WO2021022678A1 - 一种抗体药物偶联物及其应用 - Google Patents

一种抗体药物偶联物及其应用 Download PDF

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Publication number
WO2021022678A1
WO2021022678A1 PCT/CN2019/112663 CN2019112663W WO2021022678A1 WO 2021022678 A1 WO2021022678 A1 WO 2021022678A1 CN 2019112663 W CN2019112663 W CN 2019112663W WO 2021022678 A1 WO2021022678 A1 WO 2021022678A1
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Prior art keywords
antibody
drug
drug conjugate
conjugate according
adc
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PCT/CN2019/112663
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English (en)
French (fr)
Inventor
李乐乐
黄长江
孙友祥
刘丽娜
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Mabplex International Co Ltd
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Mabplex International Co Ltd
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Priority to JP2021525570A priority Critical patent/JP7303298B2/ja
Priority to SG11202101719PA priority patent/SG11202101719PA/en
Priority to EP19861250.9A priority patent/EP3797796A4/en
Priority to US16/650,226 priority patent/US11596693B2/en
Priority to AU2019341066A priority patent/AU2019341066B1/en
Priority to KR1020217018828A priority patent/KR102793948B1/ko
Priority to CA3076712A priority patent/CA3076712C/en
Priority to CN201980002409.8A priority patent/CN110997010B/zh
Publication of WO2021022678A1 publication Critical patent/WO2021022678A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • 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
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

Definitions

  • the present invention relates to the field of biomedicine, in particular to an antibody drug conjugate formed based on one or more cysteine residues or cysteine derivative residues as a drug linking carrier and its application.
  • the carrier can simultaneously couple one or more drugs to the limited attachment sites of the antibody as required.
  • Antibody-drug conjugate refers to a type of biological drug that connects a biologically active drug (Drug) and an antibody (Antibody) through a chemical linker (Linker).
  • ADC is like a precision-guided weapon system, in which biologically active drugs are used as anti-personnel ammunition to accurately strike diseased cells under the guidance of antibodies. Therefore, ADC combines the dual advantages of high efficiency of cytotoxic drug molecules and high targeting of antibodies. As of July 2019, only 6 antibody conjugate drugs have been approved globally (Table 1).
  • the actual final ADC product is an ADC mixture with different numbers of drugs connected to the antibody and different sites. Uniformity poses great challenges to the production and quality control of drugs.
  • indicator-DAR value drug-antibody ratio
  • the average DAR value is too small, there are too few drug molecules on the antibody, which will affect the overall drug efficacy. If the average DAR value is too large and too many drug molecules are loaded on the antibody, the ADC will become unstable as a whole, change its pharmacokinetic parameters, and may lead to increased plasma clearance, shortened half-life, and systemic Increased drug toxicity.
  • ADC has three classic coupling methods, one is amino coupling, one is sulfhydryl coupling, and the third is cross-link.
  • Amino coupling is to couple the drug to the lysine (Lys) residue of the antibody through a linker.
  • the first generation ADC drug Mylotarg uses the amino coupling method, but IgG includes nearly a hundred lysines , The coupling position may occur on the nearly 40 exposed lysine residues on the antibody light chain and heavy chain, and each antibody will be coupled with multiple small molecule drugs of varying numbers through lysine coupling Therefore, ADC drugs obtained by amino coupling are highly heterogeneous and have very poor product uniformity, which seriously affects the PK/PD and treatment window of the drug (see http://www.sohu.com/a/277791166_464404).
  • Sulfhydryl coupling is to first open the interchain disulfide bond in the antibody to form free cysteine (Cys) residues, and then couple with a linker-drug complex that can pair with cysteine residues. Since there are only 4 pairs of interchain disulfide bonds on IgG, 8 free cysteine residues will be formed after all the interchain disulfide bonds are opened. Therefore, the ADC formed by single sulfhydryl coupling has an average DAR value of 0 -8. Although sulfhydryl coupling can better control the number of connections on each antibody, the breaking of the interchain disulfide bond will greatly reduce the stability of the antibody.
  • Bridging coupling is a new coupling method developed on the basis of sulfhydryl coupling. Like sulfhydryl coupling, it first opens the interchain disulfide bond in the antibody to form two free cysteine residues. Since there are only 4 pairs of interchain disulfide bonds on an antibody, 8 free cysteine residues will be formed when the interchain disulfide bonds are all opened. Acid residues, so ADC formed by bridge coupling has an average DAR value of 0-4.
  • bridge coupling can not only better control the uniformity of the product, but also greatly provide coupling
  • the stability of the rear antibody, but the maximum DAR value of the bridge coupling method is 4, that is, an antibody can be coupled with a maximum of 4 drugs.
  • the antigen expression level required for effective ADC activity is based on Different antigen characteristics vary.
  • ADC needs at least 104 antigen / cell, to ensure that the number of fatal deliver cytotoxic drugs.
  • the antigen targeted by the antibody portion of the ADC should be uniformly expressed on the surface of tumor cells and have a high copy number (>10 5 /cell).
  • Tumor cell surface antigens are usually only a limited number (approximately 5000 to 106 antigen / cell), and the average of 3.5-4 DAR ADC (e.g. DAR Brentuximab vedotin average value of 4, an average value of 3.5 DAR Trastuzumab emtansine) ,
  • DAR ADC e.g. DAR Brentuximab vedotin average value of 4, an average value of 3.5 DAR Trastuzumab emtansine
  • small molecule drugs with particularly strong toxicity are often used clinically.
  • the small molecule drugs used in ADC mainly include auristatin, maytansine, calicheamicin, doxorubicin, etc. These small molecule drugs are compared with traditional chemotherapy drugs. It has a stronger killing effect on cancer cells, and usually an average dose of four to six molecules can kill target cells. However, once these particularly toxic small molecule drugs are off-target in the body, they can be fatal to patients.
  • the IC 50 of DM1 to many cells is about 10 -11 mol/L
  • the IC 50 of DM4 is about 10 -12 mol/L
  • ADC research new drugs invesligational new drugs, IND
  • IND invesligational new drugs
  • toxicity of ADC drugs in animals is mainly hematopoietic system toxicity, liver toxicity and reproductive toxicity, and some also have skin toxicity and Nephrotoxicity, in which the toxicity of the hematopoietic system, liver and reproductive system is directly related to small molecule cytotoxic drugs
  • Document 3 Saber H, Leighton JK.
  • Regul Toxicol Pharmacol, 2015, 71(3):444-452 the effective load of small molecule drugs is often too low, resulting in too low efficacy and leading to clinical failure.
  • the present invention provides a novel antibody drug conjugate capable of coupling more active units (Drug).
  • the technical scheme of the present invention is as follows:
  • the present invention provides an antibody drug conjugate as shown in formula (I)-(IV),
  • A is any antibody or functional binding fragment thereof
  • B 1 , B 2 , B, ..., B n are cysteine residues or cysteine derivative residues, which may be the same or different, B 1 and B 2 , B 2 and B 3 , ... , B n-1 and B n are connected by a peptide bond formed by dehydration condensation;
  • L 1 , L 2 , L 3 , L 4 ,..., L n+1 are any connecting units and are independent of each other, and may be the same or different.
  • the amino ends of L 1 and B 1 are covalently connected, and L 2 and B 1 , L 3 and B 2 , L 4 and B 3 , ..., L n+1 and B n are covalently connected through a mercapto group, and L 2 and D 1 , L 3 and D 2 , L 4 Covalently connected with D 3 , ..., L n+1 and D n ;
  • D 1 , D 2 , D 3 , ..., D n are any active drug unit and are independent of each other, and may be the same or different;
  • Z is any group that is covalently attached to the carbonyl group of B 1 in formula (I) or the carbonyl group of B 2 in formula (II) or the carbonyl group of B 3 in formula (III) or B in formula (IV) On the carbonyl group of n ;
  • n is an integer greater than or equal to 4, representing the number of branches connected to the active drug unit;
  • n is selected from 1, 2, 3, 4, 5, 6, 7, 8.
  • p is selected from 1, 2, 3, 4, 5, 6, 7, 8.
  • said Z is selected from:
  • the antibody-drug conjugate has a structure represented by formulas (VI-1)-(VI-5):
  • A is any antibody or functional binding fragment thereof
  • L 1 , L 2 , L 3 , L 4 , and L 5 are any connecting units and are independent of each other, and may be the same or different;
  • D 1 , D 2 , D 3 , and D 4 are any active drug unit and are independent of each other, and may be the same or different;
  • n is selected from 1, 2, 3, 4, 5, 6, 7, 8.
  • the L 1 is covalently linked to the amino residue or sulfhydryl residue on the antibody; preferably, the L 1 is covalently linked to the sulfhydryl residue on the antibody; more preferably, the L 1 is covalently linked to the sulfhydryl residue on the antibody.
  • the sulfhydryl residues formed after the opening of the interchain disulfide bonds are covalently linked.
  • L 1 , L 2 , L 3 , L 4 , ..., L n+1 are cleavable linkers, cleavable linker combinations or non-cleavable linkers.
  • the cleavable linker includes a peptide unit and a polysulfide bond, and the peptide unit contains 2-20 amino acids.
  • the peptide unit is selected from -valine-citrulline- (-Val-Cit-), -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-), -valine-alanine-(-Val-Ala-),- Valine-Lysine-(-Val-Lys-), -Valine-Arginine-(-Val-Arg-), -Phenylalanine-Citrulline-(-Phe-Cit-) , -Phenylalanine-Lys-(-Phe-Lys-), -Phenylalanine-Arginine-(-Phe-Arg-) and combinations thereof;
  • the polysulfide bond includes 2- 8 sulfur atoms, preferably the polysulfide bond is selected from
  • the L 1 may be selected from the following structures:
  • the L 2 , L 3 , L 4 ,..., L n+1 may be selected from the following structures:
  • the antibodies or functional binding fragments thereof include monoclonal antibodies, polyclonal antibodies, antibody fragments, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv ("scFv "), diabodies, linear antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or fusion proteins containing the antigen binding portion of the antibody; preferably, the antibody For humanized monoclonal antibodies or fully human antibodies.
  • the antibody is an IgG antibody or a functional binding fragment thereof. More preferably, the antibody is IgG 1 , IgG 2 , IgG 3 , and IgG 4 .
  • the active drug unit is a cytotoxic molecule, a cell differentiation factor, a stem cell nutritional factor, a steroid drug, a drug for the treatment of autoimmune diseases, an anti-inflammatory drug or a drug for the treatment of infectious diseases.
  • the cytotoxic molecules include but are not limited to tubulin inhibitors or DNA damaging agents; further preferably, the tubulin inhibitors include but are not limited to dolastatin and auristatin )-Like cytotoxic molecules, maytansine-like cytotoxic molecules;
  • the DNA damaging agents include, but are not limited to, calicheamicin, duocarmycin, and atoxomycin derivatives PBD, camptothecins and camptothecin derivatives, SN-38;
  • the auristatin cytokine molecules include, but are not limited to, MMAE or MMAF or their derivatives,
  • the maytansine-like cytotoxic molecules include, but are not limited to, DM1, DM4, or their biological species; further preferably, the cytotoxic molecules include the following molecules:
  • A is any antibody or functional binding fragment thereof
  • n is selected from 1, 2, 3, 4, 5, 6, 7, 8.
  • the present invention also provides a pharmaceutical composition, which comprises an effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of the above, and pharmaceutically acceptable excipients.
  • the present invention also provides the use of any one of the above-mentioned antibody drug conjugates in the preparation of cancer drugs.
  • the antibody drug conjugate of the present invention uses one or more cysteine residues or cysteine derivative residues as a drug connection carrier, and simultaneously couples one or more drugs to the limited connection site of the antibody, Therefore, antibody drug conjugates with higher drug payload can be easily prepared.
  • ADCs with higher drug loadings can be prepared, the range of drugs that can be coupled is larger, so that drugs with lower toxicity can be selected to prepare ADC products, thereby obtaining ADC products with a larger therapeutic window.
  • the ADC product obtained by the method of the present invention has better uniformity in the case of preparing antibody drug conjugates with the same DAR value.
  • the amount of antibody used in production can also be greatly reduced, thereby effectively reducing production costs.
  • an antibody drug conjugate includes a combination of two or more antibody drug conjugates and the like.
  • the connecting unit and linker in the present invention can be used interchangeably; the active unit, drug, and poison in the present invention can be used interchangeably.
  • the term "antigen" in the present invention refers to any molecule that triggers an immune response or can be bound by an antibody or antigen-binding molecule.
  • the immune response may involve the production of antibodies or the activation of specific immunocompetent cells or both.
  • any macromolecule including almost all proteins or peptides, can serve as an antigen.
  • the antigen can be expressed endogenously, that is, expressed from genomic DNA, or it can be expressed recombinantly, or it can be chemically synthesized.
  • the "antigens" involved in the present invention specifically refer to those tumor-associated antigens. These tumor-associated antigens are well-known in the industry and can be prepared by antibody preparation methods and information well-known in the industry.
  • Tumor-associated antigens include, but are not limited to, the following tumor-associated antigens (1)-(36).
  • the antigen-related information known in the industry is marked as follows, including name, other names, and gene bank accession number.
  • Tumor-associated antigens can be found in public databases, such as Genbank.
  • the antibody-targeted corresponding tumor-associated antigen includes all amino acid sequence variants and homologues, which have at least 70%, 80%, 85%, 90%, or 95% homology with the actual confirmed sequence, or have the same homology as the quoted sequence.
  • Tumor-associated antigen sequences have completely identical biological properties and characteristics.
  • BMPR1B (Bone Morphogenetic Protein Receptor-IB type, Genbank accession number NM_001203);
  • MPF MPF, MSLN, SMR, megakaryocyte enhancing factor, mesothelin, Genbank accession number NM_005823
  • Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate) member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession number NM_006424);
  • Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphore protein 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1), transmembrane domain (TM ) And short cytoplasmic domain, (semaphore protein) 5B, Genbank accession number AB040878);
  • PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628);
  • ETBR endothelin B receptor, Genbank accession number AY275463
  • MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession number NM_017763);
  • TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession number NM_017636);
  • CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor, Genbank accession number NP_003203 or NM_003212);
  • CD21 (CR2 (complement receptor 2) or C3DR (C3d/EB virus receptor) or Hs.73792, Genbank accession number M26004);
  • CD79b (CD79B, CD79 ⁇ , IGb (immunoglobulin-related ⁇ ), B29, Genbank accession number NM_000626);
  • FcRH2 (IFGP4, IRTA4, SPAP1A (containing the SH2 domain of phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession number NM_030764);
  • HER2 (ErbB2, Genbank accession number M11730);
  • NCA (CEACAM6, Genbank accession number M18728);
  • MDP (DPEP1, Genbank accession number BC017023);
  • IL20R ⁇ (IL20Ra, ZCYTOR7, Genbank accession number AF184971);
  • EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession number NM_004442);
  • PSCA prostate stem cell antigen precursor, Genbank accession number AJ297436
  • BAFF-R B cell activating factor receptor, BLyS receptor 3, BR3, Genbank accession number AF116456);
  • CD22 B cell receptor CD22-B isotype, Genbank accession number AK026467
  • CD79a (CD79A, CD79 ⁇ , immunoglobulin-related ⁇ , can covalently interact with Ig ⁇ (CD79B) and form a complex with Ig M molecules on the surface, transducing B cell specific proteins involved in B cell differentiation signals, Genbank accession number NP_001774.1);
  • CXCR5 Burkitt's lymphoma receptor 1
  • CXCL13 chemokines a G protein-coupled receptor activated by CXCL13 chemokines, plays a role in lymphocyte migration and humoral defense, and is involved in HIV-2 infection and possible Play a role in AIDS, lymphoma, myeloma, and leukemia, Genbank accession number NP_001701.1);
  • HLA-DOB Beta subunit (Ia antigen) of MHC class II molecules, which binds peptides and presents them to CD4+ T lymphocytes, Genbank accession number NP_002111.1);
  • P2X5 purin receptor P2X ligand gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and its defect may lead to the pathology of idiopathic detrusor instability Physiological condition, Genbank accession number NP_002552.2);
  • CD72 B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP_001773.1
  • LY64 lymphocyte antigen 64 (RP105), a family of type I membrane proteins (LRR) rich in leucine repeats, regulates B cell activation and apoptosis, and the loss of function is related to increased disease activity in patients with systemic lupus erythematosus , Genbank accession number NP_005573.1);
  • FcRH1 Fc receptor-like protein 1, putative immunoglobulin Fc domain receptor, including C2-like Ig-like and ITAM domains, may play a role in the differentiation of B lymphocytes, Genbank accession number NP_443170.1 );
  • IRTA2 Translocation Associated Immunoglobulin Superfamily Receptor 2, a putative immune receptor that may play a role in B cell development and lymphoma production; gene disorders caused by translocation occur in some B cell malignancies Above, Genbank accession number NP_112571.1);
  • TENB2 (putative transmembrane proteoglycan, related to EGF/heregulin family of growth factors and follistatin, Genbank accession number AF179274);
  • an antibody in the present invention is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments.
  • an antibody may comprise at least two heavy chains and two light chains connected to each other by disulfide bonds, or an antigen binding molecule thereof.
  • Each heavy chain contains a heavy chain variable region and a heavy chain constant region.
  • the heavy chain constant region contains three constant domains: CH1, CH2, and CH3.
  • Each light chain contains a light chain variable region and a light chain constant region.
  • the constant region of the light chain contains a constant domain: CL.
  • the heavy chain variable region and the light chain variable region can be further subdivided into hypervariable regions, called complementarity determining regions (CDR), interspersed with more conservative regions, called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each heavy chain variable region and light chain variable region contains three CDRs and four FRs, arranged in the following order from amino terminal to carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant region of Ab can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (C1q) of the classical complement system.
  • the term "antibody” in the present invention also encompasses intact immunoglobulins or antigen-binding portions thereof, which compete with intact antibodies for specific binding.
  • the antigen binding portion can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of whole antibodies.
  • the antigen-binding portion especially includes Fab, Fab', F(ab')2, Fv, domain antibodies (dAb), fragments including complementarity determining regions (CDR), single-chain antibodies (scFv), chimeric antibodies, bivalent antibodies , Trivalent antibodies, tetravalent antibodies, and polypeptides containing at least a portion of immunoglobulin sufficient to confer specific antigen binding to the polypeptide.
  • antibody in the present invention also includes both naturally occurring and non-naturally occurring (recombinantly produced) antibodies, human and non-human antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), Immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, Antibody light chain-antibody heavy chain pairs, intracellular antibodies (see, for example Stocks, (2004) Drug Discovery Today 9(22):960-66), antibody fusions (this term encompasses antibody-drug conjugates and they are used herein Sometimes referred to as "antibody conjugate”), heteroconjugate antibody, single domain antibody, monovalent antibody, single chain antibody or single chain Fv (scFv), camelized antibody, affybody , Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, antigen
  • the term "functional fragment” in the present invention refers to an antibody fragment consisting of or containing partial sequences of the heavy or light variable chain of the antibody from which it is derived.
  • the partial sequence is sufficient to retain the same binding specificity and sufficient
  • the affinity is preferably at least equal to 1/100 of the affinity of the source antibody, and in a more preferred manner at least equal to 1/10.
  • This functional fragment will contain a minimum of 5 amino acids, preferably 10, 15, 25, 50, and 100 consecutive amino acids of the antibody sequence from which it is derived.
  • humanized antibody refers to an antibody that contains CDR regions derived from a non-human antibody, and other parts of the antibody molecule are derived from one (or several) human antibodies. Moreover, in order to retain binding affinity, some residues of the backbone (called FR) segment can be modified (Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al., Science, 239:1534-1536, 1988; Riechmann et al., Nature, 332: 323-327, 1988).
  • the humanized antibodies or fragments thereof according to the present invention can be prepared by techniques known to those skilled in the art (for example, described in the document Singer et al., J. Immun. 150: 2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10: 1-142, 1992; or Bebbington et al., Bio/Technology, 10: 169-175, 1992).
  • chimeric antibody refers to an antibody in which the variable region sequence is from one species and the constant region sequence is from another species, for example, the variable region sequence is from a mouse antibody and the constant region sequence is from a human antibody.
  • the chimeric antibody or fragment thereof according to the present invention can be prepared by using gene recombination technology.
  • the chimeric antibody can be produced by cloning recombinant DNA comprising a promoter and a sequence encoding the variable region of a non-human, especially murine, monoclonal antibody according to the present invention, and a sequence encoding the constant region of a human antibody .
  • the chimeric antibody of the present invention encoded by this recombinant gene will be, for example, a mouse-human chimera whose specificity is determined by the variable region derived from murine DNA, and its isotype is determined by the variable region derived from human DNA The constant region is determined.
  • the document Verhoeyn et al. BioEssays, 8:74, 1988
  • Verhoeyn et al. BioEssays, 8:74, 1988
  • the term "monoclonal antibody” refers to a preparation of antibody molecules of single molecular composition.
  • the monoclonal antibody composition shows a single binding specificity and affinity for a specific epitope.
  • the antibodies involved in the present invention include, but are not limited to, the following: Morolizumab-CD3, abciximab, rituximab, daclizumab, palivizumab, Infliximab, trastuzumab, etanercept, basilizumab, gemtuzumab, alemtuzumab, ibrituzumab, adalimumab, alfaxet, Omalizumab, falizumab, tositumomab, cetuximab, ABT-806, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab Lizumab, Linacipr, Certuzumab, Romigrastim, AMG-531, Golimumab, Ustekinumab, ABT-874, Belacept, Belimumab, Ascycept, anti-CD20
  • linking unit in the present invention refers to a molecule with bifunctional or multifunctional groups that can react with protein/antibody molecules and drug molecules respectively, so it acts as a "bridge” to connect protein/antibody and drug molecules stand up.
  • linkers or “linkers of antibody-drug conjugates” can be divided into two categories: non-cleavable linker and cleavable linker .
  • the non-breakable linker is a relatively stable linker whose structure is difficult to degrade and break in the in vivo environment.
  • the drug release mechanism is: After the conjugate binds to the antigen and is endocytosed by the cell, the antibody is enzymatically digested in the lysosome, and the small molecule drug is released.
  • An active molecule composed of amino acid residues of an antibody. The resulting change in the structure of the drug molecule does not reduce its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate neighboring cells. Therefore, such active drugs cannot kill adjacent tumor cells that do not express the targeted antigen (antigen-negative cells) (bystander effect) (Bioconjugate Chem. 2010, 21, 5-13).
  • Common non-breakable linkers such as MC linkers and MCC linkers:
  • the cleavable linker can rupture and release the active drug (the small molecule drug itself) in the target cell.
  • Breakable linkers can be divided into two main categories: chemically labile linkers and enzyme labile linkers.
  • Chemically labile linkers can be selectively broken due to differences in the properties of plasma and cytoplasm. Such properties include pH, glutathione concentration, etc.
  • Linkers that are sensitive to pH are often referred to as acid cleavage linkers. Such linkers are relatively stable in the neutral environment of blood (pH 7.3-7.5), but will be hydrolyzed in weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). Most of the first generation of antibody drug conjugates use such linkers, such as hydrazone, carbonate, acetal, and ketal. 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 a certain extent.
  • glutathione-sensitive linkers also known as disulfide bond linkers.
  • Drug release is based on the difference between the high concentration of glutathione in the cell (millimolar range) and the relatively low concentration of glutathione in the blood (micromolar range). This is especially true for tumor cells, whose low oxygen content leads to increased reductase activity, which leads to higher glutathione concentrations.
  • Disulfide bonds are thermodynamically stable, so they have better stability in plasma.
  • Enzyme-labile linkers can better control drug release.
  • Peptide linkers can be effectively cut by lysosome proteases, such as cathepsin (Cathepsin B) or plasmin (increased in some tumor tissues).
  • This peptide linkage is considered to be very stable in the plasma circulation. This is because the unsuitable pH value outside the cell and serum protease inhibitors result in the protease usually inactive outside the cell.
  • enzyme labile linkers are widely used as cleavable linkers for antibody-drug conjugates. Typical enzyme-labile linkers such as vc linkers.
  • the suicide linker is generally embedded between the cleavable linker and the active drug, or is part of the cleavable linker itself.
  • the mechanism of action of the suicide linker is: when the rupturable linker is broken under suitable conditions, the suicide linker can spontaneously rearrange the structure and release the active drug connected to it.
  • Common suicide linkers such as p-aminobenzyl alcohol (PAB) and so on.
  • the term "active unit" in the present invention generally refers to any compound that has the desired biological activity and has a reactive functional group to prepare the conjugate of the present invention.
  • the desired biological activity includes diagnosis, cure, alleviation, treatment, and prevention of human or other animal diseases.
  • these new drugs should also be included in the drugs of the present invention.
  • the drugs include, but are not limited to, cytotoxic drugs, cell differentiation factors, stem cell nutritional factors, steroid drugs, drugs for treating autoimmune diseases, anti-inflammatory drugs or drugs for infectious diseases. More specifically, the drugs include but are not limited to tubulin inhibitors or DNA or RNA damaging agents.
  • the active units involved in the present invention include but are not limited to the following:
  • affinity ligand is a substrate, inhibitor, stimulant, neurotransmitter, radioisotope, or any combination of the foregoing;
  • Radiolabels 32P, 35S, fluorescent dyes, electron densification reagents, enzymes, biotin, streptavidin, digoxigenin, haptens, immunogenic proteins, nucleic acids with sequences complementary to the target Molecule, or any combination of the foregoing;
  • the "active unit” is selected from the group consisting of maytansine compounds, V-ATPase inhibitors, pro-apoptotic agents, Be12 inhibitors, McL1 inhibitors, HSP90 inhibitors, IAP inhibitors Agent, mTOr inhibitor, microtubule stabilizer, microtubule destabilizer, auristatin, dolastatin, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitor, DPPIV inhibitor, proteasome inhibitor, inhibitor of phosphoryl transfer reaction in mitochondria, protein synthesis inhibitor, kinase inhibitor, CDK2 inhibitor, CDK9 inhibitor, kinesin inhibitor, HDAC inhibitor, DNA damaging agent, DNA Alkylating agents, DNA intercalators, DNA minor groove binders, DHFR inhibitors, donotxin peptides, vitamin A precursors, and folic acid.
  • maytansine compounds V-ATPase inhibitors, pro-apoptotic agents, Be12 inhibitors, McL1 inhibitors, HSP90 inhibitors, I
  • the "active unit” is a cytotoxic drug (e.g., antimetabolites, antitumor antibiotics, alkaloids), immune enhancers, or radioisotopes.
  • the drug can be selected from the group consisting of amanitins, anthracyclines, baccatins, camptothecins, cemadotins, colchicine ( colchicines, colcimids, combretastatins, cryptophycins, discodermolides, docetaxel, doxorubicin, echinoderma Echinomycins, eleutherobins, epothilones, estramustines, lexitropsins, maytansines, methotrexate ( methotrexate), netropsins, puromycins, rhizoxins, taxanes, tubulysins, or vinca alkaloids.
  • the "drug" can be selected from MMAD (Monomethyl auristatin D) and its derivatives, MMAE (Monomethyl auristatin E) and its derivatives, MMAF (Monomethyl auristatin F) and its derivatives, maytansine derivatives DM1 (Mertansine derivative M1), maytansine derivatives DM4 (Mertansine derivative M4), becarcinomycin ( Duocarmycine and its derivatives, Calicheamicin and its derivatives, PBDA (Pyrrolobenzodiazepines), Doxorubicin, Vinca Alkaloids, Metrotrexate, Vinblastine, Dauno Daunorubicin and its derivatives, tubulysins and its derivatives.
  • the "active unit” is maytansine or maytansinoids.
  • Maytansine compounds inhibit cell proliferation by inhibiting the formation of microtubules of tubulin (Science 1975, 189, 1002-1005; US 5208020).
  • Maytansinoids are derivatives of maytansinoids. Both maytansinoids and maytansinoids have high-efficiency cytotoxicity, but they have great limitations in the clinical application of cancer treatment, which is mainly due to the low selectivity of such molecules to tumors. However, this high cytotoxicity makes them the preferred drug part of antibody-drug conjugates.
  • the main raw material for the synthesis of maytansinoids is maytansinol, which is mainly obtained by hydrolysis of ansamitocins.
  • Ansamicin can be prepared by fermentation.
  • Ansamicin derivatives (WO 2012/061590) and alanyl maytansinol (US 2012/0121615) are reported to also be used as drug "warheads" for antibody drug conjugates.
  • the "active unit” is auristatin peptide drug.
  • Otostatin peptide drugs are analogs of dolastatin 10 (dolastatin 10), and the latter is a biologically active polypeptide isolated from the marine mollusk, dolastatin (US 7498298).
  • Aplastin 10 inhibits tubulin polymerization by binding to tubulin (the same binding region as vincristine).
  • Aplastatin 10, auristatin peptide PE, and auristatin peptide E are all linear polypeptides, containing four amino acids (three of which are unique to donotxin compounds) and a C-terminal amide group.
  • Two representative otostatin peptide compounds, monomethyl otostatin peptide E (MMAE) and monomethyl otostatin peptide F (MMAF) are the preferred drug parts of antibody drug conjugates.
  • the "active unit” is a Tubulysins drug.
  • Tubulysins are a class of natural products extracted from myxobacteria, which can effectively inhibit the polymerization of tubulin and therefore have anti-mitotic activity.
  • Tubulysin D has the best activity.
  • Tubulysin D is a complex tetrapeptide compound with O-acyl/N,O-acetal functional groups in its structure, so it is unstable under acidic and alkaline conditions.
  • US2011/0021568 and US2013/0224228 respectively disclose a series of analogues of tubulysin, which have the above unstable functional groups removed from the structure and at the same time have high cell activity.
  • the "active unit” is a calichemicins drug.
  • the calicheamicin drugs are anti-tumor antibiotics, which lead to cell apoptosis by binding to the minor groove of DNA and promoting the break of the double helix DNA at specific sites.
  • the calicheamicin drugs have high activity at the subpicomol level in vitro, but their low therapeutic index excludes the prospect of clinical application. However, this high activity makes them ideal candidates for antibody drug conjugates (such as gemtuzumab and Inotuzumab Ozogamicin).
  • the "active unit” is doxorubicins. Adriamycin can be inserted into the double helix structure of DNA to block DNA replication, so it is used as a chemotherapy drug.
  • doxorubicin for human cancer cell lines, the half inhibitory concentration is 0.1-0.2 micromolar, while the cytotoxic drug activity for antibody drug conjugates is usually sub-nanomolar), As a result, its application in antibody-drug conjugates is not universal.
  • the "active unit” is benzodipyrrole antibiotics (duocarmycins, CC-1065, etc.) and other cyclopropapyrroloind-4-one (cyclopropapyrroloind-4-one , CPI) derivatives.
  • Such compounds are effective DNA minor groove binding-alkylating reagents.
  • Cyclopropabenzindol-4-one (cyclopropabenzindol-4-one, CBI) analogs have a more stable chemical structure, higher biological activity, and are easier to compare with their parent compounds containing natural CPI alkylated subunits synthesis.
  • a representative CBI derivative is the phenolic hydroxyl protected derivative CBI (see figure below), which has weakened prodrug toxicity and enhanced water solubility.
  • the "active unit” is pyrrolo[2,1-c][1,4]benzodi-azepines (PBDs) or PBD dimers (PBD dimers).
  • PBD is a kind of natural product produced by Streptomyces. Its unique characteristic is that it can form a non-distorted covalent adduct in the DNA minor groove, exactly at the purine-guanine-purine sequence.
  • the application of PBD as part of the small molecule strategy to target and lock DNA sequences and as a new type of anti-cancer and antibacterial drugs has attracted more and more interest (Biochemistry 2008, 47, 11818-11829).
  • a flexible carbon chain is used to connect the C8/C8' hydroxyl groups of two PBD units, and the resulting dimer has enhanced biological activity (WO 2011/130616).
  • PBD dimers are thought to produce sequence-selective DNA damage, such as reversed 5'-Pu-GATC-Py-3' inter-strand cross-links, leading to its biological activity. These compounds have been proven to be highly effective cytotoxic drugs and can be used as candidates for antibody-drug conjugates.
  • the "active unit” is not limited to the categories mentioned above, but also includes all drugs that can be used in antibody drug conjugates.
  • pharmaceutical composition in the present invention means a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient that are combined to achieve a specific purpose.
  • the pharmaceutical composition includes combinations separated in time and/or space, as long as they can work together to achieve the purpose of the present invention.
  • the ingredients contained in the pharmaceutical composition may be administered to the subject as a whole, or separately administered to the subject.
  • the ingredients contained in the pharmaceutical composition may be administered to the subject simultaneously or sequentially.
  • the pharmaceutically acceptable carrier is water, buffered aqueous solution, isotonic salt solution such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, carbonic acid Magnesium, 0.3% glycerin, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides, etc.
  • the type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • the composition according to the invention may contain wetting agents, emulsifiers or buffer substances as additives.
  • compositions, vaccine or pharmaceutical preparation of the present invention can be administered by any suitable route, for example, oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • an effective amount in the present invention includes an amount sufficient to improve or prevent the symptoms or conditions of medical diseases.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • the unit of weight-volume percentage in the present invention is well-known to those skilled in the art, for example, refers to the weight of the solute in a 100 ml solution.
  • maleimidohexanoyl-MMAF was prepared. Dissolve 40.1mg maleimidohexanoyl-MMAF in 1.5mL DMF, add 4.4mg Cys-Cys-Cys and 2.1uL DIPEA, stir at room temperature for 2h, then add 1.8mg Cys-Cys-Cys, react at room temperature for 2h, Then add 0.9mg Cys-Cys-Cys and react at room temperature for 2h. Add 13.5mg 6-(maleimido)hexanoic acid succinimidyl ester and 21uL DIPEA, and stir at room temperature for 15h. The solvent was distilled off under reduced pressure and purified by preparative liquid phase method to obtain 23.1 mg of compound 2 with a yield of 49%. LC-MS: (M + 3H ) 3+ 1648.4, (M-3H) 3- 1646.3.
  • maleimidohexanoyl-MMAF was prepared. 31.6 mg maleimidohexanoyl-MMAF was dissolved in 2 mL DMF. Add 3.9mg Cys-Cys and 2.9uL DIPEA, stir at room temperature for 4h, then add 1.2mg Cys-Cys, stir at room temperature for 3h, add 0.8mg Cys-Cys, and stir at room temperature for 16h.
  • maleimidohexanoyl-MMAF was prepared. Dissolve 55.0mg maleimidohexanoyl-MMAF in 1.5mL N,N-dimethylformamide, add 11.6mg Cys-Cys, add 4.3uL N,N-diisopropylethylamine, stir at room temperature 3h. Add 36.1mg MP2-PNP(2-(2-Maleimidoethoxy)ethyl(4-nitrophenyl)carbonate, maleimidoethoxyethyl-p-nitrophenyl carbonate) to the reaction system, then add 51uL N, N-diisopropylethylamine, stirring at room temperature for 15h. The solvent was distilled off under reduced pressure and purified by preparative liquid phase method to obtain 18.0 mg of compound 4 with a yield of 27%. LC-MS: (M+2H) 2+ 1142.3, (M-2H) 2- 1141.1.
  • maleimidohexanoyl-MMAF was prepared. Dissolve 55.0mg maleimidohexanoyl-MMAF in 1.5mL N,N-dimethylformamide, add 11.6mg Cys-Cys, add 4.3uL N,N-diisopropylethylamine, stir at room temperature 3h. Add 71.0mg of maleimide propionamide-octapolyethylene glycol propionate succinimidyl ester (Mal-Propionamide-PEG8-propionate-OSu) to the reaction system, and then add 51uL N,N-diisopropyl Ethylamine, stirring at room temperature for 15h. The solvent was distilled off under reduced pressure and purified by preparative liquid phase method to obtain 19.3 mg of compound 5. The yield was 28%, LC-MS: (M+2H) 2+ 1324.1, (M-2H) 2- 1324.0.
  • maleimidohexanoyl-MMAF was prepared. Dissolve 63.0mg maleimidohexanoyl-MMAF in 2.0mL N,N-dimethylformamide, add 13.3mg Cys-Cys, add 4.8uL N,N-diisopropylethylamine, stir at room temperature 3h. Add 86.6mg Mc-VC-PAB-PNP to the reaction system, then add 58uLN,N-diisopropylethylamine, and stir at room temperature for 15h. The solvent was distilled off under reduced pressure and purified by preparative liquid phase method to obtain 47.4 mg of compound 6 with a yield of 52%. LC-MS: (M+2H) 2+ 1336.0, (M-2H) 2- 1334.6.
  • maleimidohexanoyl-MMAF was prepared. Dissolve 46.6mg maleimidohexanoyl-MMAF in 2.0mL N,N-dimethylformamide, add 10.9mg Cys-Cys, add 3.6uL N,N-diisopropylethylamine, and stir at room temperature 3h. Add 84.4 mg of PY-MAA-VC-PAB-PNP to the reaction system, then add 48 uL of N,N-diisopropylethylamine, and stir at room temperature for 15 hours. The solvent was distilled off under reduced pressure, purified by preparative liquid to give compound 7 31.0mg, yield 43%, LC-MS: ( M + 2H) 2+ 1401.2, (M-2H) 2- 1399.4.
  • HIC-HPLC hydrophobic chromatography
  • HIC-HPLC hydrophobic chromatography
  • Digest SK-BR-3 tumor cells (human breast cancer cells), collect by centrifugation, count them, and dilute the cell liquid to 0.5-1.5 ⁇ 10 5 cells/mL, add 100 ⁇ L of the cell suspension to each well of a 96-well plate . Incubate overnight at 37°C and 5% CO 2 in an incubator; the next day, add the corresponding 9 concentration gradient ADC drugs, plus a set of cell controls with zero concentration ADC drugs. After 72 hours of incubation, Cell Counting Kit-8 (CCK-8 kit for short) was used for active color development. After color development, the 96-well plate was used to detect the OD value at 450 nm with a microplate reader. Prism software calculates IC50 value through OD value. When the fitted curve is "S-shaped curve" and R 2 ⁇ 0.95, the IC50 value is valid and reportable.
  • MMAF dosage change (Comparative Example X-Comparative Example 1)/Comparative Example 1; X represents the corresponding comparative example;
  • Comparative Example 2 the DAR value was increased to 7.18 by increasing the number of MMAF sites in the antibody. Compared with Comparative Example 1, when the same effect is achieved, Comparative Example 2 can effectively reduce the amount of antibody used (that is, the amount of antibody in ADC. Without considering the amount of antibody loss during the synthesis process, the amount of antibody used is equal to ADC concentration), but MMAF usage increased by 19.3%.
  • Comparative Example 3 has two MMAFs connected to one linker, and the DAR value (4.3) is equivalent to that of Comparative Example 1 (3.87). Compared with Comparative Example 1, when the same effect is achieved, Comparative Example 3 effectively reduces antibodies The usage was 65.9%, and the usage of MMAF was reduced by 24.3%.
  • Comparative Example 4 has three MMAFs connected to one linker, and the DAR value is equivalent to that of Comparative Example 1. Compared with Comparative Example 1, when the same effect is achieved, Comparative Example 4 effectively reduces the amount of antibody used by 85.5%. MMAF is used The amount is reduced by 51.8%.
  • ADC-10 and ADC-34 show more efficient cell activity, and the effect they bring is not only to increase the effect of ADC by increasing the number of MMAF in the antibody, but also further reflects
  • the single-point multi-warhead (cytotoxic drug) connection method has effectively reduced the amount of antibody and toxin used, and has played an unexpected technical effect.
  • the uniformity of ADC products can be extremely effectively improved through the method of single connection point and multiple warheads, which can effectively promote the production and quality control of drugs.

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Abstract

一种通过一个或多个半胱氨酸残基或半胱氨酸衍生物残基作为药物连接载体，在抗体的有限个连接位点上同时偶联一个或多个药物，从而制备药物载量更高或者可以选择毒性较低的药物制备ADC产品，从而获得治疗窗口更大的ADC产品。此外，由于可在一个连接位点偶联多个药物分子，因此在制备相同DAR值的抗体药物偶联物情况下，所获得的ADC产品均一性也更好。并且在生产中所需要使用的抗体用量也能得以大幅降低，从而有效的降低了生产成本。所制备的抗体药物偶联物，与同一位点仅能连接一个药物分子的抗体药物偶联物相比，在偶联的药物分子总量大幅降低的情况下，对肿瘤细胞仍能起到相同的抑制或杀伤效果。

Description

一种抗体药物偶联物及其应用 技术领域
本发明涉及生物医药领域，具体涉及一种基于一个或多个半胱氨酸残基或半胱氨酸衍生物残基作为药物连接载体形成的抗体药物偶联物及其应用，所述药物连接载体可根据需要在抗体的有限连接位点上同时偶联一个或多个药物。
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2019年7月，全球仅批准了6种抗体偶联药物(表1)。
表1已上市抗体药物偶联物
药品通用名 公司 上市时间 适应症
Brentuximab vedotin Seattle、武田 2011年 霍奇金淋巴瘤
Trastuzumab emtansine 基因泰克 2013年 乳腺癌
Inotuzumab ozogamicin 辉瑞 2017年 急性淋巴细胞白血病
Gemtuzumab ozogamicin 辉瑞 2017年 急性髓性白血病
Moxetumomab pasudotox 阿斯利康 2018年 毛细胞白血病
Polatuzumab vedotin-piiq 基因泰克 2019年 弥漫性大B细胞淋巴瘤
由于在抗体上选择的连接位点的非唯一性以及连接反应的复杂性，因此实际最终得到的ADC产品是一个抗体上连接药物数量不一，位点不一的ADC混合物，ADC药物制备的非均一性对药物的生产、质量控制造成很大的挑战。在考虑产品均一性时，有一项重要的指标----DAR值(drug-antibody ratio，药物抗体比)，即单位数量的抗体上所能携带的药物分子数量。如果平均DAR值偏小，则抗体上携带药物分子过少，会影响 整体药效。而如果平均DAR值偏大，抗体上负载了过多的药物分子，则会使ADC整体变得不稳定，改变其药代动力学参数，并有可能导致血浆清除率上升、半衰期缩短以及系统性药物毒性的增大。
目前ADC有三种经典的偶联方式，一种是氨基偶联、一种是巯基偶联、第三种是桥接偶联(cross-link)。
氨基偶联是将药物通过连接子偶联在抗体的赖氨酸(Lys)残基上，第一代ADC药物Mylotarg采用的就是氨基偶联的方式，但是IgG上包括了近百个赖氨酸，偶联的位置可能会发生在抗体轻链和重链上近40个外露的赖氨酸残基上，而且通过赖氨酸偶联每个抗体会偶联多个数量不等的小分子药物,因此通过氨基偶联获得的ADC药物高度异质混合，产品均一性极差，这严重影响药物的PK/PD和治疗窗口(参见http://www.sohu.com/a/277791166_464404)。
巯基偶联是先将抗体中的链间二硫键打开形成游离的半胱氨酸(Cys)残基，再和能与半胱氨酸残基配对的连接子-药物复合物进行偶联，由于IgG上只有4对链间二硫键，链间二硫键被全部打开后会形成8个游离的半胱氨酸残基，因此通过单巯基偶联形成的ADC，其平均DAR值为0-8，尽管巯基偶联能够较好地控制每个抗体上连接的数量，但是链间二硫键的断去会极大的降低抗体的稳定性。
桥接偶联是在巯基偶联的基础上演变的一种新的偶联方式，同巯基偶联一样，它是先将抗体中的链间二硫键打开形成两个游离的半胱氨酸残基，之后再同时与这两个游离的半胱氨酸残基配对，由于一个抗体上只有4对链间二硫键，链间二硫键被全部打开后会形成8个游离的半胱氨酸残基，因此通过桥接偶联形成的ADC，其平均DAR值为0-4，与巯基偶联相比，桥接偶联既能更好的控制产品的均一性，又能大大的提供偶联后抗体的稳定性，但是桥接偶联方式的DAR值最大为4，即一个抗体上最多会偶联4个药物，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)。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)，而ADC的平均DAR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。
基于肿瘤细胞表面只有有限数量的抗原，为了保证抗体所携带的有限数量的小分子药物能够有效的杀死肿瘤细胞，临床上往往采用毒性特别强的小分子药物。目前ADC使用的小分子药物主要有auristatin、美登素(maytansine)、卡奇霉素(calicheamicin)、多柔比星(doxorubicin)等几大类，这些小分子药物与传统的化疗药物相比，对癌细胞具备更强的杀伤效力，通常平均四到六个分子的剂量就可实现对靶细胞杀伤，但是，这些毒性特别强的小分子药物在体内一旦出现脱靶，对患者的伤害是致命的，除此之外，由于这些小分子药物的毒性特别强(如DM1对很多细胞的IC 50约为10 -11mol/L、DM4的IC 50约为10 -12mol/L(文献1：Widdison WC,Wilhelm SD,Cavangh EE,et al.Semisynthetic maytansine analogues for the targeted treatment of cancer[J].J Med Chem,2006,49:4392-4408；文献2：Lambert JM.Antibody-maytansinoid conjugates:a new strategy for the treatment of cancer[J].Drugs Future,2010,35:471-480.)、MMAE的IC 50约为10 -11-10 -9mol/L)，因此如果出现脱靶及药物提前释放等情况，则对患者存在极大的风险。FDA对收到的20个ADC研究中新药(invesligational new drugs，IND)进行汇总分析，发现ADC药物在动物中表现出来的毒性主要为造血系统毒性、肝毒性和生殖毒性，部分还存在皮肤毒性和肾毒性，其中造血系统、肝和生殖系统的毒性与小分子细胞毒药物直接相关(文献3：Saber H，Leighton JK.An FDA oncology analysis of antibody-drug conjugates[J].Regul Toxicol Pharmacol，2015，71(3)：444-452)。而如果选择毒性偏低的小分子药物，则往往因为有效荷载的小分子药物数量过低，产生的药效过低进而导致临床失败。
发明内容
为了解决上述问题，本发明提供了一种能够偶联更多个活性单元(Drug)的新型抗体药物偶联物，本发明的技术方案如下所示：
本发明提供了一种如式(Ⅰ)－(Ⅳ)所示的抗体药物偶联物，
Figure PCTCN2019112663-appb-000001
Figure PCTCN2019112663-appb-000002
其中：
A为任一抗体或其功能性结合片段；
B 1、B 2、B、……、B n为半胱氨酸残基或半胱氨酸衍生物残基，可相同也可以不同，B 1与B 2、B 2与B 3、……、B n-1与B n通过脱水缩合形成的肽键连接；
L 1、L 2、L 3、L 4、……、L n+1为任一连接单元且相互独立，可相同也可以不同，所述L 1与B 1中氨基端共价相连，所述L 2与B 1、L 3与B 2、L 4与B 3、……、L n+1与B n是通过巯基共价连接，所述L 2与D 1、L 3与D 2、L 4与D 3、……、L n+1与D n共价相连；
D 1、D 2、D 3、......、D n是任一活性药物单元且相互独立，可相同也可以不同；
Z为任一基团，共价连接于式(Ⅰ)中B 1的羰基上或式(Ⅱ)中B 2的羰基上或式(Ⅲ)中B 3的羰基上或式(Ⅳ)中B n的羰基上；
n为大于或等于4的整数，代表连接活性药物单元支链的数量；
m选自1、2、3、4、5、6、7、8。
进一步的，所述的B 1、B 2、B 3、......、B n的结构分别如式(V)所示：
Figure PCTCN2019112663-appb-000003
其中，p选自1、2、3、4、5、6、7、8。
进一步的，所述的Z选自：
OH、SH、NH 2、
Figure PCTCN2019112663-appb-000004
Figure PCTCN2019112663-appb-000005
进一步的，所述的抗体药物偶联物具有式(VI-1)-(VI-5)所示的结构：
Figure PCTCN2019112663-appb-000006
Figure PCTCN2019112663-appb-000007
其中：
A为任一抗体或其功能性结合片段；
L 1、L 2、L 3、L 4、L 5为任一连接单元且相互独立，可相同也可以不同；
D 1、D 2、D 3、D 4是任一活性药物单元且相互独立，可相同也可以不同；
m选自1、2、3、4、5、6、7、8。
进一步的，所述L 1与抗体上氨基残基或巯基残基共价连接；优选的，所述L 1与抗体上的巯基残基共价连接；更优选的，所述L 1与抗体上的链间二硫健打开后形成的巯基残基共价连接。
进一步的，所述L 1、L 2，L 3，L 4、……、L n+1为可裂解连接子、可裂解连接子组合或不可裂解连接子。
更进一步的，所述的可裂解的连接子包括肽单元和多硫键，所述的肽单元包含2-20个氨基酸，优选的所述的肽单元选自由-缬氨酸-瓜氨酸-(-Val-Cit-)、-甘氨酸-甘氨酸-苯丙氨酸-甘氨酸-(-Gly-Gly-Phe-Gly-)、-缬氨酸-丙氨酸-(-Val-Ala-)、-缬氨酸-赖氨酸-(-Val-Lys-)、-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-精氨酸-(-Phe-Arg-)及它们的组合；所述的多硫键包含2-8个硫原子，优选的所述的多硫键选自于二硫键(-S-S-)、三硫键(-S-S-S-)、四硫键(-S-S-S-S-)。
在某些具体的实施例中，所述的L 1可选自以下结构：
Figure PCTCN2019112663-appb-000008
Figure PCTCN2019112663-appb-000009
Figure PCTCN2019112663-appb-000010
Figure PCTCN2019112663-appb-000011
在某些具体的实施例中，所述的L 2，L 3，L 4、……、L n+1可选自以下结构：
Figure PCTCN2019112663-appb-000012
Figure PCTCN2019112663-appb-000013
Figure PCTCN2019112663-appb-000014
进一步的，所述的抗体或其功能性结合片段包括单克隆抗体、多克隆抗体、抗体片段、Fab、Fab'、Fab'-SH、F(ab')2、Fv、单链Fv(“scFv”)、双抗体、线性抗体、双特异性抗体、多特异性抗体、嵌合抗体、人源化抗体、全人抗体、或包含抗体的抗原结合部分的融合蛋白；优选的，所述的抗体为人源化单克隆抗体或全人抗体。
更进一步的，所述的抗体为IgG抗体或其功能性结合片段，更进一步优选的，所述的抗体为IgG 1、IgG 2、IgG 3、IgG 4。
进一步的，所述的活性药物单元为细胞毒分子、细胞分化因子、干细胞营养因子、类固醇类药物、治疗自身免疫疾病的药物、抗炎症药物或治疗传染性疾病的药物。
更进一步的，所述细胞毒分子包括但不限于微管蛋白抑制剂或DNA损伤剂；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类(calicheamicin)、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD、喜树碱(camptothecins)及喜树碱类衍生物、SN-38；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；进一步优选的，所述细胞毒分子包括以下分子：
Figure PCTCN2019112663-appb-000015
Figure PCTCN2019112663-appb-000016
Figure PCTCN2019112663-appb-000017
进一步的，所述抗体药物偶联物结构如下所示：
Figure PCTCN2019112663-appb-000018
Figure PCTCN2019112663-appb-000019
Figure PCTCN2019112663-appb-000020
Figure PCTCN2019112663-appb-000021
Figure PCTCN2019112663-appb-000022
Figure PCTCN2019112663-appb-000023
Figure PCTCN2019112663-appb-000024
Figure PCTCN2019112663-appb-000025
其中：
A为任一抗体或其功能性结合片段；
m选自1、2、3、4、5、6、7、8。
本发明还提供了一种药物组合物，其包含有效量的上述任一项所述抗体药物偶联物或其药学上可接受的盐或溶剂合物，以及药学上可接受的辅料。
本发明还提供了上述任一项所述的抗体药物偶联物在制备治疗癌症药物中的用途。
本发明的抗体药物偶联物通过一个或多个半胱氨酸残基或半胱氨酸衍生物残基作为药物连接载体，在抗体的有限连接位点上同时偶联一个或多个药物，从而可以容易的制备获得药物载量(payload)更高的抗体药物偶联物。理论上，由于可以制备更高药物载量的ADC，其可选择偶联的药物范围也更大，从而可以选择毒性较低的药物制备ADC产品，从而获得治疗窗口更大的ADC产品。此外，由于可在一个连接位点偶联多个药物分子，因此在制备相同DAR值的抗体药物偶联物情况下，通过本发明方法所获得的ADC产品均一性也更好。并且在生产中所需要使用的抗体用量也能得以大幅降低，从而有效的降低了生产成本。另外，在实验中，我们还惊奇的发现，采用本发明方法所制备的抗体药物偶联物，与同一位点仅能连接一个药物分子的抗体药物偶联物相比，在偶联的药物分子总量大幅降低的情况下，对肿瘤细胞仍能起到相同的抑制或杀伤效果。
具体实施方式
[定义]
与说明书的各方面相关的各种术语在说明书和权利要求书中通篇使用。除非另外指明，否则此类术语被赋予本领域的普通含义。其它具体定 义的术语应按照与本文所提供的定义相符的方式理解。
如本文所用，术语“一个”和“一种”和“所述”是按照标准惯例使用的并且意指一个或多个，除非上下文另有指示。因此例如，对“一种抗体药物偶联物”的提及包括两个或更多个抗体药物偶联物的组合等等。
应当理解，无论何处在本文中用语言“包含”描述方面，除此之外还提供了以“由......组成”和/或“基本上由......组成”描述的类似方面。
尽管本发明的广义范围所示的数字范围和参数近似值，但是具体实施例中所示的数值尽可能准确的进行记载。然而，任何数值本来就必然含有一定的误差，其是由它们各自的测量中存在的标准偏差所致。另外，本文公开的所有范围应理解为涵盖其中包含的任何和所有子范围。例如记载的“1至10”的范围应认为包含最小值1和最大值10之间(包含端点)的任何和所有子范围；也就是说，所有以最小值1或更大起始的子范围，例如1至6.1，以及以最大值10或更小终止的子范围，例如5.5至10。另外，任何称为“并入本文”的参考文献应理解为以其整体并入。
本发明中所用的
Figure PCTCN2019112663-appb-000026
是指含
Figure PCTCN2019112663-appb-000027
的基团在此处与其他基团通过化学键连接。
本发明中的连接单元、连接子可以互换使用；本发明中的活性单元、药物、毒物可以互换使用。
本发明中的术语“抗原”是指引发免疫应答或能够被抗体或抗原结合分子结合的任何分子。免疫应答可以涉及抗体产生或特异性免疫活性细胞的活化或这两者。本领域技术人员将容易理解任何大分子，包括几乎所有蛋白质或肽均可以充当抗原。通常，抗原可以内源性表达，即由基因组DNA表达，或者其可以重组表达，或者其可以化学合成。本发明涉及的“抗原”特指那些肿瘤相关抗原，这些肿瘤相关抗原是业内所熟知的，可以通过业内熟知的抗体制备方法和信息来制备。为了开发可用于癌症诊断与治疗的有效的细胞水平目标物，研究人员力图找寻跨膜或其他肿瘤相关多肽。这些目标物能够特异性地表达在一种或多种癌症细胞表面，而在一种或多种非癌细胞表面表达很少或不表达。通常，相对于非癌细胞表面而言,这样的肿瘤相关多肽在癌细胞表面更加过度表达。确认这样的肿瘤相关因子，可大大提高基于抗体治疗癌症的专一靶向特性。肿瘤相关抗原包括但不局限于，以下列出的肿瘤相关抗原(1)-(36)。为方便起见，为业内所熟知的抗原相关信息标示如下，包括名称，其它名称，基因库登录号。 与肿瘤相关抗原对应的核酸和蛋白序列可参见公开数据库，例如Genbank。抗体靶向对应的肿瘤相关抗原包括所有的氨基酸序列变种和同种，与实际确认的序列具有至少70％，80％，85％，90％，或95％的同源性，或者具备与引用的肿瘤相关抗原序列具有完全一致的生物性质和特征。肿瘤相关抗原(1)-(37)：
(1)BMPR1B(骨形态发生蛋白受体-IB型，Genbank登录号NM_001203)；
(2)E16(LAT1，SLC7A5，Genbank登录号NM_003486)；
(3)STEAP1(六次跨膜的前列腺上皮抗原，Genbank登录号NM_012449)；
(4)0772P(CA125，MUC16，Genbank登录号AF361486)；
(5)MPF(MPF，MSLN，SMR、巨核细胞强化因子，间皮素，Genbank登录号NM_005823)；
(6)Napi3b(NAPI-3B，NPTIIb，SLC34A2，溶质载运体家族34(磷酸钠)成员2，II型钠依赖型磷酸转运子3b，Genbank登录号NM_006424)；
(7)Sema 5b(FLJ10372，KIAA1445，Mm.42015，SEMA5B，SEMAG，脑信号蛋白5b Hlog，sema结构域，七个血小板反应蛋白重复序列(1型和类1型)，跨膜结构域(TM)和短胞质结构域，(脑信号蛋白)5B，Genbank登录号AB040878)；
(8)PSCA hlg(2700050C12Rik,C530008016Rik,RIKEN cDNA 2700050C12,RIKEN cDNA 2700050C12基因，Genbank登录号AY358628)；
(9)ETBR(内皮缩血管肽B型受体，Genbank登录号AY275463)；
(10)MSG783(RNF124,假拟蛋白FLJ20315,Genbank登录号NM_017763)；
(11)STEAP2(HGNC_8639,IPCA-1,PCANAP1,STAMP1,STEAP2,STMP,前列腺癌相关基因1，前列腺癌相关蛋白1，六次跨膜的前列腺上皮抗原2，六次跨膜的前列腺蛋白，Genbank登录号AF455138)；
(12)TrpM4(BR22450,FLJ20041,TRPM4,TRPM4B,瞬时受体电势阳离子通道，亚家族M，成员4，Genbank登录号NM_017636)；
(13)CRIPTO(CR,CR1,CRGF,CRIPTO,TDGF1,畸胎瘤衍生生长因子，Genbank登录号NP_003203或NM_003212)；
(14)CD21(CR2(补体受体2)或C3DR(C3d/EB病毒受体)或Hs.73792,Genbank登录号M26004)；
(15)CD79b(CD79B,CD79β,IGb(免疫球蛋白相关β),B29,Genbank登录号NM_000626)；
(16)FcRH2(IFGP4,IRTA4,SPAP1A(含有磷酸酶锚定蛋白1a的SH2结构域),SPAP1B,SPAP1C,Genbank登录号NM_030764)；
(17)HER2(ErbB2,Genbank登录号M11730)；
(18)NCA(CEACAM6,Genbank登录号M18728)；
(19)MDP(DPEP1,Genbank登录号BC017023)；
(20)IL20Rα(IL20Ra,ZCYTOR7,Genbank登录号AF184971)；
(21)Brevican(BCAN,BEHAB,Genbank登录号AF229053)；
(22)EphB2R(DRT,ERK,Hek5,EPHT3,Tyro5,Genbank登录号NM_004442)；
(23)ASLG659(B7h,Genbank登录号AX092328)；
(24)PSCA(前列腺干细胞抗原前体，Genbank登录号AJ297436)；
(25)GEDA(Genbank登录号AY260763)；
(26)BAFF-R(B细胞活化因子受体，BLyS受体3，BR3,Genbank登录号AF116456)；
(27)CD22(B细胞受体CD22-B同种型,Genbank登录号AK026467)；
(28)CD79a(CD79A,CD79α,免疫球蛋白相关α，能够与Igβ(CD79B)发生共价作用并在表面与Ig M分子形成复合物，转导参与B细胞分化信号的B细胞特异性蛋白，Genbank登录号NP_001774.1)；
(29)CXCR5(伯基特氏(Burkitt’s)淋巴瘤受体1，被CXCL13趋化因子活化的G蛋白偶联受体，在淋巴细胞迁移和体液防御中发挥作用，在HIV-2感染以及可能在艾滋病，淋巴瘤，骨髓瘤，和白血病中发挥作用，Genbank登录号NP_001701.1)；
(30)HLA-DOB(MHCII类分子的Beta亚基(Ia抗原)，其结合肽并将 其呈递到CD4+T淋巴小细胞，Genbank登录号NP_002111.1)；
(31)P2X5(嘌呤受体P2X配体门控离子通道5，由胞外ATP门控的离子通道，可能涉及突触传递和神经新生，其缺陷可能导致特发性逼尿肌不稳定的病理生理状况，Genbank登录号NP_002552.2)；
(32)CD72(B细胞分化抗原CD72,Lyb-2,Genbank登录号NP_001773.1)；
(33)LY64(淋巴细胞抗原64(RP105),富含亮氨酸重复的I型膜蛋白(LRR)家族，调节B细胞活化和凋亡，功能的丧失与系统性红斑狼疮患者疾病活动增加有关，Genbank登录号NP_005573.1)；
(34)FcRH1(Fc受体样蛋白1，推定的免疫球蛋白Fc结构域受体，包含C2型类Ig样和ITAM结构域，可能在B淋巴细胞分化中起作用，Genbank登录号NP_443170.1)；
(35)IRTA2(易位相关免疫球蛋白超家族受体2，推定的免疫受体，可能在B细胞发育和淋巴瘤产生中起作用；由易位导致的基因失调发生在一些B细胞恶性病上，Genbank登录号NP_112571.1)；
(36)TENB2(推定的跨膜蛋白聚糖，与生长因子的EGF/调蛋白(heregulin)家族和卵泡抑素(follistatin)相关，Genbank登录号AF179274)；
(37)其他相关的抗原。
本发明中的术语“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体)、和抗体片段。通常，抗体可以包含通过二硫键相互连接的至少两条重链和两条轻链，或其抗原结合分子。每条重链包含重链可变区和重链恒定区。重链恒定区包含三个恒定结构域：CH1、CH2和CH3。每条轻链包含轻链可变区和轻链恒定区。轻链恒定区包含一个恒定结构域：CL。重链可变区和轻链可变区可以进一步细分为高变的区域，称为互补决定区(CDR)，散布着更保守的区域，称为框架区(FR)。每个重链可变区和轻链可变区包含三个CDR和四个FR，以下列顺序从氨基末端到羧基末端排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。Ab的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，包括免疫系统的各种细胞(例如效应细胞)和经典补体系统的第一组分(C1q)。除非另有说明，本发明中的术语“抗体”还涵盖完整免疫球蛋白或其抗原结合部分，其与完整抗体竞争特异性结 合。抗原结合部分可以通过重组DNA技术或通过完整抗体的酶促或化学切割来产生。抗原结合部分尤其包括Fab、Fab’、F(ab’)2、Fv、结构域抗体(dAb)、包括互补决定区(CDR)的片段、单链抗体(scFv)、嵌合抗体、二价抗体、三价抗体、四价抗体和含有足以赋予与多肽结合的特异性抗原的至少一部分免疫球蛋白的多肽。本发明中的术语“抗体”还包括天然存在的和非天然存在的(重组产生的)抗体两者、人和非人抗体、单特异性抗体、多特异性抗体(包括双特异性抗体)、免疫球蛋白、合成抗体、包含两个重链和两个轻链分子的四聚体抗体、抗体轻链单体、抗体重链单体、抗体轻链二聚体、抗体重链二聚体、抗体轻链-抗体重链对、胞内抗体(参见，例如Stocks,(2004)Drug DiscoveryToday 9(22):960-66)、抗体融合物(该术语涵盖抗体-药物缀合物并且其在本文有时称为“抗体缀合物”)、异缀合抗体(heteroconjugate antibody)、单结构域抗体、单价抗体、单链抗体或单链Fv(scFv)、骆驼源化抗体、亲和体(affybody)、Fab片段、F(ab’)2片段、二硫键连接的Fv(sdFv)、抗独特型(抗Id)抗体(包括例如，抗-抗Id抗体)、微抗体、结构域抗体、合成抗体(本文有时称为“抗体模拟物”)及其抗原结合片段。
本发明中的术语“功能性片段”是指抗体片段由其来源抗体的重或轻可变链的部分序列构成或者包含它们，所述部分序列足以保留与其来源抗体相同的结合特异性和充分的亲和力，优选至少等于其来源抗体亲和力的1/100，在更优选方式中至少等于1/10。这种功能片段将包含最少5个氨基酸，优选其来源的抗体序列的10、15、25、50和100个连续氨基酸。
术语“人源化抗体”是指一种抗体，其包含来源于非人源抗体的CDR区、并且该抗体分子的其他部分来源于一种(或几种)人抗体。而且，为了保留结合亲和力，可以修饰骨架(称为FR)区段的一些残基(Jones et al.，Nature，321：522-525，1986；Verhoeyen et al.，Science，239：1534-1536，1988；Riechmann et al.，Nature，332：323-327，1988)。通过本领域技术人员已知的技术可以制备根据本发明的人源化抗体或其片段(例如，描述于文件Singer et al.，J.Immun.150：2844-2857，1992；Mountain et al.，Biotechnol.Genet.Eng.Rev.，10：1-142，1992；或Bebbington et al.，Bio/Technology，10：169-175，1992)。
术语“嵌合抗体”是指以下抗体，其中的可变区序列来自一物种而恒定区序列来自另一物种，例如可变区序列来自小鼠抗体而恒定区序列来自人抗体的抗体。通过使用基因重组技术可以制备根据本发明的嵌合抗体或其 片段。例如，所述嵌合抗体可以通过克隆重组DNA来生产，所述重组DNA包含启动子和编码根据本发明的非人尤其是鼠单克隆抗体可变区的序列、以及编码人抗体恒定区的序列。由这种重组基因编码的本发明嵌合抗体将是，例如，鼠-人嵌合体，该抗体的特异性由来源于鼠DNA的可变区确定，并且其同种型由来源于人DNA的恒定区来确定。对于制备嵌合抗体的方法，例如，可以参考文件Verhoeyn et al.(BioEssays，8：74，1988)。
术语“单克隆抗体”系指具有单一分子组成的抗体分子的制备物。单克隆抗体组合物显示出对于特定表位的单一结合特异性和亲和性。
在某些具体的实施例中，本发明涉及的抗体包括但不限于以下：莫罗单抗-CD3、阿昔单抗、利妥昔单抗、达利珠单抗、帕利珠单抗、英夫利昔单抗、曲妥珠单抗、依那西普、巴利昔单抗、吉妥珠单抗、阿仑单抗、替伊莫单抗、阿达木单抗、阿法赛特、奥马珠单抗、依法利珠单抗、托西莫单抗、西妥昔单抗、ABT-806、贝伐单抗、那他珠单抗、雷珠单抗、帕尼单抗、依库丽单抗、利纳西普、赛妥珠单抗、罗米司亭、AMG-531、戈利木单抗、优特克单抗、ABT-874、贝拉西普、贝利木单抗、阿塞西普、抗CD20抗体、卡纳单抗、托珠单抗、阿特珠单抗、美泊利单抗、帕妥珠单抗、HuMax CD20、曲美木单抗、替西木单抗、伊匹单抗、IDEC-114、英妥珠单抗、HuMax EGFR、阿柏西普、HuMax-CD4、特普利珠单抗、奥特西珠单抗、卡妥索单抗、抗EpCAM抗体IGN101、阿德木单抗、奥戈伏单抗、地妥西单抗、吉瑞妥昔单抗、地诺单抗、巴匹珠单抗、莫维珠单抗、依福谷单抗、瑞西巴库、LY2469298和维妥珠单抗。
本发明中的术语“连接单元”是指一种具有双官能团或多官能团的分子，可分别与蛋白/抗体分子和药物分子反应，因此做为一种“桥梁”将蛋白/抗体与药物分子连接起来。按照在细胞内药物释放的机制，“连接子”或“抗体药物偶联物的连接子”可被分为两类：不可断裂连接子(non-cleavable linker)和可断裂连接子(cleavable linker)。
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，释放出由小分子药物，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)。常见的不可断裂连接子例如MC连接子和MCC连接子等：
Figure PCTCN2019112663-appb-000028
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。典型的酶不稳定连接子例如vc连接子等。
Figure PCTCN2019112663-appb-000029
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。
Figure PCTCN2019112663-appb-000030
本发明中的术语“活性单元”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。具体的，所述的药物包括但不限于细胞毒性药物、细胞分化因子、干细胞营养因子、类固醇类药物、治疗自身免疫疾病的药物、抗炎症药物或传染性疾病的药物。更具体的，所述的药物包括但不限于微管蛋白抑制剂或者DNA、RNA损伤剂。优选的，本发明所涉及的活性单元包括但不限于以下：
(a)厄洛替尼、硼替佐米、氟维司群、索坦、来曲唑、甲磺酸伊马替尼、PTK787/ZK222584、奥沙利铂、5-氟尿嘧啶、亚叶酸、雷帕霉素、拉帕替尼、洛那法尼、索拉非尼、吉非替尼、AG1478、AG1571、噻替派、环磷酰胺、白消安、英丙舒凡、哌泊舒凡、苯并多巴、卡巴醌、美妥替哌、乌瑞替派、乙烯亚胺、六甲蜜胺、三乙烯三聚氰胺、三乙烯磷酰胺、三乙烯硫代磷酰胺、三羟甲基三聚氰胺、泡番荔枝辛、泡番荔枝辛酮、喜树碱、拓扑替康、苔藓抑素、卡利斯汀、CC-1065、阿多来新、卡折来新、比折来新、念珠藻素1、念珠藻素8、多拉司他汀、多卡米星、KW-2189、CB1-TM1、五加素、水鬼蕉碱、匍枝珊瑚醇、海绵抑制素、苯丁酸氮芥、萘氮芥、氯磷酰胺、雌莫司汀、异环磷酰胺、二氯甲基二乙胺、美法仑、 新恩比兴、苯芥胆甾醇、泼尼莫司汀、曲磷胺、尿嘧啶氮芥、卡莫司汀、氯脲霉素、福莫司汀、洛莫司汀、尼莫司汀、雷莫司汀、卡里奇霉素、卡里奇霉素γ1、卡里奇霉素ω1、达内霉素、达内霉素A、氯膦酸盐、埃斯培拉霉素、新制癌菌素发色团、阿克拉霉素、放线菌素、安曲霉素、重氮丝氨酸、博来霉素、放线菌素C、卡比柔星、洋红霉素、嗜癌素、色霉素、更生霉素、柔红霉素、地托比星、6-重氮-5-氧代-L-正亮氨酸、阿霉素、吗啉代阿霉素、氰基吗啉代阿霉素、2-吡咯啉代-阿霉素、脂质体阿霉素、脱氧阿霉素、表柔比星、依索比星、麻西罗霉素、丝裂霉素C、霉酚酸、诺加霉素、橄榄霉素、派来霉素、泊非霉素、嘌呤霉素、三铁阿霉素、罗多比星、链霉黑素、链佐星、杀结核菌素、乌苯美司、净司他汀、佐柔比星、5-氟尿嘧啶、二甲叶酸、甲氨蝶呤、蝶罗呤、三甲曲沙、氟达拉滨、6-巯基嘌呤、硫咪嘌呤、硫鸟嘌呤、安西他滨、阿扎胞苷、6-氮尿苷、卡莫氟、阿糖胞苷、二脱氧尿苷、去氧氟尿苷、依诺他滨、氟尿苷、卡鲁睾酮、丙酸屈他雄酮、环硫雄醇、美雄烷、睾内酯、氨鲁米特、米托坦、曲洛司坦、亚叶酸、醋葡醛内酯、醛磷酰胺糖苷、氨基乙酰丙酸、恩尿嘧啶、安吖啶、巴斯布西、比生群、依达曲沙、地磷酰胺、秋水仙胺、地吖醌、依氟鸟氨酸、依利醋铵、依托格鲁、硝酸镓、羟基脲、香菇多糖、氯尼达明、美登素、安丝菌素、米托胍腙、米托蒽醌、莫哌达醇、二胺硝吖啶、喷司他丁、蛋氨氮芥、吡柔比星、洛索蒽醌、2-乙基酰肼、甲基苄肼、多糖-k、雷佐生、根霉素、西佐糖、锗螺胺、细交链孢菌酮酸、三亚胺醌、2,2',2”-三氯三乙胺、T-2毒素、抚抱菌素A、杆孢菌素A、以及蛇形菌素、乌拉坦、长春地辛、达卡巴嗪、甘露醇氮芥、二溴甘露醇、二溴卫矛醇、哌泊溴烷、干胞嘧啶、阿拉伯糖苷、环磷酰胺、噻替派、紫杉醇、紫杉醇的白蛋白工程纳米粒子制剂、多烯紫杉醇、苯丁酸氮芥、吉西他滨、6-硫鸟嘌呤、巯基嘌呤、顺铂、卡铂、长春花碱、铂、依托泊苷、异环磷酰胺、米托蒽醌、长春新碱、长春瑞滨、米托蒽醌、替尼泊苷、依达曲沙、道诺霉素、氨基喋呤、希罗达、伊班膦酸盐、CPT-11、拓扑异构酶抑制剂RFS 2000、二氟甲基鸟氨酸、视黄酸、卡培他滨，或任何前述的药学上可接受的盐、溶剂化物或酸；
(b)单核因子、淋巴因子、传统多肽激素、甲状旁腺激素、甲状腺素、松弛素、松弛素原、糖蛋白激素、促卵泡激素、促甲状腺激素、黄体生成素、肝生长因子、成纤维细胞生长因子、催乳素、胎盘催乳素、肿瘤坏死因子-α、肿瘤坏死因子-β、缪勒抑制物质、小鼠促性腺激素相关肽、抑制素、激活素、血管内皮生长因子、血小板生成素、红细胞生成素、骨诱 导因子、干扰素、干扰素-α、干扰素-β、干扰素-γ、集落刺激因子(“CSF”)、巨噬细胞-CSF、粒细胞-巨噬细胞-CSF、粒细胞-CSF、白介素(IL)、IL-1、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、肿瘤坏死因子、TNF-α、TNF-β、多肽因子、LIF、kit配体，或任何前述的组合；
(c)白喉毒素、肉毒毒素、破伤风毒素、痢疾毒素、霍乱毒素、鹅膏蕈碱、鹅膏蕈碱衍生物、α-鹅膏蕈碱、吡咯并苯并二氮杂卓、吡咯并苯并二氮杂卓衍生物、河豚毒素、短裸甲藻毒素、雪卡毒素、篦麻毒素、AM毒素、微管蛋白裂解素、格尔德霉素、美登素类化合物、卡里奇霉素、柔红霉素、阿霉素、甲氨蝶呤、长春地辛、SG2285、多拉司他汀、多拉司他汀类似物、奥瑞他汀、念珠藻素、喜树碱、喜树碱衍生物和代谢物、根霉素、根霉素衍生物、CC-1065、CC-1065类似物或衍生物、倍癌霉素、烯二炔抗生素、埃斯培拉霉素、埃博霉素、阿咗那非得、阿普立定、类毒素，或任何前述的组合；
(d)亲和配体，其中所述亲和配体是底物、抑制剂、刺激剂、神经递质、放射性同位素，或任何前述的组合；
(e)放射性标记物、32P、35S、荧光染料、电子致密试剂、酶、生物素、链霉亲和素、洋地黄毒苷、半抗原、免疫原性蛋白质、具有与靶标互补的序列的核酸分子，或任何前述的组合；
(f)免疫调节化合物、抗癌剂、抗病毒剂、抗菌剂、抗真菌剂和抗寄生虫剂，或任何前述的组合；
(g)他莫昔芬、雷洛昔芬、屈洛昔芬、4-羟基他莫昔芬、曲沃昔芬、那洛昔芬、LY117018、奥那司酮或托瑞米芬；
(h)4(5)-咪唑、氨鲁米特、醋酸甲地孕酮、依西美坦、来曲唑或阿那曲唑；
(i)氟他胺、尼鲁米特、比卡鲁胺、亮丙瑞林、戈舍瑞林或曲沙他滨；
(j)芳香酶抑制剂；
(k)蛋白激酶抑制剂；
(l)脂质激酶抑制剂；
(m)反义寡核苷酸；
(n)核酶；
(o)疫苗；和
(p)抗血管生成剂。
在本发明的一些实施方案中，所述“活性单元”选自：美坦生类化合物、V-ATP酶抑制剂、促凋亡剂、Be12抑制剂、McL1抑制剂、HSP90抑制剂、IAP抑制剂、mTOr抑制剂、微管稳定剂、微管去稳定剂、阿里他汀(auristatin)、多拉司他汀(dolastatin)、MetAP(甲硫氨酸氨肽酶)、蛋白质CRMl的核输出抑制剂、DPPIV抑制剂、蛋白酶体抑制剂、线粒体中磷酰基转移反应的抑制剂、蛋白质合成抑制剂、激酶抑制剂、CDK2抑制剂、CDK9抑制剂、驱动蛋白抑制剂、HDAC抑制剂、DNA损伤剂、DNA烷基化剂、DNA嵌入剂、DNA小沟结合剂、DHFR抑制剂、以及海兔毒素肽、维生素A前体、叶酸。
在本发明的一些实施方案中，所述“活性单元”为细胞毒性药物(例如抗代谢药、抗肿瘤抗生素、生物碱)、免疫增强剂或放射性同位素。优选地，所述药物可选自瓢菌素(amanitins)、蒽环类物(anthracyclines)、浆果赤霉素(baccatins)、喜碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱(colchicines)、秋水树仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、或长春花生物碱(vinca alkaloids)。更优选地，所述“药物”可选自MMAD(单甲基澳瑞他汀D,Monomethyl auristatin D)及其衍生物、MMAE(单甲基澳瑞他汀E,Monomethyl auristatin E)及其衍生物、MMAF(单甲基澳瑞他汀F,Monomethyl auristatin F)及其衍生物、美登素类衍生物DM1(Mertansine derivative M1)、美登素类衍生物DM4(Mertansine derivative M4)、倍癌霉素(Duocarmycine)及其衍生物、卡其霉素(Calicheamicin)及其衍生物、PBDA(Pyrrolobenzodiazepines)、阿霉素(Doxorubicin)、长春花生物碱(Vinca Alkaloids)、Metrotrexate、长春花碱(Vinblastine)、道诺霉素(Daunorubicin)及其衍生物、微管蛋白裂解素(tubulysins)及其衍生物。
在某些特定的实施例中，所述的“活性单元”是美登素(maytansine) 或类美登素(maytansinoids)。美登素化合物通过抑制微管蛋白的微管形成来抑制细胞增殖(Science 1975，189，1002-1005；US 5208020)。类美登素是美登素的衍生物。美登素和类美登素都具有高效的细胞毒性，但是它们在癌症治疗的临床应用上具有很大的局限性，这主要是源于此类分子对肿瘤的低选择性。但是，这种高细胞毒性促使它们成为抗体药物偶联物的首选药物部分。以下列出了美登素，类美登素，以及在抗体药物偶联物应用中经常利用的三个类美登素分子结构。
Figure PCTCN2019112663-appb-000031
合成类美登素的主要原料是美登醇(maytansinol)，主要由安丝菌素(ansamitocins)水解得到。安丝菌素可以通过发酵的方式制备。安丝菌素衍生物(WO 2012/061590)和丙氨酰基美登醇(US 2012/0121615)据报道也可作为抗体药物偶联物的药物“弹头”。
Figure PCTCN2019112663-appb-000032
在某些特定的实施例中，所述的“活性单元”是耳抑素肽类(auristatins)药物。耳抑素肽类药物是海兔毒素10(dolastatin 10)的类似物，而后者是从海洋软体动物海兔体内分离出来的具有生物活性的多肽(US 7498298)。海兔毒素10通过结合微管蛋白(与长春新碱同样的结合区域)而抑制微管蛋白聚合。海兔毒素10，耳抑素肽PE，耳抑素肽E都是线性多肽，含有四个氨基酸(其中三个氨基酸是海兔毒素类化合物所独有的)和C-端酰胺基团。两个代表性的耳抑素肽类化合物，单甲基耳抑素肽E(MMAE)和单甲基耳抑素肽F(MMAF)，都是抗体药物偶联物的首选药物部分。
Figure PCTCN2019112663-appb-000033
在某些特定的实施例中，所述的“活性单元”是是Tubulysins类药物。Tubulysins是从粘细菌中提取出来的一类天然产物，能有效抑制微管蛋白的聚合，因此具有抗细胞有丝分裂的活性，其中Tubulysin D的活性最好。Tubulysin D是一个复杂的四肽化合物，其结构中含有O-酰基/N,O-缩醛官能团，因此在酸性和碱性条件下都不稳定。US2011/0021568和US2013/0224228分别披露了一系列tubulysin的类似物，其结构中去除了以上不稳定官能团，同时又具有高细胞活性。
Figure PCTCN2019112663-appb-000034
在某些特定的实施例中，所述的“活性单元”是卡奇霉素类(calichemicins)药物。卡奇霉素类药物是抗肿瘤抗生素，通过结合DNA小沟并促使特定位点双螺旋DNA断裂，而导致细胞凋亡。卡奇霉素类药物具有体外亚皮克摩尔级别的高活性，但是它们的低治疗指数排除了临床应用前景。然而这种高活性却是它们成为抗体药物偶联物的理想候选药物(如吉妥单抗和Inotuzumab Ozogamicin)。
Figure PCTCN2019112663-appb-000035
在某些特定的实施例中，所述的“活性单元”是阿霉素类(doxorubicins)。阿霉素能够嵌入DNA双螺旋结构从而阻断DNA复制，因此被用作化疗药物。但是由于阿霉素类较低的细胞毒性(针对人源癌细胞系，半数抑制浓度为0.1-0.2微摩尔，而用于抗体药物偶联物的细胞毒性药物活性通常为亚纳摩尔级)，导致其在抗体药物偶联物中的应用并不普遍。
Figure PCTCN2019112663-appb-000036
在某些特定的实施例中，所述的“活性单元”是苯并二吡咯类抗生素(duocarmycins,CC-1065等)和其它的环丙基吡咯吲哚-4-酮(cyclopropapyrroloind-4-one,CPI)衍生物。这类化合物是有效的DNA小沟结合-烷基化试剂。环丙苯并吲哚-4-酮(cyclopropabenzindol-4-one,CBI)类似物的化学结构更稳定，生物活性更高，而且与它们含有天然CPI烷基化亚基的父系化合物相比更容易合成。一个代表性的CBI衍生物是酚羟基保护衍生物CBI(见下图)，具有弱化的前药毒性和增强的水溶性。
Figure PCTCN2019112663-appb-000037
在某些特定的实施例中，所述的“活性单元”是吡咯并苯二氮卓类 (pyrrolo[2,1-c][1,4]benzodi-azepines，PBDs)或者PBD二聚体类(PBD dimers)。PBD是一类由链霉菌产生的天然产物，其独特特性在于能够在DNA小沟，确切是在嘌呤-鸟嘌呤-嘌呤序列处，形成非扭曲的共价加和物。应用PBD作为部分小分子策略靶向锁定DNA序列以及作为新型的抗癌和抗菌药物引起了越来越多的兴趣(Biochemistry 2008，47，11818-11829)。应用一个柔性碳链连接两个PBD单元的C8/C8’的羟基基团，所得的二聚体具有增强的生物活性(WO 2011/130616)。PBD二聚体被认为是可以产成序列选择性的DNA损伤，例如倒序的5′-Pu-GATC-Py-3′链间交联，从而导致其生物活性。这些化合物已被证明是高效的细胞毒性药物，可作为抗体药物偶联物的备选药物。
Figure PCTCN2019112663-appb-000038
在其他特定的实施例中，所述的“活性单元”是并不仅仅局限于上述提到的类别，还包括所有可用于抗体药物偶联物的药物。
本发明中的术语“药物组合物”表示组合在一起以实现某种特定目的的至少一种药物以及任选地可药用载体或辅料的组合。在某些实施方案中，所述药物组合物包括在时间和/或空间上分开的组合，只要其能够共同作用以实现本发明的目的。例如，所述药物组合物中所含的成分可以以整体施用于对象，或者分开施用于对象。当所述药物组合物中所含的成分分开地施用于对象时，所述成分可以同时或依次施用于对象。优选地，所述可药用载体是水、缓冲水溶液、等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、乙醇或聚亚烷基二醇如聚丙二醇、甘油三酯等。所用可药用载体的类型尤其依赖于根据本发明的组合物是否配制为用于口服、鼻、皮内、皮下、肌内或静脉施用。根据本发明的组合物可包含润湿剂、乳化剂或缓冲液物质作为添加剂。
本发明的药物组合物、疫苗或者药物制剂可通过任何适宜的途径施 用，例如可口服、鼻、皮内、皮下、肌内或静脉内施用。
本发明中的术语“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
[具体实施例]
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商建议的条件进行，未注明具体来源的试剂，为市场购买的常规试剂。除非另外说明，否则所有的百分数、比率、比例、或份数按重量计。
本发明中的重量体积百分比中的单位是本领域技术人员所熟知的，例如是指在100毫升的溶液中溶质的重量。
除非另行定义，文中所使用的所有专业与科学用于与本领域熟悉人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明办法中。文中所述的较佳实施方式与材料仅作示范之用。
实施例1化合物1的制备
Figure PCTCN2019112663-appb-000039
将145.4mg马来酰亚胺己酸溶于10mL DMF中，加入244.7mg HATU和226uL DIPEA，室温搅拌。将201.9mgMMAF溶于5mL DMF中，缓慢滴加至上述反应体系中，室温搅拌16h。减压蒸馏出溶剂，制备液相法纯化，得到产品(马来酰亚胺己酰-MMAF，Mc-MMAF)90.3mg，收率36％。LC-MS：(M+H) +924.8，(M-H) -923.2。
将36.1mg马来酰亚胺己酰-MMAF溶于1.5mL DMF中，加入5.6mg Cys-Cys和2.1uL DIPEA，室温搅拌3h，再加入1.1mg Cys-Cys，室温搅拌2h。加入17.7mg 6-(马来酰亚胺基)己酸琥珀酰亚胺酯和25uL DIPEA，室温搅拌15h。减压蒸馏出溶剂，制备液相法纯化，得到化合 物1 20.0mg，收率45％。LC-MS：(M+2H) 2+1134.1，(M-2H) 2-1132.2。
实施例2化合物2的制备
Figure PCTCN2019112663-appb-000040
按照实施例1的制备方法制备马来酰亚胺己酰-MMAF。将40.1mg马来酰亚胺己酰-MMAF溶于1.5mL DMF中，加入4.4mg Cys-Cys-Cys和2.1uL DIPEA，室温搅拌2h，再加入1.8mg Cys-Cys-Cys，室温反应2h，再加入0.9mg Cys-Cys-Cys，室温反应2h。加入13.5mg 6-(马来酰亚胺基)己酸琥珀酰亚胺酯和21uL DIPEA，室温搅拌15h。减压蒸馏出 溶剂，制备液相法纯化，得到化合物2 23.1mg，收率49％。LC-MS：(M+3H) 3+1648.4，(M-3H) 3-1646.3。
实施例3化合物3的制备
Figure PCTCN2019112663-appb-000041
按照实施例1的制备方法制备马来酰亚胺己酰-MMAF。将31.6mg马来酰亚胺己酰-MMAF溶于2mL DMF中。加入3.9mg Cys-Cys和2.9uL DIPEA，室温搅拌4h，再加入1.2mg Cys-Cys，室温搅拌3h，再加入0.8mg Cys-Cys，室温搅拌16h。另将12.1mg 1,3,5-三丙烯酰基六氢-1,3,5-三嗪-巯基乙酸和10.4mg TSTU溶于1.5mL DMF中，加入17uL DIPEA，室温搅拌2h后加入上述反应体系中，室温搅拌5h。减压蒸馏 出溶剂，制备液相法纯化，得到化合物3 18.4mg，收率29％。LC-MS：(M+2H) 2+1199.3，(M-2H) 2-1197.4。
实施例4化合物4的制备
Figure PCTCN2019112663-appb-000042
按照实施例1的制备方法制备马来酰亚胺己酰-MMAF。将55.0mg马来酰亚胺己酰-MMAF溶于1.5mL N,N-二甲基甲酰胺中，加入11.6mg Cys-Cys，加入4.3uL N,N-二异丙基乙胺，室温搅拌3h。加入36.1mg MP2-PNP(2-(2-Maleimidoethoxy)ethyl(4-nitrophenyl)carbonate，马来酰亚胺基乙氧基乙基-对硝基苯碳酸酯)于反应体系，再加入51uL N,N-二异丙基乙胺，室温搅拌15h。减压蒸馏出溶剂，制备液相法纯化，得到化合物4 18.0mg，收率27％。LC-MS：(M+2H) 2+1142.3，(M-2H) 2-1141.1。
实施例5化合物5的制备
Figure PCTCN2019112663-appb-000043
按照实施例1的制备方法制备马来酰亚胺己酰-MMAF。将55.0mg马来酰亚胺己酰-MMAF溶于1.5mL N,N-二甲基甲酰胺中，加入11.6mg Cys-Cys，加入4.3uL N,N-二异丙基乙胺，室温搅拌3h。加入71.0mg马来酰亚胺丙酰氨基-八聚乙二醇基丙酸琥珀酰亚胺酯(Mal-Propionamide-PEG8-propionate-OSu)于反应体系，再加入51uL N,N-二异丙基乙胺，室温搅拌15h。减压蒸馏出溶剂，制备液相法纯化，得到化合物5 19.3mg。收率28％，LC-MS：(M+2H) 2+1324.1，(M-2H) 2-1324.0。
实施例6化合物6的制备
Figure PCTCN2019112663-appb-000044
按照实施例1的制备方法制备马来酰亚胺己酰-MMAF。将63.0mg马来酰亚胺己酰-MMAF溶于2.0mL N,N-二甲基甲酰胺中，加入13.3mg Cys-Cys，加入4.8uL N,N-二异丙基乙胺，室温搅拌3h。加入86.6mg Mc-VC-PAB-PNP于反应体系，再加入58uLN,N-二异丙基乙胺，室温搅拌15h。减压蒸馏出溶剂，制备液相法纯化，得到化合物6 47.4mg，收率52％，LC-MS：(M+2H) 2+1336.0，(M-2H) 2-1334.6。
实施例7化合物7的制备
Figure PCTCN2019112663-appb-000045
按照实施例1的制备方法制备马来酰亚胺己酰-MMAF。将46.6mg 马来酰亚胺己酰-MMAF溶于2.0mL N,N-二甲基甲酰胺中，加入10.9mg Cys-Cys，加入3.6uL N,N-二异丙基乙胺，室温搅拌3h。加入84.4mg PY-MAA-VC-PAB-PNP于反应体系，再加入48uL N,N-二异丙基乙胺，室温搅拌15h。减压蒸馏出溶剂，制备液相法纯化，得到化合物7 31.0mg，收率43％，LC-MS：(M+2H) 2+1401.2，(M-2H) 2-1399.4。
实施例8抗体药物偶联物(ADC)的制备
抗体药物偶联物合成的通用方法为：
方法A：用pH＝7.4的PBS缓冲液，把抗Her-2抗体配制为10mg/mL的溶液，加入2.4摩尔当量的TCEP，振荡混匀1小时，再加入5.0摩尔当量的连接子-毒素，振荡混匀，反应1h，反应结束后，超滤除去残留的小分子，加载至疏水色谱(HIC-HPLC)进行DAR、药物分布、裸抗比例检测。
方法B：用pH＝9的硼酸-硼砂缓冲液，把抗Her-2抗体配制为10mg/mL的溶液，加入5摩尔当量的TCEP，振荡混匀1小时，再加入6.0摩尔当量的连接子-毒素，振荡混匀，反应3h，反应结束后，超滤除去残留的小分子，加载至疏水色谱(HIC-HPLC)进行DAR、药物分布、裸抗比例检测。
采用上述通用的ADC制备方法制备出如下化合物(A为任一抗体或其功能性结合片段，m为1、2、3、4、5、6、7、8)，下述化合物的：
Figure PCTCN2019112663-appb-000046
Figure PCTCN2019112663-appb-000047
实施例9 ADC对SK-BR-3肿瘤细胞株的抑制实验
消化SK-BR-3肿瘤细胞(人乳腺癌细胞)，离心收集后，进行计数，并将细胞液稀释至0.5-1.5×10 5个/mL，96孔板中每孔加入100μL该细胞悬液。37℃、5％CO 2培养箱孵育过夜；第二天加入相应9个浓度梯度的ADC药物，外加一组零浓度ADC药物的细胞对照。培养72h后采用Cell Counting Kit-8(简称CCK-8试剂盒)进行活性显色，显色后的96孔板通过酶标仪检测450nm处OD值。Prism软件通过OD值计算得出IC50值。当拟合的曲线呈“S型曲线”且R 2≥0.95时，IC50值有效可报告。
我们选取了平均DAR值为3.87的ADC-0(比较例1)、平均DAR值为7.18的ADC-0(比较例2)、平均DAR值为4.3的ADC-10(比较例3)、平均DAR值为4.29的ADC-34(比较例4)进行SK-BR-3肿瘤细胞株的抑制实验，其IC50值如表2所示：
表2 SK-BR-3肿瘤细胞株的抑制实验细胞评价
Figure PCTCN2019112663-appb-000048
*以比较例1作为基数，抗体用量变化＝(比较例X－比较例1)/比较例1；X代表相应比较例；
*以比较例1为基数，MMAF用量变化＝(比较例X－比较例1)/比较例1；X代表相应比较例；
比较例2通过提高抗体中连接的MMAF数量位点，将DAR值提高到7.18。与比较例1相比，在达到相同的效果的情况下，比较例2可以有效降低抗体使用量(即ADC中的抗体量，在不考虑合成过程抗体用量损耗的情况下，抗体的使用量等于ADC的浓度)，但MMAF使用量增加了19.3％。
比较例3在一个连接子上连接有两个MMAF，DAR值(4.3)与比较例1(3.87)相当，与比较例1相比，在达到相同的效果的情况下，比较例3有效降低抗体使用量65.9％，MMAF使用量降低24.3％。
比较例4在一个连接子上连接有三个MMAF，DAR值与比较例1相 当，与比较例1相比，在达到相同的效果的情况下，比较例4有效降低抗体使用量85.5％，MMAF使用量降低51.8％。
从以上比较数据来看，ADC-10和ADC-34表现出更高效的细胞活性，其所带来的效果并不仅仅是单纯的通过提高抗体中MMAF的数量来提升ADC的效果，还进一步反映了在携带相同MMAF数量的情况下，通过单点多弹头(细胞毒药物)的连接方式，起到了有效降低抗体使用量以及毒素使用量的效果，起到了意料不到的技术效果。并且通过单连接点多弹头的方式，还能极为有效的提高ADC产品的均一性，可以有效的促进药品药物生产、质量控制过程。
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 (16)

  1. 一种如式(Ⅰ)－(Ⅳ)所示的抗体药物偶联物：
    Figure PCTCN2019112663-appb-100001
    其中：
    A为任一抗体或其功能性结合片段；
    B 1、B 2、B 3、......、B n为半胱氨酸残基或半胱氨酸衍生物残基，可相同也可以不同，B 1与B 2、B 2与B 3、……、B n-1与B n通过脱水缩合形成的肽键连接；
    L 1、L 2、L 3、L 4、......、L n+1为任一连接单元且相互独立，可相同也 可以不同，所述L 1与B 1中氨基端共价相连，所述L 2与B 1、L 3与B 2、L 4与B 3、……、L n+1与B n是通过巯基共价连接，所述L 2与D 1、L 3与D 2、L 4与D 3、……、L n+1与D n共价相连；
    D 1、D 2、D 3、……、D n是任一活性药物单元且相互独立，可相同也可以不同；
    Z为任一基团，共价连接于式(Ⅰ)中B 1的羰基上或式(Ⅱ)中B 2的羰基上或式(Ⅲ)中B 3的羰基上或式(Ⅳ)中B n的羰基上；
    n为大于或等于4的整数，代表连接活性药物单元支链的数量；
    m选自1、2、3、4、5、6、7、8。
  2. 根据权利要求1所述的抗体药物偶联物，其特征在于，所述的B 1、B 2、B 3、......、B n的结构分别如式(V)所示：
    Figure PCTCN2019112663-appb-100002
    其中，p选自1、2、3、4、5、6、7、8。
  3. 根据权利要求1所述的抗体药物偶联物，其特征在于，所述的Z选自：OH、SH、NH 2、
    Figure PCTCN2019112663-appb-100003
    Figure PCTCN2019112663-appb-100004
  4. 根据以上权利要求任意一项所述的抗体药物偶联物，其特征在于，所述的抗体药物偶联物具有式(VI-1)-(VI-5)所示的结构：
    Figure PCTCN2019112663-appb-100005
    Figure PCTCN2019112663-appb-100006
    其中：
    A为任一抗体或其功能性结合片段；
    L 1、L 2、L 3、L 4、L 5为任一连接单元且相互独立，可相同也可以不同；
    D 1、D 2、D 3、D 4是任一活性药物单元且相互独立，可相同也可以不同；
    m选自1、2、3、4、5、6、7、8。
  5. 根据以上权利要求任意一项所述的抗体药物偶联物，其特征在于：所述L 1与抗体上氨基残基或巯基残基共价连接；优选的，所述L 1与抗体上的巯基残基共价连接；更优选的，所述L 1与抗体上的链间二硫健打开后形成的巯基残基共价连接。
  6. 根据以上权利要求任意一项所述的抗体药物偶联物，其特征在于：所述L 1、L 2，L 3，L 4、……、L n+1为可裂解连接子、可裂解连接子组合或不可裂解连接子。
  7. 根据权利要求6所述的抗体药物偶联物，其特征在于：所述的可裂解的连接子包括肽单元和多硫键，所述的肽单元包含2-20个氨基酸，优选的所述的肽单元选自由-缬氨酸-瓜氨酸-(-Val-Cit-)、-甘氨酸-甘氨酸-苯丙氨酸-甘氨酸-(-Gly-Gly-Phe-Gly-)、-缬氨酸-丙氨酸-(-Val-Ala-)、-缬氨酸-赖氨酸-(-Val-Lys-)、-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-精氨酸-(-Phe-Arg-)及它们的组合；所述的多硫键包含2-8个硫原子，优选的所述的多硫键选自于二硫键(-S-S-)、三硫键(-S-S-S-)、四硫键(-S-S-S-S-)。
  8. 根据以上权利要求任意一项所述的抗体药物偶联物，其特征在于，所述的L 1包括以下结构：
    Figure PCTCN2019112663-appb-100007
    Figure PCTCN2019112663-appb-100008
    Figure PCTCN2019112663-appb-100009
    Figure PCTCN2019112663-appb-100010
  9. 根据以上权利要求任意一项所述的抗体药物偶联物，其特征在于，所述的L 2，L 3，L 4、……、L n+1包括以下结构：
    Figure PCTCN2019112663-appb-100011
    Figure PCTCN2019112663-appb-100012
    Figure PCTCN2019112663-appb-100013
  10. 根据以上权利要求任意一项所述的抗体药物偶联物，其特征在于：所述的抗体或其功能性结合片段包括单克隆抗体、多克隆抗体、抗体片段、Fab、Fab'、Fab'-SH、F(ab')2、Fv、单链Fv(“scFv”)、双抗体、线性抗体、双特异性抗体、多特异性抗体、嵌合抗体、人源化抗体、全人抗体、或包含抗体的抗原结合部分的融合蛋白；优选的，所述的抗体为人源化单克隆抗体或全人抗体。
  11. 根据权利要求10所述的抗体药物偶联物，其特征在于：所述的抗体为IgG抗体或其功能性结合片段，进一步优选的，所述的抗体为IgG 1、IgG 2、IgG 3、IgG 4。
  12. 根据以上权利要求任意一项所述的抗体药物偶联物，其特征在于：所述的活性药物单元为细胞毒分子、细胞分化因子、干细胞营养因子、类固醇类药物、治疗自身免疫疾病的药物、抗炎症药物或治疗传染性疾病的药物。
  13. 根据权利要求12所述的抗体药物偶联物，其特征在于：所述细胞毒分子包括但不限于微管蛋白抑制剂或DNA损伤剂；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类(calicheamicin)、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD、喜树碱(camptothecins)及喜树碱类衍生物、SN-38；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；进一步优选的，所述细胞毒分子包括以下结构：
    Figure PCTCN2019112663-appb-100014
    Figure PCTCN2019112663-appb-100015
    Figure PCTCN2019112663-appb-100016
  14. 根据以上权利要求任意一项所述的抗体药物偶联物，其特征在于：所述抗体药物偶联物结构如下所示：
    Figure PCTCN2019112663-appb-100017
    Figure PCTCN2019112663-appb-100018
    Figure PCTCN2019112663-appb-100019
    Figure PCTCN2019112663-appb-100020
    Figure PCTCN2019112663-appb-100021
    Figure PCTCN2019112663-appb-100022
    Figure PCTCN2019112663-appb-100023
    Figure PCTCN2019112663-appb-100024
    其中：
    A为任一抗体或其功能性结合片段；
    m选自1、2、3、4、5、6、7、8。
  15. 一种药物组合物，包含有效量的权利要求1-14中任一项所述抗体药物偶联物或其药学上可接受的盐或溶剂合物，以及药学上可接受的辅料。
  16. 权利要求1-14中任一项所述的抗体药物偶联物在制备治疗癌症药物中的用途。
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