WO2023000131A1 - 一种负载双毒素的抗体药物偶联物及其应用 - Google Patents

一种负载双毒素的抗体药物偶联物及其应用 Download PDF

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WO2023000131A1
WO2023000131A1 PCT/CN2021/107079 CN2021107079W WO2023000131A1 WO 2023000131 A1 WO2023000131 A1 WO 2023000131A1 CN 2021107079 W CN2021107079 W CN 2021107079W WO 2023000131 A1 WO2023000131 A1 WO 2023000131A1
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Prior art keywords
antibody
lysine
phenylalanine
valine
phe
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PCT/CN2021/107079
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English (en)
French (fr)
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黄长江
孙友祥
熊就凯
孔娜娜
闫新新
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烟台迈百瑞国际生物医药股份有限公司
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Application filed by 烟台迈百瑞国际生物医药股份有限公司 filed Critical 烟台迈百瑞国际生物医药股份有限公司
Priority to KR1020227003469A priority Critical patent/KR20230015301A/ko
Priority to CN202180002141.5A priority patent/CN113710277B/zh
Priority to US17/434,796 priority patent/US20230270874A1/en
Priority to JP2022503434A priority patent/JP7451677B2/ja
Priority to AU2021225125A priority patent/AU2021225125B9/en
Priority to PCT/CN2021/107079 priority patent/WO2023000131A1/zh
Priority to CA3129419A priority patent/CA3129419C/en
Priority to EP21838960.9A priority patent/EP4144376A4/en
Publication of WO2023000131A1 publication Critical patent/WO2023000131A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
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    • 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
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Definitions

  • the invention relates to the field of antibody-drug conjugates, in particular to an antibody-drug conjugate loaded with a double toxin through an antibody.
  • Antibody-drug conjugate refers to a type of biological drug that connects a biologically active drug (Drug) and antibody (Antibody) through a chemical linker (Linker).
  • ADC is like a precision-guided weapon system, in which biologically active drugs are used as lethal ammunition to precisely strike diseased cells under the guidance of antibodies. Therefore, ADCs combine the dual advantages of high potency of cytotoxic drug molecules and high targeting of antibodies.
  • ADCs require at least 10 4 antigens/cell to ensure delivery of lethal quantities of cytotoxic drugs.
  • the antigen targeted by the antibody portion of the ADC should be uniformly expressed on the surface of tumor cells with a high copy number (>10 5 /cell).
  • the reality is that there are usually only a limited number of antigens on the surface of tumor cells (about 5000 to 10 6 antigens/cell), and the ideal average DAR value of ADC is 3.5-4 (for example, the average DAR value of Brentuximab vedotin is 4, the average DAR value of Trastuzumab emtansine The average DAR value is 3.5), and the amount of drug delivered to tumor cells is very low, so the drug effect on tumor cells is low, which is also considered to be one of the main reasons for the clinical failure of ADC.
  • Drug generic name company target time to market Brentuximab vedotin Seattle, Takeda CD30 listed/2011 Ado-trastuzumab emtansine Genentech Her2 Launched/2013 Inotuzumab ozogamicin Pfizer CD22 listed/2017 Gemtuzumab ozogamicin Pfizer CD33 Listed/2017 Moxetumomab pasudotox-tdfk AstraZeneca CD22 listed/2018
  • the present invention provides an antibody-drug conjugate capable of loading double toxins.
  • an antibody drug conjugate comprising the following structure:
  • Ab is an antibody or antigen-binding fragment
  • S is the sulfur atom in the sulfhydryl residue formed after the interchain disulfide bond on the Ab is opened;
  • Aa is an amino acid unit comprising one or more amino acids
  • G is an optional cleavage unit
  • D 1 is MMAF
  • D2 is a second active drug unit other than MMAF
  • p is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8.
  • the antibody or antigen-binding fragment Ab is reactive with an antigen associated with cancer, an infectious disease, or an autoimmune disease, or an epitope of said antigen; optionally, the Ab is murine, chimeric, primatized, humanized , or complete, fragment (such as Fab, Fab', F(ab) 2 , F(ab') 2 , etc.) or subfragment (such as single chain construct, etc.) form of a human monoclonal antibody comprising bispecific Antibodies, or multispecific antibodies.
  • the Ab is a complete form of a mouse, chimeric, primatized, humanized, or human monoclonal antibody, and the Ab has an Fc domain and a hinge region domain of a human IgG1 or human IgG4 antibody or partial amino acid mutations, substitutions, or deletions.
  • the first active drug unit D 1 is selected from auristatin (auristatin) cytotoxic agents; preferably, the first active drug unit D 1 contains a free carboxyl group.
  • the first active drug unit D is selected from the following structures or tautomers, mesoforms, racemates, enantiomers, diastereoisomers or Its mixture form or its pharmaceutically acceptable salt or solvate:
  • the second active drug unit D2 is selected from cytotoxic molecules, immune enhancers and radioisotopes, said cytotoxic molecules include but not limited to tubulin inhibitors, DNA damage agents, topoisomerase inhibitors, ALK inhibitors , PARP inhibitors; further preferably, the tubulin inhibitors include but not limited to dolastatin (dolastatin) and auristatin (auristatin) class cytotoxic molecules, maytansine (maytansine) class cytotoxic molecules;
  • the DNA damage agents include but are not limited to calicheamicins, duocarmycins, antramycin derivatives PBD; the topoisomerase inhibitors include camptothecins and camptothecin derivatives; further preferably, the auristatin (auristatin) class cytokine molecules include but are not limited to MMAE or MMAF or their derivatives, and the maytansinoid cytotoxic molecules include but are not limited to DM1, DM4 or their derivatives.
  • the second active drug unit D2 is selected from ALK inhibitors, PARP inhibitors, topoisomerase inhibitors.
  • the second active drug unit D2 is selected from the following structures or tautomers, mesoforms, racemates, enantiomers, diastereoisomers or a mixture thereof or a pharmaceutically acceptable salt or solvate thereof:
  • the first active drug unit D1 and the second active drug unit D2 are MMAF and SN38, respectively.
  • the first active drug unit D1 and the second active drug unit D2 are MMAF and MK4827 respectively.
  • the first active drug unit D1 and the second active drug unit D2 are MMAF and Ceritinib respectively.
  • the first active drug unit D1 and the second active drug unit D2 are MMAF and Dxd, respectively.
  • the first active drug unit D1 and the second active drug unit D2 are MMAF and Exatecan, respectively.
  • the amino acid unit Aa may comprise one amino acid selected from: -glycine-, -alanine-, -valine-, -leucine-, -isoleucine-, -proline-, -phenylalanine Amino acid-, -tryptophan-, -methionine-, -tyrosine-, -serine-, -threonine-, -cysteine-, -asparagine-, -glutamine-, - Aspartic acid-, -glutamic acid-, -lysine-, -arginine-, -histidine-, -citrulline-, -lysine (trityl)-, -lysine Amino acid (monomethoxytrityl)-,-lysine (fluorenyloxycarbonyl)-; preferably, the AA is selected from-valine-,-citrulline-,-alanine -
  • the amino acid unit Aa may contain two amino acids, and the two may be the same or different, and they are all independently selected from -glycine-, -alanine-, -valine-, -leucine-, -isoleucine Amino acid-,-proline-,-phenylalanine-,-tryptophan-,-methionine-,-tyrosine-,-serine-,-threonine-,-cysteine-, -Asparagine-, -Glutamine-, -Aspartic Acid-, -Glutamic Acid-, -Lysine-, -Arginine-, -Histidine-, -Citrulline-, - Lysine (trityl)-,-lysine (monomethoxytrityl)-,-lysine (fluorenyloxycarbonyl)-; preferably, the amino acid is selected from-valine Acid-,-citrulline-,
  • the amino acid unit Aa may contain three amino acids, and the three may be the same or different, and they are all independently selected from -glycine-, -alanine-, -valine-, -leucine-, -isoleucine Amino acid-,-proline-,-phenylalanine-,-tryptophan-,-methionine-,-tyrosine-,-serine-,-threonine-,-cysteine-, -Asparagine-, -Glutamine-, -Aspartic Acid-, -Glutamic Acid-, -Lysine-, -Arginine-, -Histidine-, -Citrulline-, - Lysine (trityl)-,-lysine (monomethoxytrityl)-,-lysine (fluorenyloxycarbonyl)-; preferably, the amino acid is selected from-valine Acid-,-citrulline-,
  • the amino acid unit Aa can contain four amino acids, and the four can be the same or different, and they are all independently selected from -glycine-, -alanine-, -valine-, -leucine-, -isoleucine Amino acid-,-proline-,-phenylalanine-,-tryptophan-,-methionine-,-tyrosine-,-serine-,-threonine-,-cysteine-, -Asparagine-, -Glutamine-, -Aspartic Acid-, -Glutamic Acid-, -Lysine-, -Arginine-, -Histidine-, -Citrulline-, - Lysine (trityl)-,-lysine (monomethoxytrityl)-,-lysine (fluorenyloxycarbonyl)-; preferably, the amino acid is selected from-valine Acid-,-citrulline-,
  • the amino acid unit Aa is selected from -valine-citrulline-(-Val-Cit-), -valine-alanine-(-Val-Ala-), -Valine-Lysine-(-Val-Lys-), -Valine-Lysine(trityl)-(-Val-Lys(Trt)-), -Valine-Lysine Acid (monomethoxytrityl)-(-Val-Lys(Mmt)-), -valine-lysine(fluorenyloxycarbonyl)-(-Val-Lys(Fmoc)-), -valine Amino Acid-Arginine-(-Val-Arg-), -Phenylalanine-Citrulline-(-Phe-Cit-), -Phenylalanine-Lysine-(-Phe-Lys-) ,-Phenylalanine-Lysine (Trityl
  • the amino acid unit Aa is -phenylalanine-arginine-arginine-(-Ala-Arg-Arg-);
  • the amino acid unit Aa is selected from the group consisting of -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-), -glycine-phenylalanine-leu Amino Acid-Glycine-(-Gly-Phe-Leu-Gly-), -Alanine-Leucine-Alanine-Leucine (-Ala-Leu-Ala-Leu-).
  • the structure of the amino acid unit Aa can be selected from the following:
  • the self-cleavage unit G is empty or selected from the following structures: Wherein said R 1 and R 2 are any substituent groups, preferably, said R 1 and R 2 are independently selected from H or C 1 to C 10 alkyl groups or are:
  • the self-cleavage unit G When the self-cleavage unit G is empty, the last amino acid group in the amino acid unit Aa is covalently linked to the second active drug unit D2.
  • the self-cleavage unit G is selected from the following structures:
  • the antibody drug conjugate is selected from the following structures, wherein p is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8:
  • the present invention also relates to the application of the antibody-drug conjugate described in any one of the foregoing in the preparation of drugs for treating or preventing cancer, infectious diseases or autoimmune diseases.
  • the cancer refers to hematopoietic tumor, carcinoma, sarcoma, melanoma or glial tumor, such as but not limited to: breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, Bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophagus cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, Solid or hematologic tumors such as neuroblastoma, glioma multiforme, sarcoma, lymphoma, and leukemia.
  • the autoimmune disease refers to a disease caused by the body's immune response against its own tissues, such as including but not limited to: immune-mediated thrombocytopenia, dermatomyositis, Sjögren's syndrome, multiple sclerosis , Sidenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, rheumatoid arthritis, polyglandular syndrome, bullous pemphigoid, diabetes, Hen-Scher Purpura II, poststreptococcal nephritis, erythema nodosum, Takayasu arteritis, Addison's disease, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, Ankylosing spondylitis, Goodpasture syndrome, thromboangiit
  • infectious diseases mainly refer to pathogenic biological infections, including but not limited to the following: human immunodeficiency virus (HIV), Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, pneumophilic Legionella, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus spp, Haemophilus influenzae type B, Treponema pallidum, Lyme disease, West Nile virus, green Pseudomonas pyogenes, Mycobacterium leprae, Bacillus abortus, Rabies virus, Influenza virus, Cytomegalovirus, Herpes simplex virus type I, Herpes simplex virus type II, Human serum parvovirus, Respiratory syncytial virus, Varicella-band Herpes zo
  • the present invention also provides an intermediate compound, the structure of which is shown in the following formula:
  • Aa is an amino acid unit comprising one or more amino acids
  • G is an optional cleavage unit
  • D 1 is MMAF
  • D2 is the second active drug unit different from MMAF.
  • the first active drug unit D 1 is selected from auristatin (auristatin) cytotoxic agents; preferably, the first active drug unit D 1 contains a free carboxyl group.
  • the first active drug unit D is selected from the following structures or tautomers, mesoforms, racemates, enantiomers, diastereoisomers or Its mixture form or its pharmaceutically acceptable salt or solvate:
  • the second active drug unit D2 is selected from cytotoxic molecules, immune enhancers and radioisotopes, said cytotoxic molecules include but not limited to tubulin inhibitors, DNA damage agents, topoisomerase inhibitors, ALK inhibitors , PARP inhibitors; further preferably, the tubulin inhibitors include but not limited to dolastatin (dolastatin) and auristatin (auristatin) class cytotoxic molecules, maytansine (maytansine) class cytotoxic molecules;
  • the DNA damage agents include but are not limited to calicheamicins, duocarmycins, antramycin derivatives PBD; the topoisomerase inhibitors include camptothecins and camptothecin derivatives; further preferably, the auristatin (auristatin) class cytokine molecules include but are not limited to MMAE or MMAF or their derivatives, and the maytansinoid cytotoxic molecules include but are not limited to DM1, DM4 or their derivatives.
  • the second active drug unit D2 is selected from ALK inhibitors, PARP inhibitors, topoisomerase inhibitors.
  • the second active drug unit D2 is selected from the following structures or tautomers, mesoforms, racemates, enantiomers, diastereoisomers or a mixture thereof or a pharmaceutically acceptable salt or solvate thereof:
  • the first active drug unit D1 and the second active drug unit D2 are MMAF and SN38, respectively.
  • the first active drug unit D1 and the second active drug unit D2 are MMAF and MK4827 respectively.
  • the first active drug unit D1 and the second active drug unit D2 are MMAF and Ceritinib respectively.
  • the first active drug unit D1 and the second active drug unit D2 are MMAF and Dxd, respectively.
  • the first active drug unit D1 and the second active drug unit D2 are MMAF and Exatecan, respectively.
  • the amino acid unit Aa may comprise one amino acid selected from: -glycine-, -alanine-, -valine-, -leucine-, -isoleucine-, -proline-, -phenylalanine Amino acid-, -tryptophan-, -methionine-, -tyrosine-, -serine-, -threonine-, -cysteine-, -asparagine-, -glutamine-, - Aspartic acid-, -glutamic acid-, -lysine-, -arginine-, -histidine-, -citrulline-, -lysine (trityl)-, -lysine Amino acid (monomethoxytrityl)-,-lysine (fluorenyloxycarbonyl)-; preferably, the AA is selected from-valine-,-citrulline-,-alanine -
  • the amino acid unit Aa may contain two amino acids, and the two may be the same or different, and they are all independently selected from -glycine-, -alanine-, -valine-, -leucine-, -isoleucine Amino acid-,-proline-,-phenylalanine-,-tryptophan-,-methionine-,-tyrosine-,-serine-,-threonine-,-cysteine-, -Asparagine-, -Glutamine-, -Aspartic Acid-, -Glutamic Acid-, -Lysine-, -Arginine-, -Histidine-, -Citrulline-, - Lysine (trityl)-,-lysine (monomethoxytrityl)-,-lysine (fluorenyloxycarbonyl)-; preferably, the amino acid is selected from-valine Acid-,-citrulline-,
  • the amino acid unit Aa may contain three amino acids, and the three may be the same or different, and they are all independently selected from -glycine-, -alanine-, -valine-, -leucine-, -isoleucine Amino acid-,-proline-,-phenylalanine-,-tryptophan-,-methionine-,-tyrosine-,-serine-,-threonine-,-cysteine-, -Asparagine-, -Glutamine-, -Aspartic Acid-, -Glutamic Acid-, -Lysine-, -Arginine-, -Histidine-, -Citrulline-, - Lysine (trityl)-,-lysine (monomethoxytrityl)-,-lysine (fluorenyloxycarbonyl)-; preferably, the amino acid is selected from-valine Acid-,-citrulline-,
  • the amino acid unit Aa can contain four amino acids, and the four can be the same or different, and they are all independently selected from -glycine-, -alanine-, -valine-, -leucine-, -isoleucine Amino acid-,-proline-,-phenylalanine-,-tryptophan-,-methionine-,-tyrosine-,-serine-,-threonine-,-cysteine-, -Asparagine-, -Glutamine-, -Aspartic Acid-, -Glutamic Acid-, -Lysine-, -Arginine-, -Histidine-, -Citrulline-, - Lysine (trityl)-,-lysine (monomethoxytrityl)-,-lysine (fluorenyloxycarbonyl)-; preferably, the amino acid is selected from-valine Acid-,-citrulline-,
  • the amino acid unit Aa is selected from -valine-citrulline-(-Val-Cit-), -valine-alanine-(-Val-Ala-), -Valine-Lysine-(-Val-Lys-), -Valine-Lysine(trityl)-(-Val-Lys(Trt)-), -Valine-Lysine Acid (monomethoxytrityl)-(-Val-Lys(Mmt)-), -valine-lysine(fluorenyloxycarbonyl)-(-Val-Lys(Fmoc)-), -valine Amino Acid-Arginine-(-Val-Arg-), -Phenylalanine-Citrulline-(-Phe-Cit-), -Phenylalanine-Lysine-(-Phe-Lys-) ,-Phenylalanine-Lysine (Trityl
  • the amino acid unit Aa is -phenylalanine-arginine-arginine-(-Ala-Arg-Arg-);
  • the amino acid unit Aa is selected from the group consisting of -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-), -glycine-phenylalanine-leu Amino Acid-Glycine-(-Gly-Phe-Leu-Gly-), -Alanine-Leucine-Alanine-Leucine (-Ala-Leu-Ala-Leu-).
  • the structure of the amino acid unit Aa can be selected from the following:
  • the self-cleavage unit G is empty or selected from the following structures: Wherein said R 1 and R 2 are any substituent groups, preferably, said R 1 and R 2 are independently selected from H or C 1 to C 10 alkyl groups or are:
  • the self-cleavage unit G When the self-cleavage unit G is empty, the last amino acid group in the amino acid unit Aa is covalently linked to the second active drug unit D2.
  • the self-cleavage unit G is selected from the following structures:
  • the compound is selected from the following structures:
  • the present invention also provides the use of any one of the compounds described above in the preparation of medicines for treating or preventing cancer, infectious diseases or autoimmune diseases.
  • the present invention also provides a synthetic method for the above-mentioned intermediate compound, characterized in that the synthetic route of the method is:
  • the method comprises the steps of:
  • Step 1 Dissolving the compound Fmoc-Aa-G, the second active drug unit D 2 , and the organic base 1 in an appropriate amount of organic solvent 1, and allowing them to react in contact to obtain the compound Fmoc-Aa-GD 2 ;
  • Step 2 removing the protection of the Fmoc group from the compound Fmoc-Aa-GD 2 to obtain the compound Aa-GD 2 ;
  • Step 3 Dissolving compound Aa-GD 2 , compound Mc-D 1 , and condensing agent 1 in an appropriate amount of organic solvent 2, and allowing them to react in contact to obtain compound Mc-D 1 -Aa-GD 2 ;
  • the organic base 1 is selected from one or more of N, N diisopropylethylamine, triethylamine, pyridine; the organic solvent 1 and the organic solvent 2 are independently selected from DMF, DMA One or two of them; the condensing agent 1 is selected from one or more of TSTU, HATU, HBTU, HCTU, PyBop, CDMT, T3P.
  • the present invention also provides the synthesis method provided above, which is characterized in that the synthetic route for preparing a specific intermediate compound, and the synthetic route is selected from the following:
  • MMAF is connected in series with another drug unit through a linker at a cysteine binding site in the antibody, and the two can exert a significant synergistic effect, thereby effectively improving the effect of killing tumor cells. It provides a new solution for the development of high-efficiency and low-toxicity ADCs.
  • antibody drug conjugate used in the present invention refers to a compound in which an antibody or antigen-binding fragment, a linking unit, and an active drug unit are linked together through a chemical reaction, and its structure usually consists of three parts : an antibody or antibody-like ligand, a drug part (ie, an active drug unit), and a linker that couples the antibody or antibody-like ligand and the drug part.
  • antibody used in the present invention refers to a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell.
  • the function of the antibody is to present the drug to the target cell population bound to the antibody.
  • These antibodies include but are not limited Protein hormones, lectins, growth factors, antibodies, or other molecules that bind to cells.
  • the antibody binds to an antigen comprising, but not limited to, the group consisting of: carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp, HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM-6, alpha-fetoprotein (AFP) , VEGF, ED-B fibronectin, EGP-1, EGP-2, EGF receptor (ErbB1), Erb
  • the antibodies include but are not limited to the following: anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR Antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg/AFP antibody, anti-cladin 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c - MET antibody, anti-SLITRK6 antibody, anti-KIT/CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7/CS1 antibody, anti-NaPi2B/SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1/CD227 antibody, anti-AXL antibody , anti-CD166 antibody, anti-B7-H3 (
  • Antibodies of the present invention include murine antibodies, chimeric antibodies, primatized antibodies, humanized antibodies (ie humanized) and fully human antibodies (ie human), preferably humanized antibodies and fully human antibodies.
  • murine antibody in this disclosure refers to an antibody prepared using a mouse according to the knowledge and skill in the art. hybridization in which test subjects are injected with a specific antigen and then isolated expressing antibodies with desired sequence or functional properties
  • chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the mouse variable region gene It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally expresses the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.
  • humanized antibody is also called CDR-grafted antibody (CDR-grafted antibody), which refers to the antibody variable region framework grafted with the mouse CDR sequence, that is, a different type of human germline antibody framework Antibodies generated in the sequence. It can overcome the heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components.
  • CDR-grafted antibody CDR-grafted antibody
  • Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com/ac.uk/vbase), as well as in Kabat, E.A. et al.
  • the humanized antibody of the present invention also includes the humanized antibody after affinity maturation of CDR by phage display. Further descriptions of methods involving the use of mouse antibodies in humanization include, for example, the methods of Queen et al., Proc., Natl. Acad. Sci.
  • the development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the invention is a fully human monoclonal antibody.
  • the relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen.
  • binding fragments included in "antigen-binding fragments” include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, comprising (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VH and VL domains of a single arm of an antibody; (v ) a single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) optionally via A combination of two or more isolated CDRs joined by a synthetic linker.
  • CDRs complementarity determining regions
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by a synthetic linker using recombinant methods, thus making it possible to produce a single protein in which the VL and VH regions pair to form a monovalent molecule. chain (referred to as single-chain Fv (scFv); see, eg, Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Nat L. Acad. Sci. USA 85:5879-5883).
  • single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody.
  • Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgAl, IgA2, IgD, IgE or IgM antibodies.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating an IgG antibody molecule with the protease papain (cleaving the amino acid residue at position 224 of the H chain), wherein the H chain N About half of the end sides and the entire L chain are held together by disulfide bonds.
  • F(ab') 2 is obtained by digesting the lower part of the two disulfide bonds in the IgG hinge region with the enzyme pepsin, having a molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions connected at the hinge position Antibody fragments.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F(ab')2.
  • the Fab' fragment can be produced by inserting DNA encoding a Fab' fragment of an antibody into a prokaryote expression vector or a eukaryote expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.
  • single-chain construct includes, but is not limited to, "single-chain antibody”, “single-chain Fv” or “scFv”, and is meant to comprise an antibody heavy chain variable domain or region (i.e., VH) and an antibody light chain connected by a linker.
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeat variants (Holliger et al. (1993), proc. Natl.
  • monoclonal antibodies can be obtained by injecting a mouse with a composition comprising an antigen, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to generate hybridomas, cloning the hybrid tumors, positive clones producing antibodies against the antigen are selected, the clones producing antibodies against the antigen are cultured, and the antibodies are isolated from the hybridoma culture.
  • Isolation and purification from hybridoma cultures can be accomplished by a number of well-established techniques. Such separation techniques include protein A or protein G sepharose affinity chromatography, size exclusion chromatography, and ion exchange chromatography.
  • linking unit refers to a chemical structural fragment or bond that is linked to the antibody/antigen-binding fragment at one end and the drug at the other end, thus serving as a "bridge” linking the antibody/antigen-binding fragment to the drug molecule. It may include linkers, spacers and amino acid units, and may be synthesized by methods known in the art, such as described in US2005-0238649A1. As used herein, “linker units” can be divided into two categories: non-cleavable linkers and cleavable linkers.
  • Non-cleavable linkers are relatively stable linkers whose structure is difficult to degrade and break in vivo.
  • the drug release mechanism is as follows: after the conjugates bind to the antigen and are endocytosed by cells, the antibody is enzymatically hydrolyzed in lysosomes, releasing the drug, linker, An active molecule composed of antibody amino acid residues. The resulting change in the molecular structure of the drug does not reduce its cytotoxicity, but since the active molecule is charged (amino acid residues), it cannot penetrate neighboring cells.
  • a cleavable linker can break within the target cell and release the active drug (the small molecule drug itself).
  • Cleavable linkers can be divided into two main categories: chemically labile linkers and enzymatically labile linkers.
  • Chemically unstable linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties. Such properties include pH, glutathione concentration, etc.
  • Linkers that are sensitive to pH are also commonly referred to as acid-cleavable linkers. Such linkers are relatively stable in the neutral environment of blood (pH 7.3-7.5), but will be hydrolyzed in the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). Most of the first-generation antibody-drug conjugates use such linkers, such as hydrazones, carbonates, acetals, and ketals. Due to the limited plasma stability of acid-cleavable linkers, antibody drug conjugates based on such linkers usually have a short half-life (2-3 days). This short half-life limits the application of pH-sensitive linkers in the new generation of antibody-drug conjugates to some extent.
  • Glutathione-sensitive linkers also known as disulfide linkers. Drug release is based on the difference between the high concentration of intracellular glutathione (millimolar range) and the relatively low concentration of glutathione in the blood (micromolar range). This is especially true for tumor cells, where low oxygen levels lead to increased reductase activity and thus higher glutathione concentrations. Disulfide bonds are thermodynamically stable, so they have good stability in plasma.
  • Enzyme-labile linkers such as peptide linkers, allow better control of drug release.
  • Peptide linkers can be efficiently cleaved by endolysosomal proteases such as cathepsin B or plasmin (these enzymes are increased in some tumor tissues). This peptide linkage is thought to be very stable in the plasma circulation because proteases are generally inactive outside the cell due to unfavorable extracellular pH and serum protease inhibitors.
  • Enzyme-labile linkers are widely used as cleavable linkers for antibody-drug conjugates due to their high plasma stability and good intracellular cleavage selectivity and efficiency.
  • the suicide linker is generally embedded between the cleavable linker and the active drug, or is itself a part of the cleavable linker.
  • the mechanism of action of the suicide linker is: when the cleavable linker is broken under suitable conditions, the suicide linker can spontaneously undergo structural rearrangement, and then release the active drug linked to it.
  • Common suicide linkers such as p-aminobenzyl alcohol (PAB) and so on.
  • toxin drug
  • drug moiety drug unit
  • drug unit used in the present invention generally refer to the same structure, and can be used with any name in the present invention. They generally refer to any compound with desired biological activity and reactive functional groups for the preparation of the conjugates of the present invention. Desired biological activities include diagnosing, curing, mitigating, treating, preventing disease in humans or other animals. As new drugs are continuously discovered and developed, these new drugs should also be included in the drugs described in the present invention.
  • amino acid refers to an amino acid with an isoelectric point of less than 7. Acidic amino acid molecules often contain one or more acidic groups such as carboxyl groups, which can be effectively ionized into negative ions in the structure to increase hydrophilicity. Acidic amino acids may be natural or unnatural amino acids.
  • Natural amino acid refers to an amino acid that is biosynthesized. Natural amino acids are generally in the L-form, but there are a few exceptions, such as glycine, including natural and biosynthetic.
  • Unnatural amino acid refers to an amino acid obtained by synthetic means.
  • the compounds and methods claimed herein may utilize any of a variety of antibodies known in the art.
  • Antibodies used can be purchased from various known sources or prepared by techniques well known to those skilled in the art.
  • the compounds and methods claimed in the present invention can utilize a variety of linking units known in the art, and the linking units used can be purchased from various known sources or prepared using techniques known to those skilled in the art. Therefore, the selection of antibodies and linking units should not be considered as a limitation of the claimed compounds and methods of the present invention.
  • Mc-MMAF Take 20.0mg of Mc-MMAF and 9.8mg of TSTU, add 14.2 ⁇ L of N,N-diisopropylethylamine and 18.8mg of VC-PAB-Ceritinib, wait until the end of the reaction at room temperature, spin to dry the solvent, and purify by preparative liquid phase to obtain Mc - Pure MMAF-VC-PAB-Ceritinib 12.0 mg.
  • Mc-MMAF 20.0mg and TSTU 9.8mg add 14.2 ⁇ L N,N-diisopropylethylamine and VC-PAB-MK4827 18.8mg, room temperature to the end of the reaction for 16h, spin to dry the solvent, and purify by preparative liquid phase method to obtain Mc-MMAF-VC-PAB-MK4827 pure 7.0mg.
  • compound LD-3 i.e. Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38
  • compound LD-4 i.e. Mc-MMAF-VC-PAB- Exatecan
  • compound LD-5 ie Mc-MMAF-GGFG-Dxd
  • ADC antibody-drug conjugate
  • TCEP Tris-2-carboxyethyl-phosphine
  • DTPA Diethylene triamine pentacetate acid
  • the amount of reducing agent can be added within a certain concentration range according to the required coupling rate, and mixed with a certain concentration of monoclonal antibody (such as: 5 ⁇ 30mg/mL) according to a certain volume ratio (1:1), TCEP
  • the molar ratio of the final concentration of the antibody to the antibody is 0.5-6.0:1, and the reaction is stirred at 25°C for 1 hour.
  • Antibodies after TCEP reduction can be directly conjugated.
  • ADC1 ie Ab-Mc-MMAF-VC-PAB-Ceritinib
  • ADC2 ie Ab-Mc-MMAF-VC-PAB-MK4827
  • ADC3 ie Ab-Mc- MMAF-VC-PAB-DMEDA-PEG(N3)-SN38
  • ADC4 ie Ab-Mc-MMAF-VC-PAB-Exatecan
  • ADC5 ie Ab-Mc-MMAF-GGFG-Dxd.
  • p is any integer selected from 1, 2, 3, 4, 5, 6, 7, 8, wherein the Ab used is HER2 antibody.
  • This embodiment adopts cell proliferation inhibition method to Her2 monoclonal antibody-Mc-MMAF-VC-PAB-MK4827 and Her2 monoclonal antibody-Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38 to breast cancer cell SK-BR -3 cell viability was evaluated.
  • test product (see Table 2 for the sample group and the control group) was serially diluted with the basal medium, and transferred to a cell culture plate with cells at 100 ⁇ l/well; 37 ° C, 5% CO 2 Incubate for 70- 74h.
  • HER2 mAb-Mc-MMAF-VC-PAB-MK4827 Control 1-1 HER2 mAb-Mc-MMAF Control 1-2 HER2 mAb-Mc-VC-PAB-MK4827 Control 1-3 HER2 monoclonal antibody-Mc-MMAF and HER2 monoclonal antibody-Mc-VC-PAB-MK4827, mass concentration 1:1 mixed Control 1-4 HER2 naked antibody sample 2 HER2 mAb-Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38 Control 2-1 HER2 mAb-Mc-MMAF Control 2-2 HER2 mAb-Mc-VC-PAB-DMEDA-PEG(N3)-SN38 Control 2-3 HER2 monoclonal antibody-Mc-MMAF and HER2 monoclonal antibody-Mc-VC-PAB-DMEDA-PEG(N3)-SN38, mass concentration 1:1 mixed Control 2-4 HER2 naked antibody
  • the cell proliferation inhibition method was used to evaluate the cell activity of Her2 monoclonal antibody-Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38 on gastric cancer cell NCI-N87.
  • NCI-N87 cells were digested with trypsin and adjusted to a cell density of 50,000 cells/ml, 100 ⁇ L/well was added to a cell culture plate, and incubated in a 5% CO 2 incubator at 37° C. for 14-20 h.
  • the test product (see Table 4 for sample group and control group) was serially diluted with basal medium, and transferred to a cell culture plate with cells at 100 ⁇ L/well ; incubated at 37°C for 70- 74h.

Abstract

一种负载双毒素的抗体药物偶联物，通过抗体上半胱氨酸结合位点上将MMAF与另外一种药物单元串联，二者可以发挥显著的协同效应，从而有效的提升杀伤肿瘤细胞的效果，这为开发高效低毒的ADC提供了新方案。

Description

一种负载双毒素的抗体药物偶联物及其应用 技术领域
本发明涉及抗体药物偶联物领域，具体涉及一种通过一个抗体负载双毒素的抗体药物偶联物。
背景技术
抗体药物偶联物(antibody-drug conjugate，ADC)，是指通过化学连接子(Linker)将具有生物活性的药物(Drug)和抗体(Antibody)连接起来的一类生物药。ADC就像是精确制导武器体系，其中具有生物活性的药物作为杀伤性弹药，在抗体的指引下，精准打击病变细胞。因此，ADC结合了细胞毒药物分子的高效能和抗体高靶向性的双重优点。
截止到2021年3月，全球仅批准了11种抗体偶联药物(如表1所示)，他们均为一个靶点抗体偶联一种毒素分子的ADC分子。由于肿瘤细胞表面上抗原数量有限，有效的ADC活性所需的抗原表达水平根据不同抗原特性而变化。ADC需要至少10 4个抗原/细胞，以确保能够递送致死数量的细胞毒性药物。理想情况下，ADC的抗体部分所针对的抗原应在肿瘤细胞表面均匀表达且拷贝数较高(>10 5/细胞)。而现实情况为肿瘤细胞表面通常只有有限数量的抗原(大约5000到10 6个抗原/细胞)，并且ADC理想的平均DAR值为3.5-4(如Brentuximab vedotin的平均DAR值为4、Trastuzumab emtansine的平均DAR值为3.5)，输送到肿瘤细胞的药物量很低，因此对于肿瘤细胞的药效较低，这也被认为是ADC临床失败的主要原因之一。
表1已上市抗体药物偶联物
药品通用名 公司 靶点 上市时间
Brentuximab vedotin Seattle、武田 CD30 上市/2011年
Ado-trastuzumab emtansine 基因泰克 Her2 上市/2013年
Inotuzumab ozogamicin 辉瑞 CD22 上市/2017年
Gemtuzumab ozogamicin 辉瑞 CD33 上市/2017年
Moxetumomab pasudotox-tdfk 阿斯利康 CD22 上市/2018年
Enfortumab vedotin-ejfv 西雅图/安斯泰来 Nectin-4 上市/2019年
Polatuzumab vedotin-piiq 基因泰克 CD79b 上市/2019年
Fam-trastuzumab deruxtecan-nxki 阿斯利康/日本第一三共 Her2 上市/2019年
Sacituzumab govitecan-hziy Immunomedics Trop2 上市/2020年
Belantamab mafodotin Glaxosmithkline(Ireland)Ltd BCMA 上市/2020年
Cetuximab sarotalocan Rakuten Medical EGFR 上市/2020年
注：Mylotarg于2000年获批上市后在2010年撤市，后于2017年重新获批上市。
发明内容
为了解决上述问题，本发明提供了一种能够负载双毒素的抗体药物偶联物。具体而言，本发明提供了一种包含下式结构的抗体药物偶联物：
Figure PCTCN2021107079-appb-000001
其中：
Ab为抗体或抗原结合片段；
S为所述Ab上的链间二硫键打开后形成的巯基残基中的硫原子；
Aa为包含一个或多个氨基酸的氨基酸单元；
G为可选择的裂解单元；
D 1为MMAF；
D 2是不同于MMAF的第二活性药物单元；
p为选自1、2、3、4、5、6、7、8的整数。
抗体或抗原结合片段Ab与癌症、感染性疾病或自身免疫性疾病相关的抗原或所述抗原的表位具有反应性；可选的，Ab为鼠、嵌合、灵长类动物化、人化、或人单克隆抗体的完整、片段(如Fab、Fab’、F(ab) 2、F(ab’) 2等)或亚片段(如单链构建体等)形式，所述抗体包括双特异性抗体、或多特异性抗体。可选的，Ab为鼠、嵌合、灵长类动物化、人化、或人单克隆抗体的完整形式，且所述的Ab具有人IgG1或者人IgG4抗体的Fc结构 域和铰链区结构域或其部分氨基酸突变、替换、缺失形式。
第一活性药物单元D 1为选自奥瑞他汀(auristatin)类细胞毒性剂；优选的，所述的第一活性药物单元D 1含有游离的羧基。
在一些优选的实施例中，第一活性药物单元D 1选自以下结构或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式或其药学上可接受的盐或溶剂化物：
Figure PCTCN2021107079-appb-000002
第二活性药物单元D 2为选自细胞毒性分子、免疫增强剂和放射性同位素，所述细胞毒性分子包括但不限于微管蛋白抑制剂、DNA损伤剂、拓扑异构酶抑制剂、ALK抑制剂、PARP抑制剂；进一步优选的，所述微管蛋白抑制剂包括但不限于海兔毒素(dolastatin)及奥瑞他汀(auristatin)类细胞毒分子，美登素(maytansine)类细胞毒分子；所述DNA损伤剂包括但不限于卡奇霉素(calicheamicin)类、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD；所述拓扑异构酶抑制剂包括喜树碱(camptothecins)及喜树碱类衍生物；进一步优选的，所述奥瑞他汀(auristatin)类细胞素分子包括但不限于MMAE或MMAF或它们的洐生物，所述美登素类细胞毒分子包括但不限于DM1、DM4或它们的洐生物。
在一些优选的实施例中，第二活性药物单元D 2选自ALK抑制剂、PARP抑制剂、拓扑异构酶抑制剂。
在一些更优选的实施例中，第二活性药物单元D 2选自以下结构或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式或其药学上可接受的盐或溶剂化物：
Figure PCTCN2021107079-appb-000003
Figure PCTCN2021107079-appb-000004
在一些优选的实施例中，第一活性药物单元D 1和第二活性药物单元D 2分别是MMAF和SN38。
在一些优选的实施例中，所述的第一活性药物单元D 1和第二活性药物单元D 2分别是MMAF和MK4827。
在一些优选的实施例中，所述的第一活性药物单元D 1和第二活性药物单元D 2分别是MMAF和Ceritinib。
在一些优选的实施例中，所述的第一活性药物单元D 1和第二活性药物 单元D 2分别是MMAF和Dxd。
在一些优选的实施例中，所述的第一活性药物单元D 1和第二活性药物单元D 2分别是MMAF和Exatecan。
氨基酸单元Aa可以包含一个氨基酸，可选自：-甘氨酸-、-丙氨酸-、-缬氨酸-、-亮氨酸-、-异亮氨酸-、-脯氨酸-、-苯丙氨酸-、-色氨酸-、-蛋氨酸-、-酪氨酸-、-丝氨酸-、-苏氨酸-、-半胱氨酸-、-天冬酰胺-、-谷氨酰胺-、-天冬氨酸-、-谷氨酸-、-赖氨酸-、-精氨酸-、-组氨酸-、-瓜氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-；优选的，所述的AA选自-缬氨酸-、-瓜氨酸-、-丙氨酸-、-赖氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-、-精氨酸-、-苯丙氨酸-、-甘氨酸-、-亮氨酸-、-异亮氨酸-。
氨基酸单元Aa可以包含两个氨基酸，且二者可以相同，也可以不同，它们均独立的选自-甘氨酸-、-丙氨酸-、-缬氨酸-、-亮氨酸-、-异亮氨酸-、-脯氨酸-、-苯丙氨酸-、-色氨酸-、-蛋氨酸-、-酪氨酸-、-丝氨酸-、-苏氨酸-、-半胱氨酸-、-天冬酰胺-、-谷氨酰胺-、-天冬氨酸-、-谷氨酸-、-赖氨酸-、-精氨酸-、-组氨酸-、-瓜氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-；优选的，所述的氨基酸选自-缬氨酸-、-瓜氨酸-、-丙氨酸-、-赖氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-、-精氨酸-、-苯丙氨酸-、-甘氨酸-、-亮氨酸-、-异亮氨酸-。
氨基酸单元Aa可以包含三个氨基酸，且三者可以相同，也可以不同，它们均独立的选自-甘氨酸-、-丙氨酸-、-缬氨酸-、-亮氨酸-、-异亮氨酸-、-脯氨酸-、-苯丙氨酸-、-色氨酸-、-蛋氨酸-、-酪氨酸-、-丝氨酸-、-苏氨酸-、-半胱氨酸-、-天冬酰胺-、-谷氨酰胺-、-天冬氨酸-、-谷氨酸-、-赖氨酸-、-精氨酸-、-组氨酸-、-瓜氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-；优选的，所述的氨基酸选自-缬氨酸-、-瓜氨酸-、-丙氨酸-、-赖氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-、-精氨酸-、-苯丙氨酸-、-甘氨酸-、-亮氨酸-、-异亮氨酸-。
氨基酸单元Aa可以包含四个氨基酸，且四者可以相同，也可以不同，它们均独立的选自-甘氨酸-、-丙氨酸-、-缬氨酸-、-亮氨酸-、-异亮氨酸-、-脯氨酸-、-苯丙氨酸-、-色氨酸-、-蛋氨酸-、-酪氨酸-、-丝氨酸-、-苏氨酸-、-半胱氨酸-、-天冬酰胺-、-谷氨酰胺-、-天冬氨酸-、-谷氨酸-、-赖氨酸-、-精氨酸-、-组氨酸-、-瓜氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-；优选的，所述的氨基酸选自-缬氨酸-、-瓜氨酸-、-丙氨酸-、-赖氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-、-精氨酸-、-苯丙氨酸-、-甘氨酸-、-亮氨酸-、-异亮氨酸-；n为选自0、1的整数，当n为0时，AA 4和D 2直接共价连接。
在一些优选的实施例中，所述的氨基酸单元Aa选自-缬氨酸-瓜氨酸-(-Val-Cit-)、-缬氨酸-丙氨酸-(-Val-Ala-)、-缬氨酸-赖氨酸-(-Val-Lys-)、-缬氨酸-赖氨酸(三苯甲基)-(-Val-Lys(Trt)-)、-缬氨酸-赖氨酸(单甲氧基三苯甲基)-(-Val-Lys(Mmt)-)、-缬氨酸-赖氨酸(芴氧羰基)-(-Val-Lys(Fmoc)-)、-缬氨酸-精氨酸-(-Val-Arg-)、-苯丙氨酸-瓜氨酸-(-Phe-Cit-)、-苯丙氨酸-赖氨酸-(-Phe-Lys-)、-苯丙氨酸-赖氨酸(三苯甲基)-(-Phe-Lys(Trt)-)、-苯丙氨酸-赖氨酸(单甲氧基三苯甲基)-(-Phe-Lys(Mmt)-)、-苯丙氨酸-赖氨酸(芴氧羰基)-(-Phe-Lys(Fmoc)-)、-亮氨酸-瓜氨酸-(-Leu-Cit-)、-异亮氨酸-瓜氨酸-(-Ile-Cit-)、-苯丙氨酸-精氨酸-(-Phe-Arg-)；
在另一些优选的实施例中，所述的氨基酸单元Aa为-苯丙氨酸-精氨酸-精氨酸-(-Ala-Arg-Arg-)；
在另一些优选的实施例中，所述的氨基酸单元Aa选自-甘氨酸-甘氨酸-苯丙氨酸-甘氨酸-(-Gly-Gly-Phe-Gly-)、-甘氨酸-苯丙氨酸-亮氨酸-甘氨酸-(-Gly-Phe-Leu-Gly-)，-丙氨酸-亮氨酸-丙氨酸-亮氨酸(-Ala-Leu-Ala-Leu-)。
可选的，所述的氨基酸单元Aa的结构可选自如下：
Figure PCTCN2021107079-appb-000005
Figure PCTCN2021107079-appb-000006
Figure PCTCN2021107079-appb-000007
在一些具体的实施例中，所述的自裂解单元G为空或选自如下结构：
Figure PCTCN2021107079-appb-000008
Figure PCTCN2021107079-appb-000009
其中所述的R 1、R 2为任一取代基团，优选的，所述的R 1和R 2独立地选自H或者C 1至C 10的烷基或为：
Figure PCTCN2021107079-appb-000010
当自裂解单元G为空时，氨基酸单元Aa中最后一个氨基酸基团和第 二活性药物单元D 2共价连接。
可选的，所述的自裂解单元G选自如下结构：
Figure PCTCN2021107079-appb-000011
在一些具体的实施例中，所述的抗体药物偶联物选自如下结构，其中p为选自1、2、3、4、5、6、7、8的整数：
Figure PCTCN2021107079-appb-000012
Figure PCTCN2021107079-appb-000013
本发明还涉及前述任一项所述的抗体药物偶联物在制备用于治疗或预防癌症、感染性疾病或自身免疫性疾病的药物中的应用。
所述的癌症指造血肿瘤、癌、肉瘤、黑素瘤或神经胶质肿瘤，例如非限制性地选自：乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、肺癌、结肠癌、直肠癌、结直肠癌、骨癌、皮肤癌、甲状腺癌、胰腺癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、多形性胶质细胞瘤、肉瘤、淋巴瘤和白血病等实体瘤或血液肿瘤。
所述的自身免疫性疾病是指身体产生针对其自身组织的免疫应答导致的病症，诸如包括但不限于：免疫介导的血小板减少症、皮肌炎、舍格伦氏综合症、多发性硬化、西登哈姆氏舞蹈病、重症肌无力、系统性红斑狼疮、狼疮性肾炎、风湿热、类风湿性关节炎、多腺体综合征、大疱性类天疱疮、糖尿病、亨-舍二氏紫癜、链球菌感染后肾炎、结节性红斑、高安氏动脉炎、阿狄森氏病、结节病、溃疡性结肠炎、多形性红斑、IgA肾病、结节性多动脉炎、强直性脊柱炎、古德帕斯丘综合征、闭塞性血栓性脉管炎、原发性胆汁性肝硬变、桥本甲状腺炎、甲状腺毒症、硬皮病、慢性活动性肝炎、多肌炎/皮肌炎、多软骨炎、寻常天疱疮、韦格纳氏肉芽肿病、膜性肾病、肌萎缩侧索硬化、脊髓痨、巨细胞动脉炎/多肌痛、恶性贫血、急进性肾小球肾炎、纤维化肺泡炎和青少年糖尿病及新发生的疾病。
所述的感染性疾病主要指的是病原性生物感染，包括但不限于如下：人免疫缺陷病毒(HIV)、结核分支杆菌、无乳链球菌、耐甲氧西林金黄色葡萄球菌、嗜肺性军团病菌、酿脓链球菌、大肠杆菌、淋病奈瑟氏菌、脑膜炎奈瑟氏菌、肺炎球菌属、B型流感嗜血杆菌、苍白密螺旋体、莱姆病螺旋体、西尼罗病毒、绿脓假单胞菌、麻风分枝杆菌、流产杆菌、狂犬病病毒、流感病毒、巨细胞病毒、I型单纯疱疹病毒、II型单纯疱疹病毒、 人血清细小样病毒、呼吸道合胞病毒、水痘-带状疱疹病毒、乙型肝炎病毒、麻疹病毒、腺病毒、人T细胞白血病病毒、埃-巴二氏病毒、鼠白血病病毒、腮腺炎病毒、水泡性口膜炎病毒、辛德比斯病毒、淋巴细胞性脉络丛脑膜炎病毒、疣病毒、蓝舌病病毒、仙台病毒、猫白血病病毒、呼肠孤病毒、脊髓灰质炎病毒、猿猴病毒40、鼠乳房肿瘤病毒、登革热病毒、风疹病毒、恶性疟原虫、间日疟原虫、鼠弓形体、让氏锥虫、克氏锥虫、罗德西亚锥虫、布氏锥虫、曼森血吸虫、日本血吸虫、牛巴贝虫、柔嫩艾美球虫、盘尾丝虫、热带利什曼原虫、旋毛线虫、小泰累尔梨浆虫、水泡绦虫、羊绦虫、牛肉绦虫、细粒棘球绦虫、科特氏中殖孔绦虫、关节炎支原体、猪鼻支原体、口腔支原体、精氨酸支原体、莱氏无胆甾原体、唾液支原体和肺炎支原体及新发生的疾病。
本发明还提供了一种中间体化合物，其结构如下式所示：
Figure PCTCN2021107079-appb-000014
其中：
Aa为包含一个或多个氨基酸的氨基酸单元；
G为可选择的裂解单元；
D 1为MMAF；
D 2是不同于MMAF的第二活性药物单元。
第一活性药物单元D 1为选自奥瑞他汀(auristatin)类细胞毒性剂；优选的，所述的第一活性药物单元D 1含有游离的羧基。
在一些优选的实施例中，第一活性药物单元D 1选自以下结构或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式或其药学上可接受的盐或溶剂化物：
Figure PCTCN2021107079-appb-000015
第二活性药物单元D 2为选自细胞毒性分子、免疫增强剂和放射性同位 素，所述细胞毒性分子包括但不限于微管蛋白抑制剂、DNA损伤剂、拓扑异构酶抑制剂、ALK抑制剂、PARP抑制剂；进一步优选的，所述微管蛋白抑制剂包括但不限于海兔毒素(dolastatin)及奥瑞他汀(auristatin)类细胞毒分子，美登素(maytansine)类细胞毒分子；所述DNA损伤剂包括但不限于卡奇霉素(calicheamicin)类、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD；所述拓扑异构酶抑制剂包括喜树碱(camptothecins)及喜树碱类衍生物；进一步优选的，所述奥瑞他汀(auristatin)类细胞素分子包括但不限于MMAE或MMAF或它们的洐生物，所述美登素类细胞毒分子包括但不限于DM1、DM4或它们的洐生物。
在一些优选的实施例中，第二活性药物单元D 2选自ALK抑制剂、PARP抑制剂、拓扑异构酶抑制剂。
在一些更优选的实施例中，第二活性药物单元D 2选自以下结构或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式或其药学上可接受的盐或溶剂化物：
Figure PCTCN2021107079-appb-000016
Figure PCTCN2021107079-appb-000017
在一些优选的实施例中，第一活性药物单元D 1和第二活性药物单元D 2分别是MMAF和SN38。
在一些优选的实施例中，所述的第一活性药物单元D 1和第二活性药物单元D 2分别是MMAF和MK4827。
在一些优选的实施例中，所述的第一活性药物单元D 1和第二活性药物单元D 2分别是MMAF和Ceritinib。
在一些优选的实施例中，所述的第一活性药物单元D 1和第二活性药物单元D 2分别是MMAF和Dxd。
在一些优选的实施例中，所述的第一活性药物单元D 1和第二活性药物单元D 2分别是MMAF和Exatecan。
氨基酸单元Aa可以包含一个氨基酸，可选自：-甘氨酸-、-丙氨酸-、-缬氨酸-、-亮氨酸-、-异亮氨酸-、-脯氨酸-、-苯丙氨酸-、-色氨酸-、-蛋氨酸-、-酪氨酸-、-丝氨酸-、-苏氨酸-、-半胱氨酸-、-天冬酰胺-、-谷氨酰胺 -、-天冬氨酸-、-谷氨酸-、-赖氨酸-、-精氨酸-、-组氨酸-、-瓜氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-；优选的，所述的AA选自-缬氨酸-、-瓜氨酸-、-丙氨酸-、-赖氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-、-精氨酸-、-苯丙氨酸-、-甘氨酸-、-亮氨酸-、-异亮氨酸-。
氨基酸单元Aa可以包含两个氨基酸，且二者可以相同，也可以不同，它们均独立的选自-甘氨酸-、-丙氨酸-、-缬氨酸-、-亮氨酸-、-异亮氨酸-、-脯氨酸-、-苯丙氨酸-、-色氨酸-、-蛋氨酸-、-酪氨酸-、-丝氨酸-、-苏氨酸-、-半胱氨酸-、-天冬酰胺-、-谷氨酰胺-、-天冬氨酸-、-谷氨酸-、-赖氨酸-、-精氨酸-、-组氨酸-、-瓜氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-；优选的，所述的氨基酸选自-缬氨酸-、-瓜氨酸-、-丙氨酸-、-赖氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-、-精氨酸-、-苯丙氨酸-、-甘氨酸-、-亮氨酸-、-异亮氨酸-。
氨基酸单元Aa可以包含三个氨基酸，且三者可以相同，也可以不同，它们均独立的选自-甘氨酸-、-丙氨酸-、-缬氨酸-、-亮氨酸-、-异亮氨酸-、-脯氨酸-、-苯丙氨酸-、-色氨酸-、-蛋氨酸-、-酪氨酸-、-丝氨酸-、-苏氨酸-、-半胱氨酸-、-天冬酰胺-、-谷氨酰胺-、-天冬氨酸-、-谷氨酸-、-赖氨酸-、-精氨酸-、-组氨酸-、-瓜氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-；优选的，所述的氨基酸选自-缬氨酸-、-瓜氨酸-、-丙氨酸-、-赖氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-、-精氨酸-、-苯丙氨酸-、-甘氨酸-、-亮氨酸-、-异亮氨酸-。
氨基酸单元Aa可以包含四个氨基酸，且四者可以相同，也可以不同，它们均独立的选自-甘氨酸-、-丙氨酸-、-缬氨酸-、-亮氨酸-、-异亮氨酸-、-脯氨酸-、-苯丙氨酸-、-色氨酸-、-蛋氨酸-、-酪氨酸-、-丝氨酸-、-苏氨酸-、-半胱氨酸-、-天冬酰胺-、-谷氨酰胺-、-天冬氨酸-、-谷氨酸-、-赖氨酸-、-精氨酸-、-组氨酸-、-瓜氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-；优选的，所述的氨基酸选自-缬氨酸-、-瓜氨酸-、-丙氨酸-、-赖氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-、-精氨酸-、-苯丙氨酸-、-甘氨酸-、 -亮氨酸-、-异亮氨酸-；n为选自0、1的整数，当n为0时，AA 4和D 2直接共价连接。
在一些优选的实施例中，所述的氨基酸单元Aa选自-缬氨酸-瓜氨酸-(-Val-Cit-)、-缬氨酸-丙氨酸-(-Val-Ala-)、-缬氨酸-赖氨酸-(-Val-Lys-)、-缬氨酸-赖氨酸(三苯甲基)-(-Val-Lys(Trt)-)、-缬氨酸-赖氨酸(单甲氧基三苯甲基)-(-Val-Lys(Mmt)-)、-缬氨酸-赖氨酸(芴氧羰基)-(-Val-Lys(Fmoc)-)、-缬氨酸-精氨酸-(-Val-Arg-)、-苯丙氨酸-瓜氨酸-(-Phe-Cit-)、-苯丙氨酸-赖氨酸-(-Phe-Lys-)、-苯丙氨酸-赖氨酸(三苯甲基)-(-Phe-Lys(Trt)-)、-苯丙氨酸-赖氨酸(单甲氧基三苯甲基)-(-Phe-Lys(Mmt)-)、-苯丙氨酸-赖氨酸(芴氧羰基)-(-Phe-Lys(Fmoc)-)、-亮氨酸-瓜氨酸-(-Leu-Cit-)、-异亮氨酸-瓜氨酸-(-Ile-Cit-)、-苯丙氨酸-精氨酸-(-Phe-Arg-)；
在另一些优选的实施例中，所述的氨基酸单元Aa为-苯丙氨酸-精氨酸-精氨酸-(-Ala-Arg-Arg-)；
在另一些优选的实施例中，所述的氨基酸单元Aa选自-甘氨酸-甘氨酸-苯丙氨酸-甘氨酸-(-Gly-Gly-Phe-Gly-)、-甘氨酸-苯丙氨酸-亮氨酸-甘氨酸-(-Gly-Phe-Leu-Gly-)，-丙氨酸-亮氨酸-丙氨酸-亮氨酸(-Ala-Leu-Ala-Leu-)。
可选的，所述的氨基酸单元Aa的结构可选自如下：
Figure PCTCN2021107079-appb-000018
Figure PCTCN2021107079-appb-000019
Figure PCTCN2021107079-appb-000020
在一些具体的实施例中，所述的自裂解单元G为空或选自如下结构：
Figure PCTCN2021107079-appb-000021
Figure PCTCN2021107079-appb-000022
其中所述的R 1、R 2为任一取代基团，优选的，所述的R 1和R 2独立地选自H或者C 1至C 10的烷基或为：
Figure PCTCN2021107079-appb-000023
当自裂解单元G为空时，氨基酸单元Aa中最后一个氨基酸基团和第二活性药物单元D 2共价连接。
可选的，所述的自裂解单元G选自如下结构：
Figure PCTCN2021107079-appb-000024
在一些具体的实施例中，所述的化合物选自如下结构：
Figure PCTCN2021107079-appb-000025
本发明还提供了上述任一项所述的化合物在制备用于治疗或预防癌症、感染性疾病或自身免疫性疾病的药物中的应用。
本发明还提供了上述所述的中间体化合物的合成方法，其特征在于，所述方法的合成路线为：
Figure PCTCN2021107079-appb-000026
所述方法包括如下步骤：
步骤1：将化合物Fmoc-Aa-G、第二活性药物单元D 2、有机碱1溶于适量有机溶剂1中，使其接触反应，得化合物Fmoc-Aa-G-D 2；
步骤2：将化合物Fmoc-Aa-G-D 2脱除Fmoc基团保护，得化合物Aa-G-D 2；
步骤3：将化合物Aa-G-D 2、化合物Mc-D 1、缩合剂1溶于适量有机溶剂2中，使其接触反应，得化合物Mc-D 1-Aa-G-D 2；
其中，所述的有机碱1选自N，N二异丙基乙胺、三乙胺、吡啶的一种或几种；所述的有机溶剂1和有机溶剂2分别独立的选自DMF、DMA的一种或两种；所述的缩合剂1选自TSTU、HATU、HBTU、HCTU、PyBop、CDMT、T3P中的一种或几种。
在一些具体的实施例中，本发明还提供了利用上述提供的合成方法，其特征在于制备特定中间体化合物的合成路线，所述的合成路线选自如下：
Figure PCTCN2021107079-appb-000027
Figure PCTCN2021107079-appb-000028
Figure PCTCN2021107079-appb-000029
本发明惊奇地发现在抗体中一个半胱氨酸结合位点上通过连接子将MMAF与另外一种药物单元串联，二者可以发挥显著的协同效应，从而有效的提升杀伤肿瘤细胞的效果，这为开发高效低毒的ADC提供了新方案。
具体实施方式
Figure PCTCN2021107079-appb-000030
除非另有限定，本文所用的所有技术和科学术语均与本发明所属领域普通技术人员的通常理解一致。虽然也可采用与本文所述相似或等同的任何方法和材料实施或测试本发明，但本文描述了优选的方法和材料。描述和要求保护本发明时，依据以下定义使用下列术语。
当本发明中使用商品名时，申请人旨在包括该商品名产品的制剂、该商品名产品的非专利药和活性药物部分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有相同的下述含义。
本发明所使用的术语“抗体药物偶联物(即Antibody drug conjugate，ADC)”表示抗体或抗原结合片段、连接单元、活性药物单元经过化学反应连接在一起的化合物，其结构通常由三部分组成：抗体或抗体类配体、药物部分(即活性药物单元)、以及将抗体或抗体类配体及药物部分偶联起来的连接单元(linker)。
本发明所使用的术语“抗体”是指能识别和结合目标细胞相关的抗原或受体的大分子化合物，抗体的作用是将药物呈递给与抗体结合的目标细胞群，这些抗体包括但不限于蛋白类激素、凝集素、生长因子、抗体或其他能与细胞结合的分子。在一些特定的实施例中，所述的抗体结合于包括但不限于以下组成的组的抗原：碳酸酐酶IX、B7、CCCL19、CCCL21、CSAp、HER-2/neu、BrE3、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD44、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD67、CD70、CD74、CD79a、CD80、CD83、CD95、CD126、CD133、CD138、CD147、CD154、CEACAM5、CEACAM-6、甲胎蛋白(AFP)、VEGF、ED-B纤连蛋白、EGP-1、EGP-2、EGF受体(ErbB1)、ErbB2、ErbB3、因子H、FHL-1、Flt-3、叶酸受体、Ga 733、GROB、HMGB-1、缺氧诱导因子(HIF)、HM1.24、HER-2/neu、胰岛素样生长因子(ILGF)、IFN-γ、IFN-α、IFN-β、IL-2R、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-25、IP-10、IGF-1R、Ia、HM1.24、神经节糖苷、HCG、HLA-DR、CD66a-d、MAGE、McRP、McP-1、MIP-1A、MIP-1B、巨噬细胞移动抑制因子(MIF)、MUC1、MUC2、MUC3、MUC4、MUC5、胎盘生长因子(PlGF)、PSA(前列腺特异性抗原)、PSMA、PSMA二聚物、PAM4抗原、NCA-95、NCA-90、A3、A33、Ep-CAM、KS-1、Le(y)、间皮素、S100、腱生蛋白、TAC、Tn抗原、Thomas-Friedenreich抗原、肿瘤坏死抗原、肿瘤血管生成抗原、TNF-α、TRAIL受体(R1和R2)、VEGFR、RANTES、T101、癌干细胞抗原、补体因子C3、C3a、C3b、C5a、C5、和致癌基因产物。在一些具体的实施例中，所述的抗体包括但不限于如下：抗EGFRvIII抗体、抗DLL-3抗体、抗PSMA抗体、抗CD70抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM-1抗体、抗LRRC15抗体、抗LIV-1抗体、抗CanAg/AFP抗体、抗cladin 18.2抗体、抗Mesothelin抗体、抗HER2(ErbB2)抗体、抗EGFR抗体、抗c-MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3(ErbB3)抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7-H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4 抗体、抗TROP-2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P-cadherin抗体、抗GD3抗体、抗Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b抗体、抗CD19抗体、抗CD33抗体、抗CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD138抗体、抗CD352抗体、抗CD25抗体或抗CD123抗体及新发现的靶点抗体。
本发明的抗体包括鼠源抗体、嵌合抗体、灵长类动物化、人源化抗体(即人化)和全人源抗体(即人)，优选人源化抗体和全人源抗体。
术语“鼠源抗体”在本公开中为根据本领域知识和技能用鼠制备抗体。制备时用特定抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。
术语“人源化抗体(humanized antibody)”也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com/ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本发明的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。进一步描述参与人源化可使用小鼠抗体的方法的文献包括，例如Queen等，Proc.,Natl.Acad.Sci.USA,88,2869,1991和Winter及其同事的方 法[Jones.,Nature,321,522,(1986)],Riechmann,等[Nature,332,323-327,1988)，Verhoeyen,等，Science,239,1534(1988)]。
术语“全人源抗体”、“全人抗体”或“完全人源抗体”、“人抗体”，也称“全人源单克隆抗体”、，其抗体的可变区和恒定区都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。本发明为全人源单克隆抗体。全人抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。
本发明所使用的术语“抗原结合片段”是指抗体的保持特异性结合抗原的能力的一个或多个片段。“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab')2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341:544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426和Huston等人(1988)Proc.NatL.Acad.Sci.USA85：5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员己知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1,IgA2，IgD，IgE或lgM抗体。
术语Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50，000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
术语F(ab') 2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下 方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
术语Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链构建体”包括但不限于“单链抗体”、“单链Fv”或“scFv”，意指包含通过接头连接的抗体重链可变结构域或区域(即VH)和抗体轻链可变结构域或区域(即VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8：725-731,Choi等人(2001)，Eur.J.Immuno 1.31:94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001),Cancer Immunol.描述。
用于制备针对实际上任何靶抗原的抗体或其抗原结合片段的技术是本领域公知的。例如，参见Kohler和Milstein，Nature 256：495(1975)，以及Coligan等(编)，CURRENTPROTOCOLS IN IMMUNOLOGY(免疫学最新实验方案)，第1卷，第2.5.1-2.6.7页(John Wiley&Sons，1991)。简言之，单克隆抗体可如下获得：即用包括抗原的组合物注射小鼠，取出脾脏以获得B淋巴细胞，使所述B淋巴细胞与骨髓瘤细胞融合以产生杂交瘤，克隆所述杂交瘤，选择产生针对所述抗原的抗体的阳性克隆，培养产生针对所述抗原的抗体的所述克隆，并从所述杂交瘤培养物中分离所述抗体。可通过许多充分建立的技术从杂交瘤培养物中分离和纯化。此类分离技术包括蛋白A或蛋白G琼脂糖亲和层析、大小排阻层析以及离子交换层析。例如，参见Coligan第2.7.1-2.7.12页及第2.9.1-2.9.3页。也参见Baines等，“Purification of Immunoglobulin G(IgG)(免疫球蛋白G(IgG)的纯化)”，于METHODS IN MOLECULAR BIOLOGY(分子生物学方法)，第10卷，第79-104页(The Humana Press，Inc.1992)。在初次引起针对免疫原的抗体后，可对抗体进行测序并随后通过重组技术制备。鼠源抗体和抗体片段的人化和嵌合是本领域技术人员众所周知的。
术语“连接单元”是指一端与抗体/抗原结合片段连接而另一端与药 物相连的化学结构片段或键，因此做为一种“桥梁”将抗体/抗原结合片段与药物分子连接起来。它可以包括连接头、间隔物和氨基酸单元，可以通过本领域己知方法合成，诸如US2005-0238649A1中所记载的。如本文所用，“连接单元”可被分为两类：不可断裂连接子和可断裂连接子。
不可断裂连接子是一种相对比较稳定的连接子，其结构很难在体内环境下降解断裂。对于含有不可断裂连接子的抗体药物偶联物，其药物释放机制为：偶联物与抗原结合并被细胞内吞后，抗体在溶酶体中被酶解，释放出由药物，连接子，和抗体氨基酸残基共同组成的活性分子。由此带来的药物分子结构改变并不减弱其细胞毒性，但由于活性分子是带电荷的(氨基酸残基)，从而导致其不能渗入邻近细胞。因此，此类活性药物不能杀死邻近不表达靶向抗原(抗原阴性细胞)的肿瘤细胞(旁观者效应,bystander effect)(Bioconjugate Chem.2010，21，5-13)。常见的连接子例如Mc连接子和Mcc连接子等，如下结构所示。
Figure PCTCN2021107079-appb-000031
可断裂连接子，顾名思义，可以在目标细胞内断裂并释放出活性药物(小分子药物本身)。可断裂连接子可分为两个主要的类别：化学不稳定连接子和酶不稳定连接子。
化学不稳定连接子可以由于血浆和细胞质性质的不同而选择性的断裂。这样的性质包括pH值，谷胱甘肽浓度等。
对pH值敏感的连接子，通常又称为酸断裂连接子。这样的连接子在血液的中性环境下相对稳定(pH 7.3-7.5)，但是在弱酸性的内涵体(pH 5.0-6.5)和溶酶体(pH 4.5-5.0)内将会被水解。第一代的抗体药物偶联物大多应用这类连接子，例如腙，碳酸酯，缩醛，缩酮类。由于酸断裂连接子有限的血浆稳定性，基于此类连接子的抗体药物偶联物通常具有较短的半衰期(2-3天)。这种较短的半衰期在一定程度上限制了pH敏感连接子在新一代抗体药物偶联物中的应用。
对于谷胱甘肽敏感的连接子，又称二硫键连接子。药物释放是基于细胞内谷胱甘肽的高浓度(毫摩尔范围)与血液中相对较低的谷胱甘肽浓度(微摩尔范围)差异引起的。对于肿瘤细胞而言尤其如此，其低含氧量导致 还原酶的活性增强，因而导致更高的谷胱甘肽浓度。二硫键具有热力学稳定性，因此在血浆中具有较好的稳定性。
酶不稳定连接子，如肽连接子，能够更好地控制药物释放。肽连接子能够被溶酶体内蛋白酶，如组织蛋白酶(Cathepsin B)或纤溶酶(在一些肿瘤组织中此类酶含量增加)有效地切断。这种肽连接被认为在血浆循环中非常稳定，这是因为细胞外不合宜的pH值及血清蛋白酶抑制剂导致蛋白酶通常在细胞外不具备活性。鉴于较高的血浆稳定性和良好的细胞内断裂选择性和有效性，酶不稳定连接子被广泛用做抗体药物偶联物的可断裂连接子。
自杀式连接子一般嵌合在可断裂连接子与活性药物之间，或者本身就是可断裂连接子的一部分。自杀式连接子的作用机制是：当可断裂连接子在合宜的条件下断裂后，自杀式连接子能够自发地进行结构重排，进而释放与之连接的活性药物。常见的自杀式连接子如对氨基苄醇类(PAB)等。
本发明所使用的术语“毒素”、“药物”以及“药物部分”、“药物单元”泛指同一结构，可以在本发明中以任一名称使用。它们泛指任何具有期望的生物活性，并具有反应性官能团以便制备本发明所述偶联物的化合物。期望的生物活性包括诊断、治愈、缓解、治疗、预防人或其它动物的疾病。随着新型药物不断被发现和发展，这些新药也应纳入本发明所述的药物中。它能够对细胞的生长或增殖产生有害效果的任何物质，可以是来自细菌、真菌、植物或动物的小分子毒素及其衍生物，包括喜树碱类衍生物如伊沙替康，美登木素生物碱及其衍生物(CN101573384)如DM1、DM3、DM4,auristatin F(AF)及其衍生物，如MMAF、MMAE、3024(WO 2016/127790A1)，白喉毒素、外毒素、蓖麻毒蛋白(ricin)A链、相思豆毒蛋白(abrin)A链、modeccin、α-帚曲霉素(sarcin)、油桐(Aleutites fordii)毒蛋白、香石竹(dianthin)毒蛋白、美洲商陆(Phytolaca americana)毒蛋白(PAPI、PAPII和PAP-S)、苦瓜(Momordica charantia)抑制物、麻疯树毒蛋白(curcin)、巴豆毒蛋白(crotin)、肥皂草(sapaonaria officinalis)抑制物、白树毒蛋白(gelonin)、丝林霉素(mitogellin)局限曲霉素(restrictocin)、酚霉素(phenomycin)、依诺霉素(enomycin)和单端孢菌素(trichothecenes)。
术语“氨基酸”的三字母代码和单字母代码如J.biol.Chem,1968,243,3558.中所述。包括单不限于“酸性氨基酸”、“天然氨基酸”、“非天然氨基酸”。“酸性氨基酸”指氨基酸的等电点小于 7，酸性氨基酸分子中往往带有1个或多个羧基等酸性基团，在结构中可有效电离为负离子形式而增加亲水性。酸性氨基酸可以为天然的，也可为非天然的氨基酸。“天然氨基酸”指由生物合成的氨基酸。天然氨基酸一般情况下是L-型的，但也有少数例外，比如甘氨酸，包括天然的和生物体合成的。“非天然氨基酸”指通过合成手段所获得的氨基酸。
最重要的是，本领域技术人员将认识到，本发明所要求保护的化合物和方法可利用本领域已知的多种抗体的任一种抗体。使用的抗体可以从多种已知来源商购买获得或者通过本领域技术人员公知技术制备。另外，本发明所要求保护的化合物和方法可利用本领域已知的多种连接单元，使用的连接单元可以从多种已知来源商购买获得或者用于本领域技术人员公知技术制备。因此，抗体和连接单元的选择不应当视为本发明所要求的保护的化合物和方法的限制。
Figure PCTCN2021107079-appb-000032
实施例1 化合物L-D(即连接基团-双毒素基团化合物)的制备
①化合物LD-1(即Mc-MMAF-VC-PAB-Ceritinib)的制备
Figure PCTCN2021107079-appb-000033
取Fmoc-VC-PAB-PNP 75.6mg和Ceritinib(即色瑞替尼)50.0mg，加入5mL N,N-二甲基甲酰胺和49μL N,N-二异丙基乙胺，室温至反应结束，旋干溶剂，Flash法纯化后得到Fmoc-VC-PAB-Ceritinib粗品80.0 mg。LC-MS：[M+H]+：1185.2；[M-H]-：1183.7。
向Fmoc-VC-PAB-Ceritinib粗品中加入8mL N,N-二甲基甲酰胺和2mL N,N-二异丙基乙胺，室温至反应结束，旋干溶剂，制备液相法纯化后得到VC-PAB-Ceritinib纯品50.0mg。LC-MS：[M+H]+：963.4，[M-H]-：961.6。
取Mc-MMAF 20.0mg和TSTU 9.8mg，加入14.2μL N,N-二异丙基乙胺和18.8mg VC-PAB-Ceritinib，室温至反应结束，旋干溶剂，制备液相法纯化，得Mc-MMAF-VC-PAB-Ceritinib纯品12.0mg。LC-MS：[M+H]+：＝1869.8，[M-H]-：＝1868.2。
②化合物LD-2(即Mc-MMAF-VC-PAB-MK4827)的制备
Figure PCTCN2021107079-appb-000034
取Fmoc-VC-PAB-PNP 131.7mg和MK4827(即Niraparib)50.0mg，加入5mL N,N-二甲基甲酰胺和78μL N,N-二异丙基乙胺，室温至反应结束，旋干溶剂，Flash法纯化后得Fmoc-VC-PAB-MK4827粗品98.0mg。LC-MS：[M+H]+：947.5；[M-H+HCOOH]-：992.4。
向上述Fmoc-VC-PAB-MK4827粗品中加入8mL N,N-二甲基甲酰胺和2mL N,N-二异丙基乙胺，室温至反应结束，旋干溶剂，制备液相法 纯化，得VC-PAB-MK4827纯品60.0mg。LC-MS：[M+H]+：725.4，[M-H+HCOOH]-：770.0。
取Mc-MMAF 20.0mg和TSTU 9.8mg，加入14.2μL N,N-二异丙基乙胺和VC-PAB-MK4827 18.8mg，室温至反应结束16h，旋干溶剂，制备液相法纯化，得Mc-MMAF-VC-PAB-MK4827纯品7.0mg。LC-MS：[M+H]+：m/z＝1632.6，[M-H+HCOOH]-：m/z＝1677.5。
③其他化合物的制备
参照化合物1和化合物2的制备方法，制备出化合物LD-3(即Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38)，化合物LD-4(即Mc-MMAF-VC-PAB-Exatecan)和化合物LD-5(即Mc-MMAF-GGFG-Dxd)。
Figure PCTCN2021107079-appb-000035
实施例2 抗体药物偶联物的制备
抗体药物偶联物(ADC)的制备采用通用的偶联方法：用纯化水分别配制还原剂和保护剂如下：1～20mM TCEP(Tris-2-carboxyethyl-phosphine)、1～20mM DTPA(Diethylene triamine pentacetate acid)母液，还原剂用量根据所需偶联率不同可在一定浓度范围内添加，与一定浓度单克隆抗体(如：5～30mg/mL)按照一定体积比(1:1)混合，TCEP与抗体的终浓度摩尔比0.5～6.0:1，于25℃搅拌反应1h。TCEP还原后的抗体可直接进行偶联。
配制一定浓度(5mM)连接子-活性药物单元化合物溶于25％的DMSO(dimethyl sulfoxide，二甲亚砜)，按照药物与巯基的摩尔比0.3～2.8:1缓慢加药，于25℃搅拌反应1-4h。反应结束后，用PBS缓冲液离心超滤3次，纯化去除残余未反应药物以及DMSO等游离小分子，并且利用SDS-PAGE电泳和疏水高效液相(HIC-HPLC)法检测偶联情况。
通过本实施例提供的方法，分别制备了ADC1(即Ab-Mc-MMAF-VC-PAB-Ceritinib)、ADC2(即Ab-Mc-MMAF-VC-PAB-MK4827)、ADC3(即Ab-Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38)、ADC4(即Ab-Mc-MMAF-VC-PAB-Exatecan)、ADC5(即Ab-Mc-MMAF-GGFG-Dxd)。其中，p为选自1、2、3、4、5、6、7、8的任一整数，其中所用的Ab为HER2抗体。
Figure PCTCN2021107079-appb-000036
Figure PCTCN2021107079-appb-000037
实施例3 体外细胞活性评价
①乳腺癌细胞SK-BR-3活性评价
本实施例采用细胞增殖抑制法对Her2单抗-Mc-MMAF-VC-PAB-MK4827及Her2单抗-Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38对乳腺癌细胞SK-BR-3的细胞活性进行评价。
将SK-BR-3细胞胰酶消化后调整细胞密度为50000个/ml，100μL/孔加入细胞培养板中，37℃，5％CO 2培养箱中孵育14-20h。用基础培养基对供试品(样品组和对照组见表2)进行梯度稀释，以100μl/孔转入铺有细胞的细胞培养板中；37℃，5％CO 2培养箱中孵育70-74h。用培养基将CCK-8稀释10倍，将96孔板中的旧培养基吸出，每孔加入100μL稀释的CCK-8溶液，5％CO 2条件下显色2-4h，离心去气泡后在酶标仪上选择测定波长450nm/655nm进行读数。
表2供试样品
序号 供试品
样品1 HER2单抗-Mc-MMAF-VC-PAB-MK4827
对照1-1 HER2单抗-Mc-MMAF
对照1-2 HER2单抗-Mc-VC-PAB-MK4827
对照1-3 HER2单抗-Mc-MMAF和HER2单抗-Mc-VC-PAB-MK4827，质量浓度1:1混合
对照1-4 HER2裸抗体
样品2 HER2单抗-Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38
对照2-1 HER2单抗-Mc-MMAF
对照2-2 HER2单抗-Mc-VC-PAB-DMEDA-PEG(N3)-SN38
对照2-3 HER2单抗-Mc-MMAF和HER2单抗-Mc-VC-PAB-DMEDA-PEG(N3)-SN38，质量浓度1:1混合
对照2-4 HER2裸抗体
表3的结果表明，HER2单抗-Mc-MMAF-VC-PAB-MK4827对SK-BR-3细胞的抑制作用优于HER2单抗-Mc-MMAF、HER2单抗-Mc-VC-PAB-MK4827、HER2单抗-Mc-MMAF和HER2单抗-Mc-VC-PAB-MK4827的混合物、HER2裸抗体；HER2单抗-Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38对SK-BR-3细胞的抑制作用优于HER2单抗-Mc-MMAF、HER2单抗-Mc-VC-PAB-DMEDA-PEG(N3)-SN38、HER2单抗-Mc-MMAF和HER2单抗-Mc-VC-PAB-DMEDA-PEG(N3)-SN38的混合物、HER2裸抗体。并且双毒素串联组的两组毒素产生了协同效应。
表3对SK-BR-3细胞的抑制作用
Figure PCTCN2021107079-appb-000038
Figure PCTCN2021107079-appb-000039
②胃癌细胞NCI-N87活性评价
本实施例采用细胞增殖抑制法对Her2单抗-Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38对胃癌细胞NCI-N87的细胞活性进行评价。
将NCI-N87细胞胰酶消化后调整细胞密度为50000个/ml，100μL/孔加入细胞培养板中，37℃，5％CO 2培养箱中孵育14-20h。用基础培养基对供试品(样品组和对照组见表4)进行梯度稀释，以100μL/孔转入铺有细胞的细胞培养板中；37℃，5％CO 2培养箱中孵育70-74h。用培养基将CCK-8稀释10倍，将96孔板中的旧培养基吸出，每孔加入100μL稀释的CCK-8溶液，5％CO 2条件下显色2-4h，离心去气泡后在酶标仪上选择测定波长450nm/655nm进行读数。
表4供试样品
序号 供试品
样品3 HER2单抗-Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38
对照3-1 HER2单抗-Mc-MMAF
对照3-2 HER2单抗-Mc-VC-PAB-DMEDA-PEG(N3)-SN38
对照3-3 HER2单抗-Mc-MMAF和HER2单抗-Mc-VC-PAB-DMEDA-PEG(N3)-SN38，质量浓度1:1混合
对照3-4 HER2裸抗体
表5的结果表明，HER2单抗-Mc-MMAF-VC-PAB-DMEDA-PEG(N3)-SN38对NCI-N87细胞的抑制作用优于HER2单抗-Mc-MMAF和HER2单抗-Mc-VC-PAB-DMEDA-PEG(N3)-SN38的混合物、HER2裸抗体。并且双毒素串联组的两组毒素产生了显著的协同效应。
表5对NCI-N87细胞的抑制作用
Figure PCTCN2021107079-appb-000040
Figure PCTCN2021107079-appb-000041
本发明已通过各个具体实施例作了举例说明。但是，本领域普通技术人员能够理解，本发明并不限于各个具体实施方式，普通技术人员在本发明的范围内可以作出各种改动或变型，并且在本说明书中各处提及的各个技术特征可以相互组合，而仍不背离本发明的精神和范围。这样的改动和变型均在本发明的范围之内。

Claims (23)

  1. 一种具有以下结构式的抗体药物偶联物或其药学上可接受的盐，
    Figure PCTCN2021107079-appb-100001
    其中：
    Ab为抗体或抗原结合片段；
    S为所述Ab上的链间二硫键打开后形成的巯基残基中的硫原子；
    Aa为包含一个或多个氨基酸的氨基酸单元；
    G为可选择的裂解单元；
    D 1为MMAF；
    D 2是不同于MMAF的第二活性药物单元；
    p为选自1、2、3、4、5、6、7、8的整数。
  2. 根据权利要求1所述的抗体药物偶联物或其药学上可接受的盐，其特征在于，所述Ab为鼠、嵌合、人源化、或全人抗体的完整或抗原结合片段。
  3. 根据权利要求2所述的抗体药物偶联物或其药学上可接受的盐，其特征在于，所述抗原结合片段包括Fab、Fab’、F(ab) 2、F(ab’) 2；所述抗体包括双特异性抗体、或多特异性抗体。
  4. 根据权利要求3所述的抗体药物偶联物或其药学上可接受的盐，其特征在于，所述Ab具有人IgG1或者人IgG4抗体的Fc结构域和/或铰链区结构域或其部分氨基酸突变、替换、缺失形式。
  5. 根据权利要求1－4任一项所述的抗体药物偶联物或其药学上可接受的盐，其特征在于，所述的第一活性药物单元D 1选自以下结构或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式或其药学上可接受的盐或溶剂化物：
    Figure PCTCN2021107079-appb-100002
  6. 根据权利要求5所述的抗体药物偶联物或其药学上可接受的盐，其特征在于，所述的第二活性药物单元D 2选自细胞毒性分子、免疫增强剂和放射性同位素，所述细胞毒性分子包括但不限于微管蛋白抑制剂、DNA损伤剂、拓扑异构酶抑制剂、ALK抑制剂、PARP抑制剂；进一步优选的，所述微管蛋白抑制剂包括但不限于海兔毒素(dolastatin)及奥瑞他汀(auristatin)类细胞毒分子，美登素(maytansine)类细胞毒分子；所述DNA损伤剂包括但不限于卡奇霉素(calicheamicin)类、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD；所述拓扑异构酶抑制剂包括喜树碱(camptothecins)及喜树碱类衍生物；进一步优选的，所述奥瑞他汀(auristatin)类细胞素分子包括但不限于MMAE或MMAF或它们的洐生物，所述美登素类细胞毒分子包括但不限于DM1、DM4或它们的洐生物。
  7. 根据权利要求6所述的抗体药物偶联物或其药学上可接受的盐，其特征在于，所述的第二活性药物单元D 2不是微管蛋白抑制剂。
  8. 根据权利要求6所述的抗体药物偶联物或其药学上可接受的盐，其特征在于，所述的第二活性药物单元D 2选自MK4827，Ceritinib，Dxd，SN38，Exatecan或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式或其药学上可接受的盐或溶剂化物。
  9. 根据权利要求1－8中任一项所述的抗体药物偶联物或其药学上可接受的盐，其特征在于，所述的氨基酸单元Aa选自：-甘氨酸-、-丙氨酸-、-缬氨酸-、-亮氨酸-、-异亮氨酸-、-脯氨酸-、-苯丙氨酸-、-色氨酸-、-蛋氨酸-、-酪氨酸-、-丝氨酸-、-苏氨酸-、-半胱氨酸-、-天冬酰胺-、-谷氨酰胺-、-天冬氨酸-、-谷氨酸-、-赖氨酸-、-精氨酸-、-组氨酸-、-瓜氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-；缬氨酸-瓜氨酸-(-Val-Cit-)、-缬氨酸-丙氨酸-(-Val-Ala-)、-缬氨酸-赖氨酸-(-Val-Lys-)、-缬氨酸-赖氨酸(三苯甲基)-(-Val-Lys(Trt)-)、-缬氨酸-赖氨酸(单甲氧基三苯甲基)-(-Val-Lys(Mmt)-)、-缬氨酸-赖氨酸(芴氧羰基)-(-Val-Lys(Fmoc)-)、-缬氨酸-精氨酸-(-Val-Arg-)、-苯丙氨酸-瓜氨酸 -(-Phe-Cit-)、-苯丙氨酸-赖氨酸-(-Phe-Lys-)、-苯丙氨酸-赖氨酸(三苯甲基)-(-Phe-Lys(Trt)-)、-苯丙氨酸-赖氨酸(单甲氧基三苯甲基)-(-Phe-Lys(Mmt)-)、-苯丙氨酸-赖氨酸(芴氧羰基)-(-Phe-Lys(Fmoc)-)、亮氨酸-瓜氨酸-(-Leu-Cit-)、-异亮氨酸-瓜氨酸-(-Ile-Cit-)、-苯丙氨酸-精氨酸-(-Phe-Arg-)；-苯丙氨酸-精氨酸-精氨酸-(-Ala-Arg-Arg-)；-甘氨酸-甘氨酸-苯丙氨酸-甘氨酸-(-Gly-Gly-Phe-Gly-)、-甘氨酸-苯丙氨酸-亮氨酸-甘氨酸-(-Gly-Phe-Leu-Gly-)、-丙氨酸-亮氨酸-丙氨酸-亮氨酸(-Ala-Leu-Ala-Leu-)。
  10. 根据权利要求1－9中任一项所述抗体药物偶联物或其药学上可接受的盐，其特征在于，所述的裂解单元G为空或选自如下结构：
    Figure PCTCN2021107079-appb-100003
    Figure PCTCN2021107079-appb-100004
    其中所述的R 1、R 2为任一取代基团，优选的，所述的R 1和R 2独立地选自H或者C 1至C 10的烷基或者为：
    Figure PCTCN2021107079-appb-100005
  11. 根据权利要求1所述的抗体药物偶联物或其药学上可接受的盐，其特征在于，所述的抗体药物偶联物选自如下结构，其中p为选自1、2、3、4、5、6、7、8的整数：
    Figure PCTCN2021107079-appb-100006
    Figure PCTCN2021107079-appb-100007
  12. 权利要求1-11中任一项所述的抗体药物偶联物或其药学上可接受的盐在制备用于治疗或预防癌症、感染性疾病或自身免疫性疾病的药物中的应用。
  13. 一种中间体化合物，其结构如下式所示：
    Figure PCTCN2021107079-appb-100008
    其中：
    Aa为包含一个或多个氨基酸的氨基酸单元；
    G为可选择的裂解单元；
    D 1为MMAF；
    D 2是不同于MMAF的第二活性药物单元。
  14. 根据权利要求13所述的化合物，其特征在于，所述的第一活性药物单元D 1选自以下结构或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式或其药学上可接受的盐或溶剂化物：
    Figure PCTCN2021107079-appb-100009
  15. 根据权利要求14所述的化合物，其特征在于，所述的第二活性药物单元D 2选自细胞毒性分子、免疫增强剂和放射性同位素，所述细胞毒性分子包括但不限于微管蛋白抑制剂、DNA损伤剂、拓扑异构酶抑制剂、ALK抑制剂、PARP抑制剂；进一步优选的，所述微管蛋白抑制剂包括但不限于海兔毒素(dolastatin)及奥瑞他汀(auristatin)类细胞毒分子，美登素(maytansine)类细胞毒分子；所述DNA损伤剂包括但不限于卡奇霉素(calicheamicin)类、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD；所述拓扑异构酶抑制剂包括喜树碱(camptothecins)及喜树碱类衍生物；进一步优选的，所述奥瑞他汀(auristatin)类细胞素分子包括但不限于MMAE或MMAF或它们的洐生物，所述美登素类细胞毒分子包括但不限于DM1、DM4或它们的洐生物。
  16. 根据权利要求14所述的化合物，其特征在于，所述的第二活性药物单元D 2不是微管蛋白抑制剂。
  17. 根据权利要求15所述的化合物，其特征在于，所述的第二活性药物单元D 2选自MK4827，Ceritinib，Dxd，SN38，Exatecan或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体或其混合物形式或其药学上可接受的盐或溶剂化物。
  18. 根据权利要求13－17中任一项所述的化合物，其特征在于，所述的氨基酸单元Aa选自：-甘氨酸-、-丙氨酸-、-缬氨酸-、-亮氨酸-、-异 亮氨酸-、-脯氨酸-、-苯丙氨酸-、-色氨酸-、-蛋氨酸-、-酪氨酸-、-丝氨酸-、-苏氨酸-、-半胱氨酸-、-天冬酰胺-、-谷氨酰胺-、-天冬氨酸-、-谷氨酸-、-赖氨酸-、-精氨酸-、-组氨酸-、-瓜氨酸-、-赖氨酸(三苯甲基)-、-赖氨酸(单甲氧基三苯甲基)-、-赖氨酸(芴氧羰基)-；-缬氨酸-瓜氨酸-(-Val-Cit-)、-缬氨酸-丙氨酸-(-Val-Ala-)、-缬氨酸-赖氨酸-(-Val-Lys-)、-缬氨酸-赖氨酸(三苯甲基)-(-Val-Lys(Trt)-)、-缬氨酸-赖氨酸(单甲氧基三苯甲基)-(-Val-Lys(Mmt)-)、-缬氨酸-赖氨酸(芴氧羰基)-(-Val-Lys(Fmoc)-)、-缬氨酸-精氨酸-(-Val-Arg-)、-苯丙氨酸-瓜氨酸-(-Phe-Cit-)、-苯丙氨酸-赖氨酸-(-Phe-Lys-)、-苯丙氨酸-赖氨酸(三苯甲基)-(-Phe-Lys(Trt)-)、-苯丙氨酸-赖氨酸(单甲氧基三苯甲基)-(-Phe-Lys(Mmt)-)、-苯丙氨酸-赖氨酸(芴氧羰基)-(-Phe-Lys(Fmoc)-)、-亮氨酸-瓜氨酸-(-Leu-Cit-)、-异亮氨酸-瓜氨酸-(-Ile-Cit-)、-苯丙氨酸-精氨酸-(-Phe-Arg-)；-苯丙氨酸-精氨酸-精氨酸-(-Ala-Arg-Arg-)；-甘氨酸-甘氨酸-苯丙氨酸-甘氨酸-(-Gly-Gly-Phe-Gly-)、-甘氨酸-苯丙氨酸-亮氨酸-甘氨酸-(-Gly-Phe-Leu-Gly-)、-丙氨酸-亮氨酸-丙氨酸-亮氨酸(-Ala-Leu-Ala-Leu-)。
  19. 根据权利要求13－18中任一项所述的化合物，其特征在于，所述的裂解单元G为空或选自如下结构：
    Figure PCTCN2021107079-appb-100010
    Figure PCTCN2021107079-appb-100011
    其中所述的R 1、R 2为任一取代基团，优选的，所述的R 1和R 2独立地选自H或者C 1至C 10的烷基或者为：
    Figure PCTCN2021107079-appb-100012
  20. 根据权利要求13所述的化合物，其特征在于，所述的化合物选自如下结构：
    Figure PCTCN2021107079-appb-100013
  21. 权利要求13－20任一项所述的化合物在制备用于治疗或预防癌症、感染性疾病或自身免疫性疾病的药物中的应用。
  22. 一种如权利要求13所述的化合物的合成方法，其特征在于，所述方法的合成路线为：
    Figure PCTCN2021107079-appb-100014
    所述方法包括如下步骤：
    步骤1：将化合物Fmoc-Aa-G、第二活性药物单元D 2、有机碱1溶于适量有机溶剂1中，使其接触反应，得化合物Fmoc-Aa-G-D 2；
    步骤2：将化合物Fmoc-Aa-G-D 2脱除Fmoc基团保护，得化合物Aa-G-D 2；
    步骤3：将化合物Aa-G-D 2、化合物Mc-D 1、缩合剂1溶于适量有机溶剂2中，使其接触反应，得化合物Mc-D 1-Aa-G-D 2；
    其中，所述的有机碱1选自N，N二异丙基乙胺、三乙胺、吡啶的一种或几种；所述的有机溶剂1和有机溶剂2分别独立的选自DMF、DMA的一种或两种；所述的缩合剂1选自TSTU、HATU、HBTU、HCTU、PyBop、CDMT、T3P中的一种或几种。
  23. 一种如权利要求22所述的合成方法，其特征在于，所述的化合物选自权利要求20所示的结构，所述的合成路线选自如下：
    Figure PCTCN2021107079-appb-100015
    Figure PCTCN2021107079-appb-100016
    Figure PCTCN2021107079-appb-100017
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