, use HIC for DAR detection, and SEC for purity detection.
  • Example 14 In vitro activity testing of different antibody-drug conjugates
  • the linker drug conjugate mc-AAN-Exatecan was respectively coupled with HER2 antibody, EGFR antibody and non-specific binding antibody (Anti-HEL) to prepare the corresponding ADC, and the linker drug conjugate mc-Pro-Exa was coupled with HER2 antibody to prepare ADC, and the cell killing activity was detected.
  • the test method is as follows:
  • the target cells are plated in a certain number into a 96-well black bottom transparent cell culture plate. After 24 hours of incubation, the samples diluted in concentration gradients are added. After another 72 hours of incubation, the color developing reagent is used, the color is developed for 60 minutes, and the plate is read using an ELISA reader. The more live cells there are, the higher the signal value is. According to the different signal values obtained at different concentration gradient points, a four-parameter fitting curve is formed on the ELISA reader to obtain the C value, i.e., the IC 50 value.
  • Example 15 Preparation of ADC using linkers or cytotoxins with different structures and detection and analysis
  • the inventors prepared the following ADC stock solution and tested and analyzed the basic quality characteristics of the obtained ADC stock solution.
  • the polymer content (HMW%) of the ADC prepared by the Linker-Payload without PAB is significantly lower than that of the ADC prepared by the ADC containing PAB fragment, indicating that the PAB fragment is not conducive to the preparation of ADC by this type of Linker-Payload.
  • the specific data are shown in Table 5;
  • Payloads containing primary amine (-NH 2 ) structures can be released by linkers that do not contain PAB fragments, while payloads containing non-primary amine groups (-NH-(i-Pr) structures) cannot be released by linkers that do not contain PAB fragments. See Table 6 for specific data.
  • mc-Pro-Belotecan and mc-Pro-Exatecan were found to have no tumor cell killing activity, indicating that its structure is stable and cannot release the payload through enzymatic hydrolysis; while the ADC conjugated with mc-Pro-Exatecan showed good killing activity, indicating that the structure can release the payload; at the same time, the ADC produced by conjugating mc-Pro-Exatecan with non-binding antibodies (without targeted endocytosis) had no specific cell killing activity, indicating that the structure was stable outside the cell and needed to be internalized into the cell before the payload could be released.

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Abstract

一种连接子药物偶联物、抗体药物偶联物、其制备方法和应用，所述连接子药物偶联物具有式Ⅰ所示的结构，M-X-D（式I），其中，M为包含马来酰亚胺(m)片段或环辛炔片段的化学结构，X为1～5个氨基酸或其衍生物组成的连接片段，D为细胞毒素。所述连接子药物偶联物可以在去除疏水性结构PAB的情况下，通过酶解反应释放Payload，既能够降低Linker-Drug的疏水性，减少制备ADC时产生的聚合物含量，也可以表现出相应的细胞杀伤活性。

Description

一类连接子药物及其抗体-药物偶联物的制备方法和应用
相关申请的交叉引用
本申请是以CN申请号为202310093383.0、申请日为2023年1月31日的申请，以及CN申请号为202310947390.2、申请日为2023年7月28日的申请为基础，并主张其优先权，所有申请的内容在此作为整体引入本申请中。
技术领域
本发明涉及药物领域，具体地，涉及连接子药物偶联物、抗体药物偶联物、其制备方法和应用。
背景技术
抗体偶联药物(antibody-drug conjugate，ADC)的研究可以追溯到1980s，但是直到2000年，首个抗体偶联药物(商品名Mylotarg，Pfizer研发)才被FDA批准用于治疗急性粒细胞白血病，但由于偶联技术、靶向性、有效性等受限，完整的抗体偶联药物在血液不稳定，导致致死性毒性的产生，于2010年撤市。这使得本就不明朗的ADC药物研究，更蒙上了一层阴影。
但是，Seagen通过对原有技术的改进，利用自己的新型抗体偶联技术开发了brentuximab vedotin(SGN-35，商品名Adcetris，)新型抗体偶联药物，并已于2011年被FDA批准用于治疗霍奇金淋巴瘤和系统性间变性大细胞淋巴瘤。2013年抗体偶联药物再次取得突破，Genentech/ImmunoGen联合开发的Ado-trastuzumab emtansine(T-DM1，商品名Kadcyla)被FDA批准用于HER2阳性乳腺癌，这是首个针对实体瘤的抗体偶联药物。随着这两个药物的研发成功，ADC药物再次以火热的状态进入人们的研究视野。
近年来，ADCs发展迅猛，目前已有15个ADC药物被批准上市，200多个ADC进入临床试验。ADC在肿瘤靶向治疗领域正发挥着越来越重要的作用。
抗体药物偶联物由三个部分组成：抗体(Ab)、连接子(linker)和小分子细胞毒药物(payload/warhead，弹头)。ADC的抗体部分应当具有肿瘤特异性、良好的内吞效率、良好的抗原亲和力，而且无或低免疫原性，IgG1是最常用的抗体部分。弹头部分一般都是作用于微管、DNA或RNA的细胞毒药 物，如喜树碱类、美登素类、奥瑞他汀类等，需要有足够强的毒性、足够的水溶性，而且作用靶点位于细胞内。连接抗体和小分子细胞毒药物的连接子则要求在血液循环中稳定、在细胞内快速解体，可以高效地释放毒素。
发明内容
本申请的发明人创造性地设计了一种新的连接子结构，由此提供了本发明。
连接子药物偶联物
本发明的第一方面涉及一种连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，所述连接子药物偶联物具有式Ⅰ所示的结构，
M-X-D
式I
其中，
M为包含马来酰亚胺(m)片段或包含环辛炔及其衍生物片段(如BCN、DIBO、DIBAC、BARAC等)的化学结构；
X为1～5个氨基酸或其衍生物组成的连接片段，优选选自Ala-Ala-Ala-Ala-Asn、Ala-Ala-Ala-Asn、Ala-Ala-Asn、Ala-Asn、Asn、Ala-Ala-Ala-Ala-(3-cyano-alanine)、Ala-Ala-Ala-(3-cyano-alanine)、Ala-Ala-(3-cyano-alanine)、Ala-(3-cyano-alanine)、3-cyano-alanine、Ala-Ala-Ala-Pro、Ala-Ala-Pro、Ala-Pro、Pro、Pro-Asn、Asn-Pro、Lys、Lys-Asn、Lys-Pro、Ala-Ala-Ala-Ala-Gln、Ala-Ala-Ala-Gln、Ala-Ala-Gln、Ala-Gln、Ala-Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid)、Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid)、Ala-Ala-(2-amino-4-cyanobutanoic acid)、Ala-(2-amino-4-cyanobutanoic acid)、2-amino-4-cyanobutanoic acid、Gly-Gly-Gly-Gln、Gly-Gly-Gln、Gly-Gln、Gln及它们的氘代化物；
D为细胞毒素。
本发明的连接子药物偶联物中，连接子由包含马来酰亚胺(m)片段或包含环辛炔及其衍生物片段(如BCN、DIBO、DIBAC、BARAC等)的化学结构和1～5个氨基酸或其衍生物组成的连接片段构成，其具有以下特点：血液循环中稳定、可以在细胞内或肿瘤微环境中快速解体，并高效释放毒素。
包含马来酰亚胺(m)片段的化学结构可以选自4-(N-马来酰亚氨基甲 基)-环己烷-1-甲酰基(MCC)、6-马来酰亚氨基己酰基(MC)、M-(PEG)n、MC-(PEG)n、MCC-(PEG)n等，各结构如下所示：
其中，n代表PEG的聚合度，优选为2-12之间的整数。
在某些实施方案中，包含马来酰亚胺(m)片段的化学结构为6-马来酰亚氨基己酰基(MC)。在某些实施方案中，包含马来酰亚胺(m)片段的化学结构为MC-(PEG)n，其中，n选自4、8、12。发明人发现，较低的PEG聚合度在一定程度上有利于降低连接子药物偶联物制备的ADC中的聚合物含量(HMW％)，因此，PEG的聚合度n优选为4。
包含环辛炔片段的化学结构可以选自BCN、DIBO、DIBAC、BARAC等，各结构如下所示：

对于大多数目前在研的ADC，PAB结构(对氨基苄基-氨甲酰基)作为连接子中的自释放片段，是有效释放Payload的必需结构。然而，PAB结构的疏水性容易使制备ADC时产生较多的聚合物。因此，还需进一步研究开发新的连接子结构以克服上述问题。
为此，本申请发明人经过实验研究发现，若D为含有羟基(-OH)、伯胺(-NH2)或仲胺(-NHR)基团的细胞毒素，则本申请的连接子药物偶联物包含的氨基酸片段与含有羟基(-OH)、伯胺(-NH2)或仲胺(-NHR)结构Payload连接，可以在去除疏水性结构PAB的情况下，通过酶解反应释放Payload，既能够降低Linker-Drug的疏水性，减少制备ADC时产生的聚合物含量，也可以表现出相应的细胞杀伤活性。
由此，在某些实施方案中，D为含有羟基(-OH)、伯胺(-NH2)或仲胺(-NHR)基团的细胞毒素。
在某些实施方案中，所述含有羟基(-OH)、伯胺(-NH2)或仲胺(-NHR)基团的细胞毒素可以选自喜树碱类化合物及其衍生物(如依喜替康、14-氨基喜树碱、9-氨基喜树碱、贝洛替康等)等拓扑异构酶I抑制剂、阿霉素类化合物及其衍生物(如Daunorubicin)等拓扑异构酶II抑制剂、美登素类化合物及其衍生物、卡里奇霉素类化合物及其衍生物、多卡米星类化合物及其衍生物(如Duocarmycin衍生物)和吡咯并苯二氮卓类化合物及其衍生物(如PBD)等DNA烷基化试剂、金盏花素类化合物及其衍生物(如MMAE及其衍生物或MMAF及其衍生物等)微管蛋白抑制剂、Protac类化合物和分子胶类化合物(如来那度胺等)、雷帕霉素及其衍生物等免疫抑制剂以及其他各种具有细胞杀伤活性的小分子化合物。在某些实施方案中，所述含有羟基(-OH)、伯胺(-NH2)或仲胺(-NHR)基团的细胞毒素选自依喜替康(Exatecan)、贝洛替康(belotecan)、雷帕霉素(Rapamycin)。在某些实施方案中，所述含有羟基(-OH)基团的细胞毒素为雷帕霉素(Rapamycin)。在某些实施方案中，所述含有仲胺(-NHR)基团的细胞毒素为贝洛替康(belotecan)。在某些实施方案中，所述含有伯胺(-NH2)基团的细胞毒素为依喜替康(Exatecan)。
在某些实施方案中，式I中的所述1～5个氨基酸或其衍生物组成的连接片段选自Ala-Ala-Ala-Ala-Asn、Ala-Ala-Ala-Asn、Ala-Ala-Asn、Ala-Asn、Asn、Ala-Ala-Ala-Ala-(3-cyano-alanine)、Ala-Ala-Ala-(3-cyano-alanine)、Ala-Ala-(3-cyano-alanine)、Ala-(3-cyano-alanine)、3-cyano-alanine、Ala-Ala-Ala-Pro、Ala-Ala-Pro、Ala-Pro、Pro、Pro-Asn、Asn-Pro、Lys、Lys-Asn、Lys-Pro、Ala-Ala-Ala-Ala-Gln、Ala-Ala-Ala-Gln、Ala-Ala-Gln、Ala-Gln、Ala-Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid)、Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid)、Ala-Ala-(2-amino-4-cyanobutanoic acid)、Ala-(2-amino-4-cyanobutanoic acid)、2-amino-4-cyanobutanoic acid、Gly-Gly-Gly-Gln、Gly-Gly-Gln、Gly-Gln、Gln及它们的氘代化物。
式I中的氨基酸单体或其衍生物或由多个氨基酸或其衍生物组合成的片段能够被肿瘤细胞溶酶体内的酶识别，并解离该类化合物中的羧基与含有羟基(-OH)、伯胺(-NH2)或仲胺(-NHR)的化合物形成的酯键或酰胺键(肽键)，不需要额外的自裂解片段(self-immolative moiety)。
在某些实施方案中，式I中的所述1～5个氨基酸或其衍生物组成的连接片段选自Ala-Ala-Ala-Ala-Asn、Ala-Ala-Ala-Asn、Ala-Ala-Asn、Ala-Asn、Asn、Ala-Ala-Ala-Ala-(3-cyano-L-alanine)、Ala-Ala-Ala-(3-cyano-L-alanine)、Ala-Ala-(3-cyano-L-alanine)、Ala-(3-cyano-L-alanine)、3-cyano-L-alanine、Ala-Ala-Ala-Pro、Ala-Ala-Pro、Ala-Pro、Pro、Pro-Asn、Asn-Pro、Lys、Lys-Asn、Lys-Pro、Ala-Ala-Ala-Ala-Gln、Ala-Ala-Ala-Gln、Ala-Ala-Gln、Ala-Gln、Ala-Ala-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)、Ala-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)、Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)、Ala-((S)-2-amino-4-cyanobutanoic acid)、(S)-2-amino-4-cyanobutanoic acid、Gly-Gly-Gly-Gln、Gly-Gly-Gln、Gly-Gln、Gln。
在某些实施方案中，所述连接片段选自带有天冬酰胺(Asn)的连接片段，例如Ala-Ala-Ala-Ala-Asn、Ala-Ala-Ala-Asn、Ala-Ala-Asn、Ala-Asn、Asn、Pro-Asn、Lys-Asn。在某些实施方案中，所述连接片段为Ala-Ala-Asn(AAN)。
在某些实施方案中，本发明的连接子药物偶联物为MCC-AAQ-Exatecan，其具有如下所示的结构：
抗体药物偶联物
本发明的第二方面涉及一种抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，所述抗体药物偶联物由本发明第一方面所述的连接子药物偶联物与抗体偶联形成。
在某些实施方案中，所述抗体药物偶联物具有式II所示的结构，
Ab-(L-D)p
式II
其中，
Ab为抗体；
L为接头，其结构为M’-X，其中，M’与Ab相连，X与D相连；
M’为如上定义的M通过马来酰亚胺基团连接至Ab所形成的化学结构；
X为如上定义的1～5个氨基酸或其衍生物组成的连接片段；
D为如上定义的细胞毒素；
p为2-8之间的任意数值(如2、2.5、3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9或8，或者如2-2.5、2.5-3、3-3.5、3.5-4、4-4.5、4.5-5、5-5.5、5.5-6、6-6.5、6.5-7、7-7.5或7.5-8)。
式II中，L-D表示接头与细胞毒素通过共价键相连，形成L-D分子；Ab-(L-D)p表示p个L-D分子通过共价键偶联至Ab。
本发明中，药物抗体比(DAR)是指偶联到抗体的药物分子的个数(例如，式II中的p)。本文所述的抗体药物偶联物中包含的药物分子的个数可以是整数，也可以是小数。无论是整数还是小数，其指的均是每个抗体偶联的药物分子的平均数量。“p为2-8之间的任意数值”，其表示，p可以是选自2-8之间 的任意整数(包括端点2和8)，也可以是选自2-8之间的任意小数，例如2.3、3.9、4.0、4.2。同时，本领域技术人员可以理解，即使采用相同的制备方法，不同的批次制备得到的抗体药物偶联物的DAR值也不一定完全相同，例如，可以在上下不超过0.5的范围内浮动。
药物抗体比(DAR)可以通过常规分析手段来验证，诸如质谱、ELISA测定法、HIC和HPLC。还可以测定ADC在p方面的定量分布。在有些情况中，将p为某数值的同质ADC从具有其它药物载荷的ADC中分离、纯化和验证可以通过诸如反相HPLC或电泳的手段来实现。
本发明中，连接子药物偶联物通过共价键偶联至抗体Ab上时，一种方式可以通过马来酰亚胺与抗体中的巯基偶联形成-S-丁二酰亚胺结构。例如MCC-AAQ-Exatecan通过共价键偶联至Ab上时，形成的ADC的结构式如下所示：
上述形成的马来酰亚胺接头的ADC中，-S-既可以是通过工程化改造引入的外接巯基，也可以是抗体Ab被还原进而被打开二硫键后的抗体自身所含有的巯基。
另一种方式可以通过环辛炔与修饰抗体中引入的叠氮基团(-N3)偶联形成三氮唑结构结构。例如BCN-AAQ-Exatecan通过共价键偶联至Ab上时，形成的ADC的结构式如下所示：
可用于本发明的抗体没有特别的限制，可以选自鼠源抗体、兔源抗体、噬菌体展示来源抗体、酵母展示来源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体。
在某些实施方案中，所述抗体为单克隆抗体，非限制性地选自：HER2抗体、EGFR抗体。
在某些实施方案中，所述抗体为HER2抗体。在某些实施方案中，所述抗体由轻链和重链组成，所述轻链包含CDR-L1、CDR-L2和CDR-L3，其氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3所示。在某些实施方案中，所述重链包含CDR-H1、CDR-H2、CDR-H3，其氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6所示。
在某些实施方案中，所述轻链包含氨基酸序列为SEQ ID NO：7的轻链可变区。在某些实施方案中，所述轻链还包含氨基酸序列为SEQ ID NO：8的轻链恒定区。在某些实施方案中，所述轻链的氨基酸序列为SEQ ID NO：9。
在某些实施方案中，所述重链包含氨基酸序列为SEQ ID NO：10的重链可变区。在某些实施方案中，所述重链还包含氨基酸序列为SEQ ID NO：11的重链恒定区。在某些实施方案中，所述重链的氨基酸序列为SEQ ID NO：12。
在某些实施方案中，所述抗体为EGFR抗体。在某些实施方案中，所述抗体由轻链和重链组成，所述轻链包含CDR-L1、CDR-L2和CDR-L3，其氨基酸序列分别如SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15所示。在某些实施方案中，所述重链包含CDR-H1、CDR-H2、CDR-H3，其氨基酸序列分别如SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18所示。
在某些实施方案中，所述轻链包含氨基酸序列为SEQ ID NO：19的轻链可变区。在某些实施方案中，所述轻链还包含氨基酸序列为SEQ ID NO：20 的轻链恒定区。在某些实施方案中，所述轻链的氨基酸序列为SEQ ID NO：21。
在某些实施方案中，所述重链包含氨基酸序列为SEQ ID NO：22的重链可变区。在某些实施方案中，所述重链还包含氨基酸序列为SEQ ID NO：23的重链恒定区。在某些实施方案中，所述重链的氨基酸序列为SEQ ID NO：24。
本发明涉及的部分序列的信息提供于表1中。
表1：序列的描述

药物组合物和制药用途
本发明的第三方面提供了药物组合物，其包含前述的连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，或前述的抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物。
在一些实施方案中，所述药物组合物还包含用于治疗肿瘤的化疗药物、免疫治疗药物和免疫抑制剂中的至少一种。
在一些实施方案中，所述化疗药物例如为阿霉素(Adriamycin)、环磷酰胺、紫杉烷类(如紫杉醇(Taxol)、多西他赛(Taxotere))、卡培他滨(Xeloda)、吉西他滨(Gemzar)、长春瑞滨(Navelbine)、他莫昔芬、芳香酶抑制剂(瑞宁得、弗隆、阿诺新)、5-FU加亚叶酸、伊立替康(camptosar)、奥沙利铂、顺铂、卡铂、雌莫司汀、米托蒽醌(Novantrone)、泼尼松、长春新碱(Oncovin)、多柔比星、强的松等，或它们的组合。
在一些实施方案中，所述免疫治疗药物例如为PD-1抗体、PD-L1抗体、EGFR单抗、CD20单抗、HER2单抗(例如曲妥珠单抗、Trastuzumab biosimilar、Trastuzumab-dkst)等，或它们的组合。
在一些实施方案中，所述免疫抑制剂选自：(1)糖皮质激素类，如可的松和强的松；(2)微生物代谢产物，如环孢菌素和藤霉素等；(3)抗代谢物，如硫唑嘌呤和6-巯基嘌呤等；(4)多克隆和单克隆抗淋巴细胞抗体，如抗淋巴细胞球蛋白和OKT3等；(5)烷化剂类，如环磷酰胺。在一些具体的实施方案中，所述免疫抑制剂例如为甲基强的松龙、强的松、硫唑嘌呤、普乐可复、赛尼哌、舒莱、环孢菌素、他克莫司、雷帕霉素、霉酚酸酯、咪唑立宾、环磷酰胺、芬戈莫德等。
在一些实施方案中，所述药物组合物还包含至少一种药用辅料。
本发明的第四方面提供了前述的连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，或前述的抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物在制备药物中的用途，所述药物用于预防和/或治疗肿瘤。
本发明的第五方面提供了治疗和/或预防肿瘤的方法，其包括：给予有需要的受试者治疗和/或预防有效量的前述的连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，或前述的抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物。
本发明中所述的“肿瘤”是指器官或组织局部细胞过度增生后形成的病变,包括血液肿瘤和实体瘤，包括良性肿瘤和恶性肿瘤。在某些实施方案中，所述肿瘤选自以下标志物阳性或高表达的肿瘤：HER2、EGFR。
在某些实施方案中，所述肿瘤选自：乳腺癌、黑色素瘤、脑膜瘤、软组织肉瘤、唾液腺肿瘤、原发性肝癌、椎管内肿瘤、纵隔肿瘤、脑癌、骨癌、阴茎癌、骨肉瘤、颅内肿瘤、舌癌、上颌窦癌、甲状腺癌、恶性淋巴瘤、多发性骨髓瘤、脑垂体腺瘤、睾丸肿瘤、非何杰金氏淋巴癌、膀胱癌、白血病、胃癌、鼻咽癌、喉癌、口腔癌、食管癌、肺癌(例如非小细胞肺癌和小细胞肺癌)、肾癌、宫颈癌、绒毛膜癌、外阴癌、皮肤癌、子宫内膜癌、卵巢癌、前列腺癌、胰腺癌、结肠癌、直肠癌、大肠癌、卡波西肉瘤、非黑色素瘤皮肤癌(包括鳞状细胞癌和基底细胞癌)、血管瘤、神经胶质瘤、脑胶质瘤。
制备方法
本发明的第六方面提供了前述连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物的制备方法，所述方法包括以下步骤：
1.制备由1～5个氨基酸或其衍生物组成的连接片段X；
2.将X与包含马来酰亚胺(m)片段或环辛炔片段的化学结构M连接，得到M-X；
3.将细胞毒素D与M-X连接，得到连接子药物偶联物M-X-D。
以下以MCC-AAQ-Exatecan为例进一步对上述制备方法进行说明。MCC-AAQ-Exatecan的示例性的制备过程如下：
1.将Fmoc-Gln与Exatecan反应得到中间体一，其中中间体一结构式如下：
2.将中间体一经DEA脱Fmoc保护基，到中间体二，其中中间体二结构式如下：
3.将中间体二与Fmoc-Ala-Ala反应得到中间体三，其中中间体三结构式如下：
4.将中间体三脱Fmoc保护基得到中间体四，其中中间体四结构式如下：
5.将中间体四与MCC缩合得到产品，其中产品结构式如下：
本发明的第七方面提供了前述抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物的制备方法，所述方法包括以下步骤：
1.取一定重量的单抗，用PBS buffer稀释并调节抗体pH至7.5左右；检测蛋白浓度，并称量抗体溶液净重，计算蛋白总量；向抗体中加入的TCEP溶液，室温下反应，连续摇匀；
2.向还原后的抗体溶液加入过量的细胞毒药物溶液，混匀后在室温下反应，连续混匀；反应结束后向反应液中加入过量的N-乙酰半胱氨酸溶液，室温下反应，连续混匀；
3.使用超滤离心管将偶联产物进行纯化，并置换到储存液中，然后用除菌过滤器进行过滤，即得到抗体药物偶联物。
另外，本发明同时还提供了以下技术方案：
连接子药物偶联物
本发明的第八方面涉及一种连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，所述连接子药物偶联物具有式Ⅰ所示的结构，
M-X-D
式I
其中，
M为包含马来酰亚胺(m)片段或包含环辛炔及其衍生物片段(如BCN、DIBO、DIBAC、BARAC等)的化学结构；
X为1～4个氨基酸组成的连接片段，选自Ala-Ala-Ala-Asn、Ala-Ala-Asn、 Ala-Asn、Asn、Ala-Ala-Ala-Pro、Ala-Ala-Pro、Ala-Pro、Pro、Pro-Asn、Asn-Pro、Lys、Lys-Asn、Lys-Pro、Ala-Ala-Ala-Gln、Ala-Ala-Gln、Ala-Gln、Gly-Gly-Gly-Gln、Gly-Gly-Gln、Gly-Gln、Gln；
D为含有伯胺(-NH2)基团的细胞毒素。
本发明的连接子药物偶联物中，连接子由包含马来酰亚胺(m)片段或包含环辛炔及其衍生物片段(如BCN、DIBO、DIBAC、BARAC等)的化学结构和1～4个氨基酸组成的连接片段构成，其具有以下特点：血液循环中稳定、可以在细胞内或肿瘤微环境中快速解体，并高效释放毒素。
包含马来酰亚胺(m)片段的化学结构可以选自4-(N-马来酰亚氨基甲基)-环己烷-1-甲酰基(mcc)、6-马来酰亚氨基己酰基(mc)、m-(PEG)n、mc-(PEG)n、mcc-(PEG)n等，各结构如下所示：
其中，n代表PEG的聚合度，优选为2-12之间的整数。
在某些实施方案中，包含马来酰亚胺(m)片段的化学结构为6-马来酰亚氨基己酰基(mc)。在某些实施方案中，包含马来酰亚胺(m)片段的化学结构为mc-(PEG)n，其中，n选自4、8、12。发明人发现，较低的PEG聚合度在一定程度上有利于降低连接子药物偶联物制备的ADC中的聚合物含量(HMW％)，因此，PEG的聚合度n优选为4。
包含环辛炔片段的化学结构可以选自BCN、DIBO、DIBAC、BARAC等，各结构如下所示：
式I中的所述1～4个氨基酸组成的连接片段选自Ala-Ala-Ala-Asn、Ala-Ala-Asn、Ala-Asn、Asn、Ala-Ala-Ala-Pro、Ala-Ala-Pro、Ala-Pro、Pro、Pro-Asn、Asn-Pro、Lys、Lys-Asn、Lys-Pro、Ala-Ala-Ala-Gln、Ala-Ala-Gln、Ala-Gln、Gly-Gly-Gly-Gln、Gly-Gly-Gln、Gly-Gln、Gln。上述氨基酸单体或由多个氨基酸组合成的片段能够被肿瘤细胞溶酶体内特定的酶识别，并解离该类化合物中的羧基与含有伯胺的化合物形成的酰胺键(肽键)，不需要额外的自裂解片段(self-immolative moiety)。
在某些实施方案中，所述连接片段选自带有天冬酰胺(Asn)的连接片段，例如Ala-Ala-Ala-Asn、Ala-Ala-Asn、Ala-Asn、Asn、Pro-Asn、Lys-Asn。天冬酰胺(Asn)可被莱古酶(Legumain)识别，莱古酶除了存在于肿瘤细胞的溶酶体内，还大量分布于肿瘤细胞外，因此能够胞外释放小分子，发挥by-stander效应，对低内吞靶点细胞具有良好的杀伤作用。在某些实施方案中，所述连接片段为Ala-Ala-Asn(AAN)。
所述含有伯胺(-NH2)基团的细胞毒素可以选自喜树碱类化合物及其衍生物(如依喜替康、14-氨基喜树碱、9-氨基喜树碱等)等拓扑异构酶I抑制剂、阿霉素类化合物及其衍生物(如Daunorubicin)等拓扑异构酶II抑制剂、美登素类化合物及其衍生物、卡里奇霉素类化合物及其衍生物、多卡米星类化合物及其衍生物(如Duocarmycin衍生物)和吡咯并苯二氮卓类化合物及其衍生物(如PBD)等DNA烷基化试剂、金盏花素类化合物及其衍生物(如去 甲基MMAE或去甲基MMAF等)微管蛋白抑制剂、Protac类化合物和分子胶类化合物(如来那度胺等)等各种具有细胞杀伤活性的小分子化合物。在某些实施方案中，所述含有伯胺(-NH2)基团的细胞毒素为依喜替康(Exatecan)。
在某些实施方案中，所述含有伯胺(-NH2)基团的细胞毒素选自Exatecan(依喜替康)、14-氨基喜树碱(14-AC)、9-氨基喜树碱(9-AC)、去甲基MMAE、去甲基MMAF、Daunorubicin、来那度胺。
在某些实施方案中，本发明的连接子药物偶联物为mc-AAN-Exatecan，其具有如下所示的结构：
抗体药物偶联物
本发明的第九方面涉及一种抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，所述抗体药物偶联物由本发明第八方面所述的连接子药物偶联物与抗体偶联形成。
在某些实施方案中，所述抗体药物偶联物具有式II所示的结构，
Ab-(L-D)p
式II
其中，
Ab为抗体；
L为接头，其结构为M’-X，其中，M’与Ab相连，X与D相连；
M’为如上定义的M通过马来酰亚胺基团连接至Ab所形成的化学结构；
X为如上定义的1～4个氨基酸组成的连接片段；
D为如上定义的含有伯胺(-NH2)基团的细胞毒素；
p为2-8之间的任意数值(如2、2.5、3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5、5.1、5.2、 5.3、5.4、5.5、5.6、5.7、5.8、5.9、6、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9或8，或者如2-2.5、2.5-3、3-3.5、3.5-4、4-4.5、4.5-5、5-5.5、5.5-6、6-6.5、6.5-7、7-7.5或7.5-8)。
式II中，L-D表示接头与细胞毒素通过共价键相连，形成L-D分子；Ab-(L-D)p表示p个L-D分子通过共价键偶联至Ab。
本发明中，药物抗体比(DAR)是指偶联到抗体的药物分子的个数(例如，式I中的p)。本文所述的抗体药物偶联物中包含的药物分子的个数可以是整数，也可以是小数。无论是整数还是小数，其指的均是每个抗体偶联的药物分子的平均数量。“p为2-8之间的任意数值”，其表示，p可以是选自2-8之间的任意整数(包括端点2和8)，也可以是选自2-8之间的任意小数，例如2.3、3.9、4.0、4.2。同时，本领域技术人员可以理解，即使采用相同的制备方法，不同的批次制备得到的抗体药物偶联物的DAR值也不一定完全相同，例如，可以在上下不超过0.5的范围内浮动。
药物抗体比(DAR)可以通过常规分析手段来验证，诸如质谱、ELISA测定法、HIC和HPLC。还可以测定ADC在p方面的定量分布。在有些情况中，将p为某数值的同质ADC从具有其它药物载荷的ADC中分离、纯化和验证可以通过诸如反相HPLC或电泳的手段来实现。
本发明中，连接子药物偶联物通过共价键偶联至抗体Ab上时，一种方式可以通过马来酰亚胺与抗体中的巯基偶联形成-S-丁二酰亚胺结构。例如mc-AAN-Exatecan通过共价键偶联至Ab上时，形成的ADC的结构式如下所示：
上述形成的马来酰亚胺接头的ADC中，-S-既可以是通过工程化改造引入的外接巯基，也可以是抗体Ab被还原进而被打开二硫键后的抗体自身所含有的巯基。
另一种方式可以通过环辛炔及其衍生物片段(如BCN、DIBO、DIBAC、BARAC等)的化学结构与抗体链接，其中环辛炔与修饰抗体中引入的叠氮基团(-N3)偶联形成三氮唑结构结构。例如BCN-AAN-Exatecan通过共价键偶联至Ab上时，形成的ADC的结构式如下所示：
可用于本发明的抗体没有特别的限制，可以选自鼠源抗体、兔源抗体、噬菌体展示来源抗体、酵母展示来源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体。
在某些实施方案中，所述抗体为单克隆抗体，非限制性地选自：HER2抗体、EGFR抗体。
在某些实施方案中，所述抗体为HER2抗体。在某些实施方案中，所述抗体由轻链和重链组成，所述轻链包含CDR-L1、CDR-L2和CDR-L3，其氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3所示。在某些实施方案中，所述重链包含CDR-H1、CDR-H2、CDR-H3，其氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6所示。
在某些实施方案中，所述轻链包含氨基酸序列为SEQ ID NO：7的轻链可变区。在某些实施方案中，所述轻链还包含氨基酸序列为SEQ ID NO：8的轻链恒定区。在某些实施方案中，所述轻链的氨基酸序列为SEQ ID NO：9。
在某些实施方案中，所述重链包含氨基酸序列为SEQ ID NO：10的重链可变区。在某些实施方案中，所述重链还包含氨基酸序列为SEQ ID NO：11的重链恒定区。在某些实施方案中，所述重链的氨基酸序列为SEQ ID NO：12。
在某些实施方案中，所述抗体为EGFR抗体。在某些实施方案中，所述抗体由轻链和重链组成，所述轻链包含CDR-L1、CDR-L2和CDR-L3，其氨基酸序列分别如SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15所示。 在某些实施方案中，所述重链包含CDR-H1、CDR-H2、CDR-H3，其氨基酸序列分别如SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18所示。
在某些实施方案中，所述轻链包含氨基酸序列为SEQ ID NO：19的轻链可变区。在某些实施方案中，所述轻链还包含氨基酸序列为SEQ ID NO：20的轻链恒定区。在某些实施方案中，所述轻链的氨基酸序列为SEQ ID NO：21。
在某些实施方案中，所述重链包含氨基酸序列为SEQ ID NO：22的重链可变区。在某些实施方案中，所述重链还包含氨基酸序列为SEQ ID NO：23的重链恒定区。在某些实施方案中，所述重链的氨基酸序列为SEQ ID NO：24。
本发明涉及的部分序列的信息提供于上表1中。
药物组合物和制药用途
本发明的第十方面提供了药物组合物，其包含前述的连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，或前述的抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物。
在一些实施方案中，所述药物组合物还包含用于治疗肿瘤的化疗药物、免疫治疗药物和免疫抑制剂中的至少一种。
在一些实施方案中，所述化疗药物例如为阿霉素(Adriamycin)、环磷酰胺、紫杉烷类(如紫杉醇(Taxol)、多西他赛(Taxotere))、卡培他滨(Xeloda)、吉西他滨(Gemzar)、长春瑞滨(Navelbine)、他莫昔芬、芳香酶抑制剂(瑞宁得、弗隆、阿诺新)、5-FU加亚叶酸、伊立替康(camptosar)、奥沙利铂、顺铂、卡铂、雌莫司汀、米托蒽醌(Novantrone)、泼尼松、长春新碱(Oncovin)、多柔比星、强的松等，或它们的组合。
在一些实施方案中，所述免疫治疗药物例如为PD-1抗体、PD-L1抗体、EGFR单抗、CD20单抗、HER2单抗(例如曲妥珠单抗、Trastuzumab biosimilar、Trastuzumab-dkst)等，或它们的组合。
在一些实施方案中，所述免疫抑制剂选自：(1)糖皮质激素类，如可的松和强的松；(2)微生物代谢产物，如环孢菌素和藤霉素等；(3)抗代谢物，如硫唑嘌呤和6-巯基嘌呤等；(4)多克隆和单克隆抗淋巴细胞抗体，如抗淋巴细胞球蛋白和OKT3等；(5)烷化剂类，如环磷酰胺。在一些具体的实施方案 中，所述免疫抑制剂例如为甲基强的松龙、强的松、硫唑嘌呤、普乐可复、赛尼哌、舒莱、环孢菌素、他克莫司、雷帕霉素、霉酚酸酯、咪唑立宾、环磷酰胺、芬戈莫德等。
在一些实施方案中，所述药物组合物还包含至少一种药用辅料。
本发明的第十一方面提供了前述的连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，或前述的抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物在制备药物中的用途，所述药物用于预防和/或治疗肿瘤。
本发明的第十二方面提供了治疗和/或预防肿瘤的方法，其包括：给予有需要的受试者治疗和/或预防有效量的前述的连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，或前述的抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物。
本发明中所述的“肿瘤”是指器官或组织局部细胞过度增生后形成的病变,包括血液肿瘤和实体瘤，包括良性肿瘤和恶性肿瘤。在某些实施方案中，所述肿瘤选自以下标志物阳性或高表达的肿瘤：HER2、EGFR。
在某些实施方案中，所述肿瘤选自：乳腺癌、黑色素瘤、脑膜瘤、软组织肉瘤、唾液腺肿瘤、原发性肝癌、椎管内肿瘤、纵隔肿瘤、脑癌、骨癌、阴茎癌、骨肉瘤、颅内肿瘤、舌癌、上颌窦癌、甲状腺癌、恶性淋巴瘤、多发性骨髓瘤、脑垂体腺瘤、睾丸肿瘤、非何杰金氏淋巴癌、膀胱癌、白血病、胃癌、鼻咽癌、喉癌、口腔癌、食管癌、肺癌(例如非小细胞肺癌和小细胞肺癌)、肾癌、宫颈癌、绒毛膜癌、外阴癌、皮肤癌、子宫内膜癌、卵巢癌、前列腺癌、胰腺癌、结肠癌、直肠癌、大肠癌、卡波西肉瘤、非黑色素瘤皮肤癌(包括鳞状细胞癌和基底细胞癌)、血管瘤、神经胶质瘤、脑胶质瘤。
制备方法
本发明的第十三方面提供了前述连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物的制备方法，所述方法包括以下步骤：
1.制备由1～4个氨基酸组成的连接片段X；
2.将X与包含马来酰亚胺(m)片段或环辛炔片段的化学结构M连接，得到M-X；
3.将含有伯胺(-NH2)基团的细胞毒素D与M-X连接，得到连接子药物偶联物M-X-D。
以下以mc-AAN-Exatecan为例进一步对上述制备方法进行说明。mc-AAN-Exatecan的示例性的制备过程如下：
1.将Fmoc-Ala-OH经HOSu活化后，与L-Ala反应得到中间体一，其中中间体一结构式如下：
2.将中间体一经HOSu活化后，与L-Asn反应得到中间体二，其中中间体二结构式如下：
3.将中间体二进行脱Fmoc反应得到中间体三，其中中间体三结构式如下：
4.将中间体三与6-(马来酰亚胺基)己酸琥珀酰亚胺酯反应得到中间体四，其中中间体四结构式如下：
5.将中间体四与依喜替康(Exatecan)缩合得到产品，其中产品结构式如下：
本发明的第十四方面提供了前述抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物的制备方法，所述方法包括以下步骤：
1.取一定重量的单抗，用PBS buffer稀释并调节抗体pH至7.5左右；检测蛋白浓度，并称量抗体溶液净重，计算蛋白总量；向抗体中加入的TCEP溶液，室温下反应，连续摇匀；
2.向还原后的抗体溶液加入过量的细胞毒药物溶液，混匀后在室温下反应，连续混匀；反应结束后向反应液中加入过量的N-乙酰半胱氨酸溶液，室温下反应，连续混匀；
3.使用超滤离心管将偶联产物进行纯化，并置换到储存液中，然后用除菌过滤器进行过滤，即得到抗体药物偶联物。
有益效果
在某些实施方案中，本发明提供的连接子可以在去除疏水性结构PAB的情况下，通过酶解反应释放Payload，既能够降低Linker-Drug的疏水性，减少制备ADC时产生的聚合物含量，也可以表现出相应的细胞杀伤活性。
术语定义
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本 领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本发明中，除非另有说明，否则任何数值范围应理解为包括范围内的任何值或任何子范围。
在本发明中，术语“抗体”是指通常由两对相同的多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体的轻链可分为κ和λ两类。重链可分为μ、δ、γ、α或ε五种，依据重链的不同可将抗体分为IgM、IgD、IgG、IgA和IgE五类。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和补体系统的组分C1q的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为骨架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987 and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
在某些实施方案中，在偶联反应中少于理论最大值的药物模块偶联至抗体。一般而言，抗体不包含许多游离的和反应性的半胱氨酸硫醇基，其可连接药物模块；事实上，抗体中的大多数半胱氨酸硫醇基以二硫桥形式存在。在某些实施方案中，可以在部分或完全还原性条件下用还原剂诸如二硫苏糖醇(DTT)或三羰基乙基膦(TCEP)还原抗体以产生反应性半胱氨酸硫醇基。
本发明中，术语“药学上可接受的盐”是指，(i)本发明所提供的偶联物中 存在的酸性官能团与适当的无机或者有机阳离子(碱)形成的盐，并且包括但不限于，碱金属盐，如钠盐、钾盐、锂盐等；碱土金属盐，如钙盐、镁盐等；其他金属盐，如铝盐、铁盐、锌盐、铜盐、镍盐、钴盐等；无机碱盐，如铵盐；有机碱盐，如叔辛基胺盐、二苄基胺盐、吗啉盐、葡糖胺盐、苯基甘氨酸烷基酯盐、乙二胺盐、N-甲基葡糖胺盐、胍盐、二乙胺盐、三乙胺盐、二环己基胺盐、N,N’-二苄基乙二胺盐、氯普鲁卡因盐、普鲁卡因盐、二乙醇胺盐、N-苄基-苯乙基胺盐、哌嗪盐、四甲基胺盐、三(羟甲基)氨基甲烷盐。以及，(ii)本发明所提供的偶联物中存在的碱性官能团与适当的无机或者有机阴离子(酸)形成的盐，并且包括但不限于，氢卤酸盐，如氢氟酸盐、盐酸盐、氢溴酸盐、氢碘酸盐等；无机酸盐，如硝酸盐、高氯酸盐、硫酸盐、磷酸盐等；低级烷磺酸盐，如甲磺酸盐、三氟甲磺酸盐、乙磺酸盐等；芳基磺酸盐，如苯磺酸盐、对苯磺酸盐等；有机酸盐，如醋酸盐、苹果酸盐、富马酸盐、琥珀酸盐、柠檬酸盐、酒石酸盐、草酸盐、马来酸盐等；氨基酸盐，如甘氨酸盐、三甲基甘氨酸盐、精氨酸盐、鸟氨酸盐、谷氨酸盐、天冬氨酸盐等。
药学上可接受的盐可使用本领域熟知的标准程序获得，例如，通过将足量的碱性物质和提供药学上可以接受的阴离子的合适的酸反应，或者，通过将足量的酸性物质和提供药学上可以接受的阳离子的合适的碱反应。
在本发明中，溶剂合物表示这些形式的本发明的抗体药物偶联物：所述抗体药物偶联物通过与溶剂分子配位而形成的固态或液态形式的复合物。水合物是溶剂合物的一种具体形式，其具有配位的水分子。在本发明中，水合物是优选的溶剂合物。
制备各种含有一定量的活性成分的药物组合物的方法是已知的，或根据本发明的公开内容对于本领域技术人员是显而易见的。如REMINGTON’S PHARMACEUTICAL SCIENCES,Martin,E.W.,ed.,Mack Publishing Company,19th ed.(1995)所述，制备所述药物组合物的方法包括掺入适当的药学赋形剂、载体、稀释剂等，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。
本发明中，所述药用辅料是指生产药品和调配处方时，使用的赋形剂和附加剂，是指除活性成分外，在安全性方面已进行了合理的评估，并且包含在药物制剂中的物质。药用辅料除了赋型、充当载体、提高稳定性外，还具 有增溶、助溶、缓控释等重要功能，是可能会影响到药品的质量、安全性和有效性的重要成分。根据其来源可分为天然物、半合成物和全合成物。根据其作用与用途可分为：溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、湿润剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏着剂、抗氧剂、螯合剂、渗透促进剂、pH调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂、释放阻滞剂等；根据其给药途径可分为口服、注射、黏膜、经皮或局部给药、经鼻或口腔吸入给药和眼部给药等。同一药用辅料可用于不同给药途径的药物制剂，且有不同的作用和用途。
本发明中，所述药物组合物可根据给药途径制成各种适宜的剂型。例如片剂、胶囊剂、颗粒剂、口服溶液剂、口服混悬剂、口服乳剂、散剂、酊剂、糖浆剂、注射剂、栓剂、软膏剂、乳膏剂、糊剂、眼用制剂、丸剂、植入剂、气雾剂、粉雾剂、喷雾剂等。其中，所述的药物组合物或适宜的剂型可以含有0.01mg至1000mg的本发明的抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物。
本文使用的术语“治疗”一般是指获得需要的药理和/或生理效应。该效应根据完全或部分地预防疾病或其症状，可以是预防性的；和/或根据部分或完全稳定或治愈疾病和/或由于疾病产生的副作用，可以是治疗性的。本文使用的“治疗”涵盖了对患者疾病的任何治疗，包括：(a)预防易感染疾病或症状但还没诊断出患病的患者所发生的疾病或症状；(b)抑制疾病的症状，即阻止其发展；或(c)缓解疾病的症状，即，导致疾病或症状退化。
在本发明中，“受试者”指脊椎动物。在某些实施方案中，脊椎动物指哺乳动物。哺乳动物包括，但不限于，牲畜(诸如牛)、宠物(诸如猫、犬、和马)、灵长类动物、小鼠和大鼠。在某些实施方案中，哺乳动物指人。
在本发明中，“有效量”指在必需的剂量和时间上有效实现期望的治疗或预防效果的量。本发明的物质/分子的“治疗有效量”可根据诸如个体的疾病状态、年龄、性别和体重及该物质/分子在个体中引发期望应答的能力等因素而变化。治疗有效量还涵盖该物质/分子的治疗有益效果胜过任何有毒或有害后果的量。“预防有效量”指在必需的剂量和时间上有效实现期望的预防效果的量。通常而非必然，由于预防剂量是在疾病发作之前或在疾病的早期用于受 试者的，因此预防有效量会低于治疗有效量。在癌症的情况中，药物的治疗有效量可减少癌细胞数；缩小肿瘤体积；抑制(即一定程度的减缓，优选停止)癌细胞浸润到周围器官中；抑制(即一定程度的减缓，优选停止)肿瘤转移；一定程度的抑制肿瘤生长；和/或一定程度的减轻与癌症有关的一种或多种症状。
在本发明中，20种常规氨基酸和其缩写遵从常规用法。参见Immunology-A Synthesis(第2版,E.S.Golub和D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其通过引用合并入本文。
在本发明中，若X为Ala-Ala-Gln，则表示X为由Ala、Ala和Gln依次连接形成肽键后组成的连接片段，且本领域技术人员可以理解，其并不限定Ala、Ala和Gln具体的立体构型，如L型或D型。类似地，若X为Ala-Ala-(3-cyano-alanine)，则表示X为由Ala、Ala、(3-cyano-alanine)依次连接形成肽键后组成的连接片段，且本领域技术人员同样可以理解，其并不限定Ala、Ala、(3-cyano-alanine)具体的立体构型，如L型或D型，或者S型或R型。本发明中X其他类似的限定请参照前述内容进行理解。
在本发明中，若X选自Ala-Ala-Gln、Ala-Ala-(3-cyano-alanine)及它们的氘代化物，其表示，X可以为Ala-Ala-Gln，也可以为Ala-Ala-(3-cyano-alanine)，也可以为Ala-Ala-Gln的氘代化物，还可以为Ala-Ala-(3-cyano-alanine)的氘代化物。
术语“氘代化物”表示某个化合物中的一个或多个(如2个、3个、4个或更多个)氢原子(氢原子的位置不做具体限定，可以是烷基上的氢原子，也可以是氨基上的氢原子等等)被氘原子替代后所形成的化合物。
术语“嵌合抗体”是指这样的抗体，其中可变区序列源自一个物种，恒定区序列源自另一物种，如其中可变区序列源自小鼠抗体及恒定区序列源自人抗体的抗体。
“人源化”抗体是指非人(例如小鼠)抗体形式，其是嵌合的免疫球蛋白、免疫球蛋白链或者其片段(如Fv、Fab、Fab'、F(ab')2或者抗体的其它抗原结合亚序列)，含有源自非人免疫球蛋白的最小序列。优选地，人源化抗体是人免疫球蛋白(接受者抗体)，其中接受者抗体的互补决定区(CDR)的残基由来自具有希望的特异性、亲和性和能力的非人物种(供体抗体)如小鼠、大鼠或者兔的CDR残基置换。
此外，在人源化中，还可能对VH和/或VL的CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，由此改善抗体的一或多种结合特性(例如亲和性)。可进行例如PCR介导的突变引入突变，其对抗体结合或其它功能特性的影响可利用本文所述的体外或体内测试评估。通常，引入保守性突变。此类突变可为氨基酸取代、添加或缺失。另外，CDR内的突变通常不超过一个或两个。
下面结合具体实施例对本发明进行进一步的解释说明，但这些实施例并非限制本发明的范围。
实施例一、连接子药物MCC-AAQ-Exatecan的制备
1.中间体一的合成Fmoc-Gln-Exatecan
将Fmoc-Gln-OH(N-芴甲氧羰基-L-谷氨酰胺)(76.3mg 1.1eq.)、Exatecan(100mg 1.0eq.)加入反应瓶中，加入DMF(1ml)，DIEA(29mg 1.5eq.)加入TBTU(72.5mg 1.1eq.)，室温反应2小时，HPLC监控，无原料剩余。反应结束后，中压过柱纯化(DCM/MeoH)。收集产品，浓缩，得到中间体一。
反应式如下：
2.中间体二的合成Gln-Exatecan
将中间体一Fmoc-Gln-Exatecan(130mg 1.0eq.)加入反应瓶中，加入DCM(2ml)，室温搅拌。加入DEA(0.5ml)，室温反应。4小时后，HPLC监控，无原料剩余。停止反应，缓慢加入MTBE(10ml)，析出大量固体，搅拌30min。过滤，MTBE洗涤，得到灰白色固体，干燥，得到95mg中间体二。
反应式如下：
3.中间体三的合成Fmoc-Ala-Ala-Gln-Exatecan
将中间体二Gln-Exatecan(100mg 1.0eq.)加入反应瓶中，DMF(1mL)，Fmoc-Ala-Ala-OH(67.8mg 1.0eq)，TBTU(68.4mg 1.2eq.),DIEA(34.4mg 1.5eq.),室温反应3小时，HPLC监控，反应结束。中压过柱纯化(DCM/MeOH)，收集产品得到中间体三。
反应式如下：
4.中间体四的合成Ala-Ala-Gln-Exatecan
将中间体三Fmoc-Ala-Ala-Gln-Exatecan(140mg 1.0eq.)加入反应瓶中，加入DCM(2ml)，室温搅拌。加入DEA(0.5ml)，室温反应，4小时后，HPLC监控，反应结束。缓慢加入MTBE(10ml)，析出大量固体，搅拌30min。过滤，MTBE洗涤，得到灰白色固体，干燥，得到中间体四。
反应式如下：
5.产品合成MCC-Ala-Ala-Gln-Exatecan
将中间体四Ala-Ala-Gln-Exatecan(50mg 1.0eq.)加入反应瓶中，DMF(1mL)，再加入MCC(18mg 1.1eq)，HATU(80.8mg,3.0eq),DIEA(36.6mg,4.0eq.),室温反应2小时后，HPLC监控，至中间体四无剩余，停止反应。Prep-HPLC制备纯化，收集产品，浓缩干，得到产品(核磁如下)。
反应式
1H NMR(400MHz,DMSO-d6)δ8.17-8.26(d,J＝2.0Hz,1H),7.83-7.91(d,J＝2.0Hz,2H)，7.65-7.74(d,J＝2.0Hz,2H)，7.21-7.28(d,J＝8.0Hz,2H)，6.98-7.04(m,2H)，5.43-5.50(m,1H)，5.36-5.43(m,2H)，5.15-5.24(m,1H)，5.01-5.11(m,1H)，4.14-4.24(m,1H)，3.97-4.08(m,1H)，3.47-3.57(m,1H)，3.20-3.27(m,1H)，3.05-3.16(m,2H)，2.30-2.36(m,2H)，2.00-2.15(m,4H)，1.77-1.92(m,3H)，1.65-2.00(m,5H)，1.45-1.64(m,4H)，1.09-1.24(m,5H)，0.95-1.07(m,4H)，0.79-0.94(m,5H).
实施例二、连接子药物MCC-Ala-Ala-Asn-Exatecan的制备
1.中间体一的合成
将Fmoc-Ala-Ala-OH(N-[芴甲氧羰基]-L-丙氨酰基-L-丙氨酸)经HOSu(N-羟基丁二酰亚胺)活化后，与L-Asn(L-天冬酰胺)反应得到中间体一。
具体的步骤是：室温下反应瓶中加入Fmoc-Ala-Ala-OH(1.0eq)，HOSu(1.3eq)，THF(10v/w)，控制温度在室温，搅拌条件下缓慢加入DCC(1.3eq)，室温进行反应，用HPLC检测，至Fmoc-Ala-Ala-OH残留较少(面积归一法)，将反应液过滤，滤饼用THF(2.5v/w)淋洗。滤液中加入纯化水(11.5v/w)，加入L-Asn(1.1eq)，碳酸氢钠固体(1.0eq)，室温搅拌反应，用HPLC检测至Fmoc-Ala-Ala-OSu残留较少，然后加入一水合柠檬酸(1.0eq)搅拌，浓缩反应液除去大部分溶剂，加入DMF(4v/w)溶解产品，过滤后通过制备液相进行反相制备，浓缩制备液至无明显液滴流出后，用乙酸乙酯萃取4次，将 乙酸乙酯相浓缩至干得到中间体一，反应式如下：
2.中间体二的合成
将中间体一加DEA(二乙胺)脱Fmoc后，得到中间体二。
具体的步骤是：室温下反应瓶中加入中间体一(1.0eq)，加入DMF(15v/w)，控制温度在室温，滴加DEA(3v/w)，室温进行反应，用HPLC检测，至中间体一无残留，浓缩除去DMF，加入DCM(40v/w)，纯化水(40v/w)，搅拌后分液，将水相浓缩至干得到中间体二，反应式如下：
3.中间体三的合成
将中间体二与4-(N-马来酰亚胺甲基)环己烷羧酸-N-琥珀酰亚胺酯反应得到中间体三。
具体的步骤是：室温下反应瓶中加入中间体二(1.0eq)，4-(N-马来酰亚胺甲基)环己烷羧酸-N-琥珀酰亚胺酯(1.1eq)，加入DMF(22v/w)，水(9.5v/w)，DIPEA(1.0eq)，后室温进行反应，至中间体二残留较少(QDA检测)，待反应完成，浓缩除去大部分溶剂，加入水(50v/w)，EA(50v/w)，加入1.0eq DIEA搅拌溶清后静置分液，水相用EA(50v/w*2)萃取两次，将水相浓缩至干，得到中间体三，反应式如下：
4.产品的合成
将中间体三与依喜替康甲磺酸盐(CAS号：169869-90-3)经HATU经缩合反应得到中间体四。
具体的步骤是：室温条件下，反应瓶中加入中间体三(1.2eq)，加入DMF(15v/w)，依次加入依喜替康甲磺酸盐(1.0eq)，TBTU(1.4eq)，DIPEA(4.5eq)，后室温进行反应，HPLC中控检测，反应完成后，通过prep-HPLC纯化，浓缩制备液，DCM萃取制备液，浓缩，得到产品(核磁如下)，反应式如下：
1H NMR(400MHz,DMSO-d6)δ8.15-8.25(d,J＝2.0Hz,1H),7.85-8.0(d,J＝7.5Hz,2H)，7.7-7.85(d,J＝2.0Hz,1H)，7.33-7.4(d,J＝8.0Hz,1H)，7.28-7.32(m,1H)，6.98-7.05(d,J＝8.0Hz,2H)，6.85-6.95(m,1H)，6.45-6.57(m,1H)，5.45-5.55(m,1H)，5.40-5.45(m,1H)，5.20-5.27(m,1H)，4.40-4.50(m,1H)，3.95-4.05(m,1H)，3.20-3.27(m,2H)，3.10-3.18(m,2H)，2.86-2.97(m,2H)，2.36-2.43(m,3H)，2.15-2.25(m,1H)，1.95-2.10(m,3H)，1.80-1.93(m,3H)，1.73-1.78(m,1H)，1.55-1.70(m,3H)，1.45-1.55(m,3H)，1.0-1.3(m,10H)，0.80-0.95(m,5H).
实施例三、连接子药物MCC-Ala-Ala-(3-cyano-L-alanine)-Exatecan的制备
1.中间体一的合成(3-cyano-L-alanine)-Exatecan
将Fmoc-Asn-OH(40mg 1.2eq)加入反应瓶中，加入DMF(1.5ml)，TBTU(45mg，1.5eq)，DIEA(60mg 2.5eq)搅拌，溶解完全。搅拌30min后，再加入伊喜替康甲磺酸盐(50mg 1.0eq)，搅拌4hrs，HPLC检测不反应。补加TBTU(45mg，1.5eq)，DIEA(60mg 2.5eq)继续室温反应。HPLC监控，原料Exatecan无剩余，Fmoc-(3-cyano-L-alanine)-Exatecan占比约70％。停止反应，中压制备纯化。收集产品。30℃减压浓缩干，得到产品。
将上述产品加入反应瓶中，再加入DCM(4ml)搅拌。加入DEA(1ml)， 室温搅拌，溶解完全。HPLC监测，无原料剩余。加入MTBE 10ml，室温搅拌，有大量固体析出。过滤，得到灰白色固体。MTBE洗涤滤饼，干燥得到35mg固体，即中间体一。
反应式如下：
2.中间体二的合成MCC-Ala-Ala-OH
将SMCC(460mg，1.1eq)加入反应瓶中，再加入NH2-Ala-Ala-OH(200mg，1.0eq)，加入DMF(4ml，20V)，搅拌，再加入DIPEA(161.4mg，1.0eq)。加热至60℃反应。随反应进行反应液逐渐溶清。反应至少8小时。取样LC-MS检测，反应结束，降温至室温。35℃减压浓缩除去DMF，再加入纯化水(4ml，20V)，DIPEA(161.4mg，1.0eq)。加入EA(4ml，20V)，搅拌洗涤。有机相弃去。再加入EA(2ml，10V)萃取两次。有机相弃去。水相保留，用2M盐酸调节酸性至pH为1～2，析出大量白色固体。搅拌一小时。过滤，得到白色固体。纯化水洗涤固体。干燥。得到白色固体320mg，即中间体二。
反应式如下：
3.产品合成MCC-Ala-Ala-(3-cyano-L-alanine)-Exatecan
将中间体一(3-cyano-L-alanine)-Exatecan(35mg 1.0eq)加入反应瓶中，DMF(1mL)，再加入中间体二MCC-Ala-Ala-OH(28mg,1.1eq),TBTU(25.3mg,1.2eq),DIPEA(12.8mg,1.5eq)室温反应2小时后，HPLC监控，中间体一无剩余，停止反应。Prep-HPLC纯化，收集产品。浓缩干。得到黄色玻璃状固体即产品(核磁如下)。
反应式如下：
1H NMR(400MHz,DMSO-d6)δ8.45-8.55(d,J＝2.0Hz,1H),8.23-8.29(d,J＝2Hz,2H)，7.95-8.03(d,J＝2.0Hz,1H)，7.84-7.92(d,J＝8.0Hz,1H)，7.76-7.84(m,1H)，7.27-7.33(d,J＝8.0Hz,1H)，6.98-7.02(m,1H)，5.47-5.56(m,1H)，5.40-5.45(m,1H)，5.26-5.36(m,1H)，5.1-5.2(m,1H)，4.44-4.55(m,1H)，4.07-4.19(m,1H)，3.88-4.0(m,2H)，3.19-3.26(m,2H)，3.13-3.18(m,1H)，2.90-3.0(m,2H)，2.76-2.88(m,2H)，2.36-2.43(m,3H)，2.00-2.12(m,2H)，1.80-1.93(m,3H)，1.80-1.92(m,2H)，1.55-1.70(m,3H)，1.13-1.26(m,5H)，1.05-1.12(m,3H)，0.80-0.95(m,5H).
实施例四、连接子药物MCC-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)-Exatecan的合成路线
1.中间体一的合成((S)-2-amino-4-cyanobutanoic acid)-Exatecan
将(S)-2-((((9H-芴-9-基)甲氧基)羰基)氨基)-4-氰基丁酸(79.8mg 1.1eq)、伊喜替康甲磺酸盐(110mg 1.0eq)加入反应瓶中，加入DMF(1.5ml)，TBTU(79.73mg，1.5eq)，DIEA(80mg 2.5eq)，室温反应3小时，HPLC监控。反应结束后，加入ACN 9ml，室温搅拌。再加入DEA 1.0ml，继续室温反应1小时。取样监测，无原料剩余。加入MTBE 10ml。搅拌，无明显固体析出。 搅拌1小时，无明显变化。
30℃减压浓缩除去溶剂。浓缩过程中析出大量固体，此时溶剂仅除去少量，约3ml。停止浓缩，继续室温搅拌1小时。过滤，再用MTBE洗涤固体。干燥，得到灰白色固体即中间体一。
反应式如下：
2.中间体二的合成MCC-Ala-Ala-OH
将SMCC(460mg，1.1eq)加入反应瓶中，再加入NH2-Ala-Ala-OH(200mg，1.0eq)，DMF(4ml，20V)，搅拌，再加入DIPEA(161.4mg，1.0eq)。加热至60℃反应8小时取样LC-MS检测，反应结束，降温至室温。35℃减压浓缩除去DMF，再加入纯化水(4ml，20V)，DIPEA(161.4mg，1.0eq)。加入EA(4ml，20V)，搅拌洗涤。有机相弃去。再加入EA(2ml，10V)洗涤两次。有机相弃去。水相保留，用2M盐酸调节酸性至pH为1～2，析出大量白色固体。搅拌一小时。过滤，得到白色固体。纯化水洗涤固体。干燥。得到白色固体320mg即中间体二。
反应式如下：
3.产品合成MCC-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)-Exatecan
将中间体一((S)-2-amino-4-cyanobutanoic acid)-Exatecan(25mg 1.0eq)加入反应瓶中，DMF(1mL)，再加入中间体二MCC-Ala-Ala-OH(19mg,1.1eq),TBTU(17.7mg,1.2eq),DIPEA(8.88mg,1.5eq)室温反应2小时后，HPLC监控，反应结束。Prep-HPLC制备纯化，收集产品。浓缩除去大部分乙腈。冻干，得到黄色固体即产品(核磁如下)。
反应式如下：
1H NMR(400MHz,DMSO-d6)δ8.30-8.37(d,J＝2.0Hz,1H),7.87-7.96(d,J＝2.0Hz,2H)，7.75-7.86(d,J＝2.0Hz,2H)，7.27-7.33(d,J＝8.0Hz,1H)，6.98-7.02(m,2H)，5.45-5.54(m,1H)，5.40-5.45(m,2H)，5.23-5.32(m,1H)，5.04-5.13(m,1H)，4.25-4.37(m,1H)，3.97-4.09(m,1H)，3.56-3.66(m,4H)，3.10-3.26(m,4H)，2.35-2.46(m,4H)，2.00-2.13(m,3H)，1.77-1.92(m,3H)，1.66-1.75(m,2H)，1.56-1.64(m,3H)，1.45-1.53(m,1H)，1.12-1.26(m,4H)，1.01-1.13(m,4H)，0.80-0.94(m,4H).
实施例五、连接子药物MCC-Ala-Ala-Ala-Ala-Gln-Exatecan的合成路线
1.中间体一的合成MCC-Ala-Ala-Ala-Ala-Gln-OH
将H-Gln(Trt)-OH 2-CTC resin(1.0g，0.5mmol，0.5mmol/g)加入固相反应器中，加入DCM(20mL),在振荡器中振荡20min充分溶胀树脂。过滤，用DMF洗涤树脂3次。加入含Fmoc-Ala-OH(3.0eq，467mg，1.5mmol)，HBTU(3.0eq，569mg，1.5mmol)及HOBT(3.0eq，202mg，1.5mmol)的DMF溶液20mL，摇匀后加入DIPEA(3.0eq，0.26mL，1.5mmol)，在振荡器中反应1hr。Kaiser检测树脂颜色为阴性。过滤，用DMF洗涤树脂6次。加入20％哌啶/DMF溶液20mL，在振荡器中反应15min，过滤，并再次加入20％哌啶/DMF溶液20mL，在振荡器中反应15min，Kaiser检测树脂颜色为阳性，用DMF洗涤树脂6次，得到H-Ala-Gln(Trt)-2-CTC-resin。
向含H-Ala-Gln(Trt)-2-CTC-resin的反应管内，加入含Fmoc-Ala-OH(3.0eq，467mg，1.5mmol)，HBTU(3.0eq，569mg，1.5mmol)及HOBT(3.0eq，202mg，1.5mmol)的DMF溶液20mL，摇匀后加入DIPEA(3.0eq，0.26mL，1.5mmol)，在振荡器中反应1hr。Kaiser检测树脂颜色为阴性。过滤，用DMF洗涤树脂6次。加入20％哌啶/DMF溶液20mL，在振荡器中反应15min，过滤，并再次加入20％哌啶/DMF溶液20mL，在振荡器中反应15min，Kaiser检测树脂颜色为阳性，用DMF洗涤树脂6次，得到H-Ala-Ala-Gln(Trt)-2-CTC-resin。
向含H-Ala-Ala-Gln(Trt)-2-CTC-resin的反应管内，加入含Fmoc-Ala-OH(3.0eq，467mg，1.5mmol)，HBTU(3.0eq，569mg，1.5mmol)及HOBT(3.0eq，202mg，1.5mmol)的DMF溶液20mL，摇匀后加入DIPEA(3.0eq，0.26mL，1.5mmol)，在振荡器中反应1hr。Kaiser检测树脂颜色为阴性。过滤，用DMF洗涤树脂6次。加入20％哌啶/DMF溶液20mL，在振荡器中反应15min，过滤，并再次加入20％哌啶/DMF溶液20mL，在振荡器中反应15min，Kaiser检测树脂颜色为阳性，用DMF洗涤树脂6次，得到H-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin。
向含H-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin的反应管内，加入含Fmoc-Ala-OH(3.0eq，467mg，1.5mmol)，HBTU(3.0eq，569mg，1.5mmol)及HOBT(3.0eq，202mg，1.5mmol)的DMF溶液20mL，摇匀后加入DIPEA(3.0eq，0.26mL，1.5mmol)，在振荡器中反应1hr。Kaiser检测树脂颜色为阴性。过滤，用DMF洗涤树脂6次。加入20％哌啶/DMF溶液20mL，在振荡器中反应15min，过滤，并再次加入20％哌啶/DMF溶液20mL， 在振荡器中反应15min，Kaiser检测树脂颜色为阳性，用DMF洗涤树脂6次，得到H-Ala-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin。
向含H-Ala-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin的反应管内，加入含SMCC(3.0eq，501mg，1.5mmol)的DMF溶液20mL，摇匀后加入DIPEA(3.0eq，0.26mL，1.5mmol)，在振荡器中反应1hr。Kaiser检测树脂颜色为阴性。过滤，依次用DMF洗涤树脂6次，MeOH洗涤树脂3次，MTBE洗涤3次。真空干燥箱30℃干燥1hr，得到MCC-Ala-Ala-Ala-Ala-Gln(Trt)-2-CTC-树脂(1.42g,0.5mmol)。
向上述得到的树脂转移至50mL离心管中,加入冷的95％TFA/Tis溶液(15mL)，在振荡器中反应1hr。过滤，并将滤液缓慢加入到冷的MTBE溶液中(150mL)，静置1hr，析出白色沉淀物，离心并用冷的MTBE溶液洗涤沉淀3次。收集洗涤后的沉淀，真空干燥箱30℃干燥4hr，得到白色固体即中间体一MCC-Ala-Ala-Ala-Ala-Gln-OH。
反应式如下：
2.产品合成MCC-Ala-Ala-Ala-Ala-Gln-Exatecan
将中间体一MCC-Ala-Ala-Ala-Ala-Gln-OH(244mg，2.0eq)加入反应瓶中，再加入DMF(2mL)，伊喜替康加磺酸盐(100mg，1.0eq)，TBTU(132mg，2.2eq)，DIPEA(73mg，3.0eq)。室温搅拌反应1小时。HPLC检测，Exatecan剩余10％。
Prep-HPLC纯化，收集产品。浓缩干制备液，得到黄色固体(核磁如下)。
反应式如下：
1H NMR(400MHz,DMSO-d6)δ8.25-8.33(d,J＝2.0Hz,1H),7.87-7.95(d,J＝2.0Hz,2H)，7.76-7.86(d,J＝2.0Hz,2H)，7.28-7.34(d,J＝8.0Hz,1H)，7.20-7.26(m,1H)，6.98-7.02(m,1H)，6.73-6.76(m,1H)，5.47-5.55(m,1H)，5.40-5.41(m,1H)，5.26-5.36(m,2H)，5.05-5.15(m,2H)，4.12-4.20(m,2H)，4.03-4.09(m,1H)，3.87-3.94(m,1H)，3.20-3.27(m,2H)，3.12-3.18(m,1H)，2.65-2.68(m,1H)，2.36-2.43(m,2H)，2.31-2.35(m,1H)，2.02-2.13(m,2H)，1.78-1.93(m,3H)，1.6-1.76(m,2H)，1.56-1.64(m,2H)，1.21-1.27(m,1H)，1.06-1.20(m,6H)，0.85-0.94(m,3H).
实施例六：连接子药物MCC-Ala-Ala-Gln-Belotecan的合成路线
MCC-Ala-Ala-Gln-Belotecan合成路线
1.中间体3 Fmoc-Gln-Belotecan合成
将中间体1(212mg，0.575mmol，5.0eq)，Belotecan(50mg，0.115mmol，1.0eq)，HATU(219mg，0.575mmol，5eq)加入反应瓶中，加入DMF(3mL)震荡使其溶解，10℃下加入DIEA(190μL，1.15mmol，10eq)，10℃搅拌反应1hr。HPLC检测Belotecan反应完全。制备纯化，收集产品。浓缩冻干，得到黄色固体产物3 Fmoc-Gln-Belotecan(51mg，纯度：98.71％，收率：55.84％)。MS(ESI)：m/z 784.3[M+H]+。
2.中间体4 Gln-Belotecan合成
将中间体3 Fmoc-Gln-Belotecan(51mg，0.065mmol，1.0eq)加入反应瓶中，加入DMF(4mL)，二乙胺(1mL)，20℃搅拌2hr，HPLC检测原料无剩余。真空浓缩反应液，加入DMF(2mL)，通过制备纯化，将制备液浓缩后冻干得黄色固体4Gln-Belotecan(24mg，纯度：91.96％，收率：60.40％)。MS(ESI)：m/z 562.34[M+H]+。
3中间体5 MCC-Ala-Ala-OH的合成
将原料5-1 MCC-OSu(9.5g，59.31mmol，1.0eq)，5-2 Ala-Ala-OH(21.8g，65.24mmol，1.1eq)加入反应瓶中，25℃加入DMF(190mL)，然后加入DIEA(9.8mL，59.31mmol，1.0eq),60℃搅拌反应8hr。HPLC检测反应完全。反应液降至25℃，真空浓缩，然后依次加入H2O(190mL)，DIEA(9.8mL)及EA(190mL)，25℃搅拌1hr后加入分液漏斗中静置分液。将水相分出，用EA(95mL)萃取两次。分去EA层，在水相中缓慢滴加2M盐酸溶液(71.3mL)，25℃搅拌1hr后过滤，得到的固体经真空干燥得到白色固体5 MCC-Ala-Ala-OH(16.55g，纯度：95％，收率：73.54％)。MS(ESI)：m/z 380.2[M+H]+。
4 MCC-Ala-Ala-Gln-Belotecan合成
将中间体4(15mg，0.027mmol，1.0eq)，5MCC-Ala-Ala-OH(15.2mg，0.04mmol，1.5eq)，HOAT(7.3mg，0.054mmol，2.0eq)加入反应瓶中，加入DMF(2mL)震荡使其溶解，10℃下加入DIC(8.0μL，0.054 mmol，2.0eq)，20℃搅拌反应3hr。HPLC检测中间体4反应完全。制备纯化，收集产品。浓缩冻干，得到黄色固体产物MCC-Ala-Ala-Gln-Belotecan(11mg，纯度：96.42％，收率：43.03％)。MS(ESI)：m/z 462.5[M+2H]/2+。
1H NMR(400MHz,DMSO-d6)δ8.58(d,J＝10.4Hz,1H),8.19(d,J＝8.0Hz,1H),8.15(d,J＝8.0Hz,1H),7.92-7.87(m,4H),7.78(t,J＝8.0Hz,1H),7.35(s,1H),7.21(brs,1H),6.99(s,2H),6.77(brs,1H),5.46-5.43(m,4H),4.79-4.76(m,1H),4.35-4.28(m,1H),4.26-4.21(m,2H),3.42-3.38(m,2H),3.23(d,J＝6.8Hz,2H),3.22-3.18(m,1H),2.22-2.07(m,3H),1.93-1.84(m,3H),1.75-1.66(m,3H),1.62-1.59(m,2H),1.27-1.26(m,4H),1.23-1.21(m,5H),1.18-1.16(m,6H),0.90-0.86(m,6H).
另外，本领域技术人员还可以参照以上实施例一～实施例六的制备方法获得本发明其他连接子药物偶联物，例如，MCC-AAQ-Rapamycin，
实施例七、EGFR抗体药物偶联物的制备
一、抗体偶联物EGFR抗体-MCC-AAQ-Exatecan的制备
1.取EGFR抗体，用Tris-EDTA溶液调节抗体pH至7.5左右；用Nanodrop检测蛋白浓度，并称量抗体溶液净重，计算蛋白总量。向抗体中加入TCEP溶液，置于3D摇床上，室温下，反应120min以上，连续混匀将抗体链间二硫键完全还原。
2.向还原后的抗体溶液加入过量的MCC-AAQ-Exatecan溶液(溶于DMSO)，混匀后置于3D摇床上，室温下，反应30min以上，连续混匀。反应结束后向反应液中加入过量的N-乙酰半胱氨酸溶液，置于3D摇床上，室温下，反应30min以上，连续混匀。
3.使用30KD超滤离心管将偶联产物进行纯化，并置换到储存液(10mM Histidine，pH5.5左右)中，置换倍数大于1000倍。然后用0.22um的除菌过滤器进行过滤，即得到抗体药物偶联物EGFR抗体-MCC-AAQ-Exatecan于4℃进行保存。
4.用UV/BCA的方法检测抗体药物偶联物的蛋白浓度，用HIC进行DAR检测，SEC进行纯度检测。
二、其他EGFR抗体药物偶联物的制备
本发明的其他连接子药物偶联物(如式I所示的连接子药物偶联物)与EGFR抗体制备抗体药物偶联物的方法可参照上述抗体偶联物EGFR抗体-MCC-AAQ-Exatecan的制备方法。
实施例八、HER2抗体药物偶联物的制备
本发明的连接子药物偶联物(如式I所示的连接子药物偶联物)与HER2抗体制备抗体药物偶联物(如抗体药物偶联物HER2抗体-MCC-AAN-Exatecan)的方法可参照上述实施例七的抗体偶联物EGFR抗体-MCC-AAQ-Exatecan的制备方法。
实施例九、不同抗体药物偶联物的体外活性测试
对本发明的不同抗体药物偶联物进行体外活性测试，以检测其细胞杀伤活性。
测试方法如下：
将靶细胞按一定数量铺板入96孔黑色底透的细胞培养板，经过24小时孵育后，加入浓度梯度稀释的样品，再经过72小时或144小时孵育后，使用显色试剂，显色60分钟，使用酶标仪进行读板。活细胞数越多，信号值越高，根据不同的浓度梯度点所得到的不同信号值，在酶标仪上形成四参数拟合曲线，得到C值即IC50值。
部分数据如下。
表1 MCC-AAQ-Exatecan ADC在MDA-MB-468细胞系中的活性数据
另外，本发明还提供了以下实施例。
实施例十、连接子药物偶联物mc-AAN-Exatecan的制备
1.中间体一的合成
将Fmoc-Ala-OH(N-芴甲氧羰基-L-丙氨酸，CAS号：35661-39-3)经HOSu(N-羟基丁二酰亚胺，CAS号：6066-82-6)活化后，与L-Ala(L-丙氨酸，CAS号：56-41-7)反应得到中间体一；具体的，反应瓶内加入Fmoc-Ala-OH(3g，1.0eq)、HOSu(1.45g，1.3eq)，加入21mL的THF，控制温度在室温，搅拌条件下缓慢加入DCC(2.59g，1.3eq)，后室温进行反应，用HPLC监控，反应结束后，将反应液过滤，滤饼用THF(6mL)淋洗。滤液中加入纯化水(15mL)，后加入L-Ala(1.12g，1.3eq)，碳酸氢钠固体(0.81g，1.0eq)，室温搅拌反应，HPLC监控，反应结束后加入柠檬酸(2.02g，1.0eq)搅拌，然后反应液用乙酸乙酯萃取，浓缩有机相，加入DMF(12mL)溶解产品，过滤后通过pre-HPLC，浓缩制备液至无明显液滴流出后，用乙酸乙酯萃取三次，将乙酸乙酯相浓缩至干得到中间体一1.75g，该步反应收率为57％，反应式如下：
2.中间体二的合成
将中间体一(N-[芴甲氧羰基]-L-丙氨酰基-L-丙氨酸，CAS号：87512-31-0)经HOSu(N-羟基丁二酰亚胺，CAS号：6066-82-6)活化后，与L-Asn(L-天冬酰胺，CAS号：70-47-3)反应得到中间体二；具体的，反应瓶中加入中间体一(0.87g，1.0eq)、HOSu(0.34g，1.3eq)、THF(9mL)，控制温度在室温，搅拌条件下缓慢加入DCC(0.61g，1.3eq)，室温进行反应，用HPLC监控，反应结束后，将反应液过滤，滤饼用THF(2mL)淋洗。滤液中加入纯化水(10mL)，加入L-Asn(0.34g，1.1eq)，碳酸氢钠固体(0.19g，1.0eq)，室温搅拌反应，HPLC监控，反应结束后，加入一水合柠檬酸(0.48g，1.0eq)搅拌，浓缩反应液除去大部分溶剂，制备纯化残余物，浓缩制备液至无明显液滴流出后，用乙酸乙酯萃取，有机相浓缩至干得到中间体二826mg，该步反应收率为73％，反应式如下：
3.中间体三的合成
中间体二加DEA(二乙胺，CAS号：109-89-7)脱Fmoc后，得到中间体三；具体的，反应瓶中加入中间体二(100mg)、DMF(1.5mL)，控制温度在室温，滴加DEA(300μL)，室温进行反应，HPLC监控，至中间体二无剩余，浓缩除去DMF，加入DCM(4mL)，纯化水(4mL)，搅拌后分液，将水相浓缩至干得到中间体三92mg，该步反应收率为166％(包裹部分溶剂)，反应式如下：
4.中间体四的合成
中间体三与6-(马来酰亚胺基)己酸琥珀酰亚胺酯(CAS号：55750-63-5) 反应得到中间体四；具体的，反应瓶中加入中间体三(92mg，1.0eq)、6-(马来酰亚胺基)己酸琥珀酰亚胺酯(135mg，1.3eq)、DMF(1.5mL)、DIPEA(0.059mL，1.0eq)，HPLC监控，反应完成后，制备纯化，浓缩制备液，得到中间体四56mg，该步反应收率为35.7％，反应式如下：
5.产品的合成
中间体四与依喜替康甲磺酸盐(CAS号：169869-90-3)经缩合反应得到中间体四；具体的，室温条件下，反应瓶中加入中间体四(26mg，1.0eq)，加入DMF(1.5mL)，依次加入依喜替康甲磺酸盐(29.6mg，1.0eq)、EEDQ(20.7mg，1.5eq)，HATU(31.8mg，1.5eq)、DMAP(0.7mg，0.1eq)、DIPEA(29.2μL，3.0eq)，室温进行反应。HPLC监控，反应完成后，制备纯化，浓缩制备液，得到产品16.5mg(核磁如下)，该步反应收率为33.5％，反应式如下：
1H NMR(400MHz,DMSO-d6)δ8.22-8.29(d,J＝2.0Hz,1H),7.94-8.05(d,J＝2.0Hz,3H)，7.83-7.88(d,J＝2.0Hz,1H)，7.75-7.82(d,J＝2.0Hz,1H)，7.34-7.40(m,1H)，7.28-7.33(m,1H)，6.96-7.02(d,J＝8.0Hz,2H)，6.87-6.94(m,1H)，5.42-5.55(m,1H)，5.41-5.46(m,2H)，5.21-5.26(m,1H)，4.41-4.49(m,1H)，4.00-4.12(m,2H)，3.32-3.40(m,2H)，3.12-3.17(m,2H)，2.86-2.96(m,2H)，2.71-2.76(m,2H)，2.36-2.43(m,2H)，2.14-2.24(m,1H)，1.97-2.07(m,3H)，1.81-1.93(m,2H)，1.36-1.52(m,4H)，1.05-1.25(m,8H)，0.83-0.93(m,3H).
实施例十一、连接子药物偶联物mc-Ala-Ala-Gln-Exatecan的制备
中间体一的合成Fmoc-Gln-Exatecan
将Fmoc-Gln-OH(N-芴甲氧羰基-L-谷氨酰胺)(76.3mg 1.1eq.)、Exatecan(100mg 1.0eq.)加入反应瓶中，加入DMF(1ml)，DIEA(29mg 1.5eq.)，TBTU(72.5mg 1.1eq.)，室温反应。HPLC监控，无原料剩余。反应结束后， 加入硅胶浓缩拌样，过柱纯化。收集产品，浓缩干，得到产品140mg，纯度95％(有约4％TBTU的副产物Hobt未除尽)，收率94％。
反应式如下：
1.中间体二的合成Gln-Exatecan
将中间体一Fmoc-Gln-Exatecan(130mg 1.0eq.)加入反应瓶中，加入DCM(2ml)，室温搅拌，不能溶清。加入DEA(0.5ml)，室温反应。HPLC监控，无原料剩余。停止反应，缓慢加入MTBE(10ml)，析出大量固体，搅拌30min。过滤，得到灰白色固体，干燥，得到95mg，纯度96％，收率94％。
反应式如下：
2.中间体三的合成Fmoc-Ala-Ala-Gln-Exatecan
将中间体二Gln-Exatecan(100mg 1.0eq.)加入反应瓶中，DMF(1mL)，Fmoc-Ala-Ala-OH(67.8mg 1.0eq)，TBTU(68.4mg 1.2eq.)DIEA(34.4mg 1.5eq.),室温反应。HPLC监控，无中间体二剩余，停止反应。加入硅胶拌样，过柱纯化，收集产品得到产品140mg，纯度95％，收率85％。
反应式如下：
3.中间体四的合成Ala-Ala-Gln-Exatecan
将中间体三Fmoc-Ala-Ala-Gln-Exatecan(140mg 1.0eq.)加入反应瓶中，加入DCM(2ml)，DEA(0.5ml)，室温反应。HPLC监控，无原料剩余。停止反应，缓慢加入MTBE(10ml)，析出大量固体，搅拌30min。过滤，得到灰白色固体，干燥，得到70mg产品。纯度96％收率66％。
反应式如下：
4.产品合成mc-Ala-Ala-Gln-Exatecan
将中间体四Ala-Ala-Gln-Exatecan(50mg 1.0eq.)加入反应瓶中，DMF(1mL)，再加入6-(马来酰亚胺基)己酸琥珀酰亚胺酯(21.8mg 1.1eq)，DIEA(12.1mg 1.5eq.),室温反应。HPLC监控，至中间体四无剩余，停止反应。制备纯化，收集产品，浓缩干，得到产品35mg(核磁如下)。纯度98％收率55％。
反应式如下：
1H NMR(400MHz,DMSO-d6)δ8.28-8.36(d,J＝2.0Hz,1H),7.90-7.96(d,J＝2.0Hz,2H)，7.75-7.87(d,J＝2.0Hz,2H)，7.27-7.33(d,J＝2.0Hz,1H)，7.20-7.25(m,1H)，6.97-7.03(d,J＝8.0Hz,2H)，6.69-6.75(m,1H)，6.49-6.53(m,1H)， 5.46-5.55(m,1H)，5.40-5.45(m,2H)，5.24-5.34(m,1H)，5.04-5.14(m,1H)，4.31-4.38(m,1H)，4.02-4.23(m,2H)，3.86-3.96(m,1H)，3.40-3.49(m,1H)，3.33-3.40(m,2H)，3.12-3.20(m,1H)，2.36-2.42(m,2H)，2.15-2.25(m,1H)，2.00-2.12(m,4H)，1.71-1.93(m,3H)，1.36-1.52(m,4H)，1.01-1.20(m,10H)，0.83-0.92(m,3H).
实施例十二、连接子药物偶联mc-Pro-Exatecan的制备
1.中间体一的合成Fmoc-Pro-Exatecan
将Fmoc-Pro-OH(N-芴甲氧羰基-L-脯氨酸)(41mg 1.3eq.)、Exatecan(50mg 1.0eq.)加入反应瓶中，加入DMF(1ml)，DIEA(49mg 4eq.)溶解.加入HATU(46mg 1.3eq.)，HOBt(12.7mg 1.0eq.)室温反应，HPLC监控，无原料剩余。反应结束后，加入硅胶浓缩拌样，过柱纯化。收集产品，浓缩干，得到产品50mg，纯度95％收率73％。
反应式如下：
2.中间体二的合成Pro-Exatecan
将中间体一Fmoc-Pro-Exatecan(50mg 1.0eq.)加入反应瓶中，加入DCM(1ml)，DEA(0.2ml)，室温反应，HPLC监控，无原料剩余。停止反应，缓慢加入MTBE(10ml)，析出大量固体，搅拌30min。过滤，得到灰白色固体，干燥，得到30mg纯度96％收率86％。
反应式如下：
3.产品的合成mc-Pro-Exatecan
将中间体二Pro-Exatecan(30mg 1.0eq.)加入反应瓶中，DMF(1mL)，再加入6-(马来酰亚胺基)己酸琥珀酰亚胺酯(21.2mg 1.1eq)，DIEA(12.1mg 1.5eq.),室温反应，HPLC监控，至中间体二无剩余，停止反应。加入硅胶拌样，过柱纯化，收集产品得到产品25mg(核磁如下)。纯度96％收率56％。
反应式如下：
1H NMR(400MHz,DMSO-d6)δ8.17-8.26(d,J＝2.0Hz,1H),7.80-7.90(d,J＝2.0Hz,2H)，7.21-7.28(d,J＝8.0Hz,1H)，6.98-7.04(m,1H)，5.01-5.11(m,1H)，4.14-4.24(m,1H)，3.97-4.08(m,1H)，3.47-3.57(m,1H)，3.20-3.27(m,1H)，3.05-3.16(m,2H)，2.30-2.36(m,2H)，2.00-2.15(m,4H)，1.77-1.92(m,3H)，1.65-2.00(m,6H)，1.45-1.64(m,4H)，1.09-1.24(m,4H)，0.95-1.07(m,2H)，0.79-0.94(m,3H).
实施例十三、抗体偶联物HER2抗体-mc-AAN-Exatecan(DAR8)的制备
1.取10mg的HER2抗体，用PBS buffer稀释至浓度5mg/mL，用Tris-EDTA溶液调节抗体pH至7.5左右；用Nanodrop检测蛋白浓度，并称量抗体溶液净重，计算蛋白总量。向抗体中加入8倍摩尔数的TCEP溶液，置于3D摇床上，室温下，反应120min以上，连续混匀。
2.向还原后的抗体溶液加入过量的mc-AAN-Exatecan溶液(溶于DMSO)，混匀后置于3D摇床上，室温下，反应30min以上，连续混匀。反应结束后向反应液中加入过量的N-乙酰半胱氨酸溶液，置于3D摇床上，室温下，反应30min以上，连续混匀。
3.使用15mL的30KD超滤离心管将偶联产物进行纯化，并置换到储存液(10mM Histidine，pH5.5左右)中，置换倍数大于1000倍。然后用0.22um的除菌过滤器进行过滤，即得到抗体药物偶联物HER2抗体-mc-AAN-Exatecan于4℃进行保存。
4.用UV/BCA的方法检测抗体药物偶联物的蛋白浓度，用HIC进行DAR检测，SEC进行纯度检测。
实施例十四、不同抗体药物偶联物的体外活性测试
参照实施例十三的工艺，利用连接子药物偶联物mc-AAN-Exatecan分别与HER2抗体、EGFR抗体及非特异结合抗体(Anti-HEL)偶联制备相应ADC，利用连接子药物偶联物mc-Pro-Exa与HER2抗体偶联制备ADC，并检测细胞杀伤活性。
测试方法如下：
将靶细胞按一定数量铺板入96孔黑色底透的细胞培养板，经过24小时孵育后，加入浓度梯度稀释的样品，再经过72小时孵育后，使用显色试剂，显色60分钟，使用酶标仪进行读板。活细胞数越多，信号值越高，根据不同的浓度梯度点所得到的不同信号值，在酶标仪上形成四参数拟合曲线，得到C值即IC50值。
详细数据如下。
表2 mc-AAN-Exatecan ADC在不同细胞系中的活性数据

表3 Exatecan ADC与Payload活性数据对比
表4 mc-Pro-Exa ADC在乳腺癌细胞系中的活性数据
实施例十五、使用不同结构的连接子或细胞毒素制备ADC并检测分析
参照实施例十Linker-Payload和实施例十三ADC原液的制备过程，发明人制备了以下的ADC原液，并对所获得ADC原液的基本质量特性进行检测分析。
在使用相同的抗体HER2抗体进行偶联后，DAR值均接近于8；基于对这些ADC的检测数据分析，可获得如下结论：
1.相较于含有PAB片段，不含有PAB的Linker-Payload制备的ADC其聚合物含量(HMW％)显著低于含有PAB片段制备的ADC，说明PAB片段不利于该类Linker-Payload制备ADC，具体数据见表5；
2.含有伯胺(-NH2)结构Payload能够通过不包含PAB片段的Linker释放，而含有非伯胺基团(-NH-(i-Pr)结构)的Payload无法通过不包含PAB片段的Linker释放，具体数据见表6。
发明人研究了mc-Pro-PAB-Exatecan与抗体TF(即CN201610705557.4中的TF-mAb-H39)偶联、mc-Pro-Exatecan与HER2抗体偶联所获得的ADC中聚合物的含量，发现PAB结构影响非常明显，从Linker中去除PAB后，聚合物能够从70～80％左右降低至1％。
表5 PAB片段对HMW％残留量的影响分析数据
在研究mc-Pro-Belotecan和mc-Pro-Exatecan分别与HER2抗体偶联的ADC体外细胞杀伤活性时，发现偶联mc-Pro-Belotecan的ADC无肿瘤细胞杀伤活性，说明其结构稳定，不能通过酶解反应释放Payload；而偶联mc-Pro-Exatecan的ADC体现出较好的杀伤活性，表明该结构能够释放Payload；同时，mc-Pro-Exatecan与non-binding抗体(无靶向内吞作用)偶联产生的ADC没有特异性细胞杀伤活性，表明该结构在胞外稳定，需内吞至细胞内后，才能释放出Payload。除此以外，通过对比mc-Pro-Belotecan和mc-Pro-Exatecan的化学结构，可发现两者关键性的区别在于Belotecan以-NH-(i-Pr)结构与Proline连接，而Exatecan则是以-NH2结构与Proline连接；因此推定，如果 Linker中的氨基酸片段与含有伯胺(-NH2)结构Payload连接，可以在去除疏水性结构PAB的情况下，通过酶解反应释放Payload(对于大多数目前在研的ADC，PAB结构作为自释放片段，是有效释放Payload的必需结构)；这样，既能够降低Linker-Drug的疏水性，减少制备ADC时产生的聚合物含量，也可以表现出相应的细胞杀伤活性。
表6不同Linker-Payload分别与HER2抗体偶联分析数据
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (12)

  1. 一种连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，所述连接子药物偶联物具有式Ⅰ所示的结构，
    M-X-D
    式I
    其中，
    M为包含马来酰亚胺(m)片段或环辛炔片段的化学结构；
    X为1～5个氨基酸或其衍生物组成的连接片段，优选选自Ala-Ala-Ala-Ala-Asn、Ala-Ala-Ala-Asn、Ala-Ala-Asn、Ala-Asn、Asn、Ala-Ala-Ala-Ala-(3-cyano-alanine)、Ala-Ala-Ala-(3-cyano-alanine)、Ala-Ala-(3-cyano-alanine)、Ala-(3-cyano-alanine)、3-cyano-alanine、Ala-Ala-Ala-Pro、Ala-Ala-Pro、Ala-Pro、Pro、Pro-Asn、Asn-Pro、Lys、Lys-Asn、Lys-Pro、Ala-Ala-Ala-Ala-Gln、Ala-Ala-Ala-Gln、Ala-Ala-Gln、Ala-Gln、Ala-Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid)、Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid)、Ala-Ala-(2-amino-4-cyanobutanoic acid)、Ala-(2-amino-4-cyanobutanoic acid)、2-amino-4-cyanobutanoic acid、Gly-Gly-Gly-Gln、Gly-Gly-Gln、Gly-Gln、Gln及它们的氘代化物；
    D为细胞毒素。
  2. 权利要求1的连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中，所述包含马来酰亚胺(m)片段的化学结构选自4-(N-马来酰亚氨基甲基)-环己烷-1-甲酰基(MCC)、6-马来酰亚氨基己酰基(MC)、M-(PEG)n、MC-(PEG)n、MCC-(PEG)n，各结构如下所示：

    其中，n代表PEG的聚合度，优选为2-12之间的整数。
  3. 权利要求1的连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中，所述包含环辛炔片段的化学结构选自BCN、DIBO、DIBAC、BARAC等，各结构如下所示：
  4. 权利要求1-3任一项的连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中，所述1～5个氨基酸或其衍生物组成的连接片段选自Ala-Ala-Ala-Ala-Asn、Ala-Ala-Ala-Asn、Ala-Ala-Asn、Ala-Asn、Asn、Ala-Ala-Ala-Ala-(3-cyano-alanine)、Ala-Ala-Ala-(3-cyano-alanine)、Ala-Ala-(3-cyano-alanine)、Ala-(3-cyano-alanine)、3-cyano-alanine、Ala-Ala-Ala-Pro、Ala-Ala-Pro、Ala-Pro、Pro、Pro-Asn、Asn-Pro、Lys、Lys-Asn、Lys-Pro、Ala-Ala-Ala-Ala-Gln、Ala-Ala-Ala-Gln、Ala-Ala-Gln、Ala-Gln、Ala- Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid)、Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid)、Ala-Ala-(2-amino-4-cyanobutanoic acid)、Ala-(2-amino-4-cyanobutanoic acid)、2-amino-4-cyanobutanoic acid、Gly-Gly-Gly-Gln、Gly-Gly-Gln、Gly-Gln、Gln及它们的氘代化物。
  5. 权利要求1-4任一项的连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中，D为含有羟基(-OH)、伯胺(-NH2)或仲胺(-NHR)基团的细胞毒素；
    优选地，所述含有羟基(-OH)、伯胺(-NH2)或仲胺(-NHR)基团的细胞毒素选自喜树碱类拓扑异构酶I抑制剂、Daunorubicin类拓扑异构酶II抑制剂、金盏花素衍生物、分子胶类化合物、免疫抑制剂；
    更优选地，所述含有羟基(-OH)、伯胺(-NH2)或仲胺(-NHR)基团的细胞毒素选自依喜替康(Exatecan)、14-氨基喜树碱(14-AC)、9-氨基喜树碱(9-AC)、贝洛替康(belotecan)、MMAE及其衍生物、MMAF及其衍生物、来那度胺、雷帕霉素(Rapamycin)及其衍生物；
    最优选地，所述含有羟基(-OH)、伯胺(-NH2)或仲胺(-NHR)基团的细胞毒素选自依喜替康(Exatecan)、贝洛替康(belotecan)、雷帕霉素(Rapamycin)。
  6. 权利要求1-5任一项的连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中，所述连接子药物偶联物为MCC-AAQ-Exatecan，其具有如下所示的结构：
  7. 一种抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，所述抗体药物偶联物由权利要求1-6任一项所述的连接子药物偶联物与抗体偶联形成；
    优选地，所述抗体药物偶联物具有式II所示的结构，
    Ab-(L-D)p
    式II
    其中，
    Ab为抗体；
    L为接头，其结构为M’-X，其中，M’与Ab相连，X与D相连；
    M’为如权利要求1-6任一项定义的M通过马来酰亚胺基团连接至Ab所形成的化学结构；
    X为如权利要求1-6任一项定义的1～5个氨基酸或其衍生物组成的连接片段；
    D为如权利要求1-6任一项定义的细胞毒素；
    p为2-8之间的任意数值。
  8. 权利要求7的抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，所述抗体选自鼠源抗体、兔源抗体、噬菌体展示来源抗体、酵母展示来源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体；
    优选地，所述抗体为单克隆抗体，选自：HER2抗体、EGFR抗体。
  9. 药物组合物，其包含权利要求1-6任一项所述的连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，或权利要求6或7的抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物；
    优选地，所述药物组合物还包含用于治疗肿瘤的化疗药物、免疫治疗药物和免疫抑制剂中的至少一种；
    优选地，所述药物组合物还包含至少一种药用辅料。
  10. 权利要求1-6任一项所述的连接子药物偶联物、或其药学上可接受的 盐、溶剂合物或所述盐的溶剂合物，或权利要求7或8的抗体药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物在制备药物中的用途，所述药物用于预防和/或治疗肿瘤。
  11. 制备权利要求1-6任一项所述的连接子药物偶联物、或其药学上可接受的盐、溶剂合物或所述盐的溶剂合物的方法，所述方法包括以下步骤：
    1.制备由1～5个氨基酸或其衍生物组成的连接片段X；
    2.将X与包含马来酰亚胺(m)片段或环辛炔片段的化学结构M连接，得到M-X；
    3.将细胞毒素D与M-X连接，得到连接子药物偶联物M-X-D。
  12. 权利要求11的方法，所述连接子药物偶联物为MCC-AAQ-Exatecan，所述方法包括以下步骤：
    1.将Fmoc-Gln与Exatecan反应得到中间体一，其中中间体一结构式如下：
    2.将中间体一经DEA脱Fmoc保护基，到中间体二，其中中间体二结构式如下：
    3.将中间体二与Fmoc-Ala-Ala反应得到中间体三，其中中间体三结构式如下：
    4.将中间体三脱Fmoc保护基得到中间体四，其中中间体四结构式如下：
    5.将中间体四与MCC缩合得到产品，其中产品结构式如下：
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