WO2018095422A1 - 用于抗体-药物偶联的双取代马来酰胺类连接子及其制备方法和用途 - Google Patents

用于抗体-药物偶联的双取代马来酰胺类连接子及其制备方法和用途 Download PDF

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WO2018095422A1
WO2018095422A1 PCT/CN2017/112958 CN2017112958W WO2018095422A1 WO 2018095422 A1 WO2018095422 A1 WO 2018095422A1 CN 2017112958 W CN2017112958 W CN 2017112958W WO 2018095422 A1 WO2018095422 A1 WO 2018095422A1
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Prior art keywords
compound
antibody
synthesis
group
drug conjugate
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PCT/CN2017/112958
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English (en)
French (fr)
Inventor
沈竞康
孟韬
马兰萍
王昕�
彭红丽
张永良
于霆
陈驎
杜志彦
王英
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上海青润医药科技有限公司
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Priority claimed from CN201611093699.6A external-priority patent/CN108101825B/zh
Priority claimed from CN201711169847.2A external-priority patent/CN109810039B/zh
Priority to JP2019548514A priority Critical patent/JP7058666B2/ja
Priority to CA3044898A priority patent/CA3044898C/en
Priority to ES17873634T priority patent/ES2921236T3/es
Priority to PL17873634.4T priority patent/PL3546448T3/pl
Application filed by 上海青润医药科技有限公司 filed Critical 上海青润医药科技有限公司
Priority to DK17873634.4T priority patent/DK3546448T3/da
Priority to CN201780072626.5A priority patent/CN110088086B/zh
Priority to EP17873634.4A priority patent/EP3546448B1/en
Priority to KR1020197017841A priority patent/KR102562760B1/ko
Priority to US16/464,211 priority patent/US10987430B2/en
Publication of WO2018095422A1 publication Critical patent/WO2018095422A1/zh

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    • 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
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Definitions

  • the invention relates to a novel class of disulfide bridge bridging crosslinking reagents, macromolecules, therapeutic conjugates and synthetic methods thereof. More specifically, the present invention relates to a conjugate obtained by crosslinking a cytotoxic drug and a macromolecule based on a dithiol bridge bridging reagent substituted with a maleimide, a preparation method thereof and use thereof.
  • the primary goal of the development (drug delivery) method is to specifically target drugs to cells and tissues.
  • the benefits of such treatment include avoiding the systemic physiological effects of improper delivery of such drugs to other cells and tissues, such as uninfected cells.
  • Intracellular targeting can be achieved by methods, compounds, and formulations that bioaccumulate, i.e., active metabolites, accumulate or retain in cells.
  • an antibody-drug conjugate ie, an immunoconjugate
  • a cytotoxic agent or a cytostatic agent ie, a drug that kills or inhibits tumor cells in cancer therapy
  • a cytostatic agent ie, a drug that kills or inhibits tumor cells in cancer therapy
  • Intracellular accumulation occurs in which systemic administration of these unconjugated agents also produces an unacceptable level of toxicity to normal cells in addition to the tumor cells that are attempted to be eliminated.
  • Efforts to improve the therapeutic index of ADCs ie, highest efficacy with minimal toxicity
  • ADC Antibody Drug Conjguate
  • MylotargTM first antibody-drug conjugate
  • AML acute myeloid leukemia
  • Adcetris TM (2011) and Genentech, a new drug developed by Seattle Genetics for the treatment of Hodgkin's lymphoma (HL)/recurrent anaplastic large cell lymphoma (ALCL).
  • HL Hodgkin's lymphoma
  • ALCL recurrent anaplastic large cell lymphoma
  • Antibody drug conjugates generally consist of three components: an antibody or antibody ligand, a small molecule drug, and a linker that couples the ligand to the drug.
  • highly active cytotoxic drugs are usually lysine residues attached to the surface of the ligand via a linker, or cysteine residues in the hinge region of the antibody (by the chain)
  • the partial drug-ligand ratio (DAR) is 2-4 on the partial disulfide bond reduction.
  • the large amount of lysine residues (more than 80) on the surface of the antibody and the non-selectivity of the coupling reaction lead to uncertainty in the number and location of the coupling, which in turn leads to heterogeneity of the resulting antibody drug conjugate.
  • the DAR value distribution of T-DM1 (average DAR value of 3.5) is 0-8. Again, despite the resistance There are only four pairs of interchain disulfide bonds in the body hinge region, but to achieve the optimal average DAR value (2-4), partial reduction of interchain disulfide bonds is required. Since the existing reducing agent (DTT, TCEP, etc.) cannot selectively reduce the interchain disulfide bond, the resulting conjugate is not a uniform product, and is composed of a plurality of components, and the DAR value of the main component thereof is 0, 2, 4, 6, 8, and the components corresponding to each of the specific DAR values have isomers formed due to the difference in the attachment sites.
  • the heterogeneity of the antibody drug conjugate product can result in pharmacokinetic properties, potency, and toxicity heterogeneity between the various member components. For example, components with higher DAR values are cleared faster in the body and result in higher toxicity.
  • the purpose of the fixed-point coupling of the existing antibodies by a simple chemical method is to save a lot of manpower, material and financial resources, and thus is more attractive.
  • related studies include: a coupling technology reported by Polylites Co., Ltd. CN200480019814.4; WO2014197871A2 by Igenica Biotherapeutics; CN201380025774.3 by Sorrento Medical Co., Ltd.; Shanghai New Concept Bio Patent documents such as CN201310025021.4 applied by Pharmaceutical Technology Co., Ltd.
  • R is X or ArS-
  • X is selected from the group consisting of halogen, preferably bromine or iodine;
  • Ar is selected from the group consisting of substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted 5-12 membered heteroaryl;
  • Ar is selected from the group consisting of phenyl, halobenzene, C 1 -C 4 alkylphenyl, C 1 -C 4 alkoxyphenyl, 2-pyridyl, 2-pyrimidinyl, 1-methylimidazole- 2-base,
  • W is an amine group R 1 attached to a carbonyl group, and R 1 is selected from -NH 2 , And the like; wherein: the C 1 -C 4 alkylphenyl group is further preferably a 4-methylphenyl group; and the C 1 -C 4 alkoxyphenyl group is further preferably a 4-methoxyphenyl group.
  • Ar' is selected from the group consisting of substituted or unsubstituted C 6 -C 10 arylene, substituted or unsubstituted 5-12 membered heteroarylene; preferably, Ar' is selected from substituted or unsubstituted phenylene Or pyridyl, said substituent means that the hydrogen atom on the group is substituted by one or more substituents selected from the group consisting of halogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, three Fluoromethyl, nitrile, amide, and the like.
  • L 1 is -O(CH 2 CH 2 O) n - attached to the Ar' group, wherein n is selected from any integer from 1 to 20, preferably any integer from 1 to 10.
  • the linker fragment has a structure selected from the group consisting of:
  • a second aspect of the invention provides a substituted maleic acid amide linker-drug conjugate comprising a linker fragment of formula Ia according to the first aspect of the invention, and a pharmaceutically acceptable salt or solvent thereof
  • the structure is as shown in the formula Ib:
  • R, Ar', and L 1 have the same definitions as above;
  • L 2 is a chemical bond or an AA-PAB structure; wherein AA is a dipeptide or a tripeptide fragment (ie, a fragment formed by ligating 2-3 amino acids through a peptide bond), preferably including Val-Cit (valine-citrulline) , Val-Ala (valine-glycine), Phe-Lys (phenylalanine-lysine), Ala-Ala-Asn (glycine-glycine-asparagine), D-Ala-Phe-Lys (D) Type glycine-phenylalanine-lysine), etc., PAB is p-aminobenzylcarbamoyl;
  • CTD is a cytotoxic small molecule drug bonded to L 2 via an amide bond and/or a drug for treating autoimmune diseases and anti-inflammatory; preferably CTD is selected from the group consisting of a tubulin inhibitor, a topoisomerase inhibitor, More preferably, the tubulin inhibitor is selected from the group consisting of a maytansin derivative, Monomethyl auristatin E, Monomethylauristatin F, Monomethyl Dolastatin 10, a Tubulysin derivative, a Cryptophycin derivative, and Taltobulin.
  • the DNA binding agent is selected from the group consisting of PBD derivatives and duocarmycin derivatives.
  • the topoisomerase inhibitor is selected from the group consisting of a doxorubicin metabolite PNU-159682 derivative, an irinotecan metabolite SN38 derivative, and exenatide.
  • the CTD has a molecular structure selected from the group consisting of D1-D13 and D13', selected from the group consisting of:
  • the compound of formula Ib is selected from the group consisting of
  • an antibody-drug conjugate comprising a maleimide linker drug conjugate and an antibody substituted with a formula Ib according to the second aspect of the invention Coupling is formed.
  • the conjugate is covalently linked to one or more pharmaceutical components.
  • the antibody and drug are coupled by covalent means (e.g., by covalent attachment to a linker, respectively).
  • an antibody-drug conjugate having the structure of Formula Ic and/or Id;
  • Ar', L 1 , L 2 , CTD have the same definitions as above;
  • m 1.0 to 5.0, preferably 3.0 to 4.2;
  • Ab is selected from the group consisting of proteins, enzymes, antibodies, antibody fragments, and polypeptides.
  • the formula Id is a ring-opened product of the N-phenylmaleamide of the formula Ic.
  • the antibody or Ab is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, an antibody fragment (preferably an antibody Fab fragment).
  • a disulfide chain is reduced by the disulfide chain of the hinge region of the antibody or antibody fragment, and a cysteine residue is formed by the cysteine
  • the thiol group in the residue undergoes a substitution reaction with the aryl thioether of the formula Ib, thereby attaching the compound of the formula Ib to the antibody or antibody fragment.
  • the CTD is a cytotoxic small molecule drug, preferably a tubulin inhibitor, a topoisomerase inhibitor or a DNA binding agent.
  • tubulin inhibitor is selected from the group consisting of maytansine derivatives, Monomethyl auristatin E (MMAE), Monomethylauristatin F (MMAF), Monomethyl Dolastatin 10, Tubulysin derivatives, Cryptophycin a derivative, Taltobulin.
  • the DNA binding agent is selected from the group consisting of PBD derivatives, duocarmycin derivatives.
  • the topoisomerase inhibitor is selected from the group consisting of the Doxorubicin metabolite PNU-159682 derivative, the irinotecan (CPT-11) metabolite SN38 derivative.
  • the antibody is an antibody capable of binding to a tumor-associated antigen selected from the group consisting of:
  • the antibody is a HER2 antibody, further preferably trastuzumab or pertuzumab.
  • the antibody is an EGFR antibody, further preferably Erbitux or Vectibix.
  • the antibody is a Tissue factor (TF) antibody.
  • a pharmaceutical composition comprising: (a) an antibody-drug conjugate according to the third aspect of the invention; and (b) a pharmaceutically acceptable dilution Agent, carrier or excipient.
  • a fifth aspect of the invention provides the use of the antibody-drug conjugate according to any one of the third aspects of the invention for the preparation of a medicament for treating a tumor
  • the tumor is selected from the group consisting of breast cancer, ovarian cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple Myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumor, pancreatic cancer, prostate cancer, colon, stomach cancer, Glioma, mesothelioma.
  • a method of treating a tumor comprising the step of administering to a subject in need thereof a therapeutically effective amount of an antibody-drug conjugate according to the third aspect of the invention.
  • the subject is a mammal, preferably a human.
  • a sixth aspect of the invention there is provided a method of preparing an antibody-drug conjugate according to the fifth aspect of the invention, the method comprising the steps of:
  • the antibody in step (1) is reduced by a reducing reagent such that the interchain disulfide bond of the antibody is reduced to produce a thiol group.
  • the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride (TCEP), beta-mercaptoethanol, beta-mercaptoethylamine hydrochloride, or dithiothreitol ( DTT).
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • beta-mercaptoethanol beta-mercaptoethylamine hydrochloride
  • DTT dithiothreitol
  • the buffer is selected from the group consisting of potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH) / sodium chloride (NaCl) / diethyltriamine pentaacetic acid (DTPA) Buffer, disodium hydrogen phosphate-citric acid/sodium chloride (NaCl)/diethyltriaminepentaacetic acid (DTPA), boric acid-borax/sodium chloride (NaCl)/diethyltriaminepentaacetic acid (DTPA) ), histidine-sodium hydroxide/sodium chloride (NaCl)/diethyltriaminepentaacetic acid (DTPA), and PBS/diethyltriaminepentaacetic acid (DTPA).
  • KH 2 PO 4 -NaOH potassium dihydrogen phosphate-sodium hydroxide
  • NaCl sodium chloride
  • DTPA diethyltriamine penta
  • the volume of the organic solvent in the reaction liquid does not exceed 15%.
  • the organic solvent in the step (2) is selected from the group consisting of acetonitrile (ACN), dimethylformamide (DMF), dimethylacetamide (DMA), and dimethylene. Sulfone (DMSO).
  • the coupling reaction is carried out at 0 to 37 °C.
  • step (1) is carried out using beta-mercaptoethanol, beta-mercaptoethylamine hydrochloride or DTT reduction
  • a step is further included between step (1) and step (2). After the reduction reaction is completed, the product is subjected to a desalting column or ultrafiltration to remove the reducing agent.
  • reaction route of the method is as follows:
  • R, Ab, Ar', L 1 , L 2 , CTD, m are as defined above.
  • the method comprises the steps of:
  • the antibody stock solution is diluted to 2-10 mg/mL with a reaction buffer, 140-200 times excess molar ratio of dithiothreitol (DTT), or 6.0-20 times excess molar ratio of three (2) - carboxyethyl)phosphine hydrochloride (TCEP), the reaction solution is stirred at 10-35 ° C for 2-48 hours;
  • DTT dithiothreitol
  • TCEP carboxyethyl)phosphine hydrochloride
  • the method further comprises the steps of: when step 1) is reduced by DTT, after the reduction reaction is completed in step 1), the reaction solution is passed through a desalting column or ultrafiltration to remove excess DTT;
  • the substituted maleimide compound may be previously dissolved in an organic solvent, and the organic solvent is preferably selected from the group consisting of: acetonitrile (ACN), dimethyl sulfoxide (DMSO), and dimethyl ketone. Amide (DMF) or diethylacetamide (DMA); further preferably, the substituted maleimide compound and the organic solvent are dissolved at 10 mg/ml, and the volume ratio of the organic solvent is not more than 15% of the reaction liquid.
  • ACN acetonitrile
  • DMSO dimethyl sulfoxide
  • DMA diethylacetamide
  • the method further comprises the step of: after step 2) completion of the coupling reaction, the reaction mixture is purified by filtration with sodium succinate/NaCl buffer or histidine-acetic acid/sucrose gel according to UV280 ultraviolet absorption. Values were collected for peak samples.
  • the method further comprises the steps of: after the completion of the coupling reaction in step 2), the reaction mixture is ultrafiltered, and then sterilized by filtration, and the obtained product is stored at a low temperature; further preferably, the storage temperature is -100 to 60 ° C; further Preferably, the ultrafiltration uses a device having a pore size of 0.15 to 0.3 microns.
  • said step 1) is carried out using TCEP. Excess TCEP may not be removed.
  • the reaction buffer of step 1) may be: 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyl Triamine pentaacetic acid (DTPA), pH 6-9; 50 mM disodium hydrogen phosphate-citric acid/150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 6-9; 50 mM boric acid - Borax / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 6-9; 50 mM histidine-sodium hydroxide / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine Pentaacetic acid (DTPA), pH 6-9 and PBS//1 m
  • the drug-drug coupling ratio (DAR) of the obtained antibody-drug conjugate is relatively uniform, and antibody-drug coupling with different product uniformity can also be obtained by using different substituted maleimide linkers described in the present invention.
  • DAR drug-drug coupling ratio
  • it can be further purified, but not limited to the following methods: hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), ion exchange chromatography (IEC).
  • R and n are the same as defined above, and X represents a halogen, preferably Br, Cl; and U and V each independently represent N or C.
  • the C can be obtained by reduction of B, and the reaction formula is as follows:
  • the B can be obtained by a substitution reaction of A with a fluoronitrobenzene, and the reaction formula is as follows:
  • the B can be prepared by:
  • A is obtained by reacting n-glycol with t-butyl haloacetate.
  • the reaction formula is as follows:
  • n and X are the same as above.
  • a process for the preparation of the substituted maleimide linker-drug conjugate of the second aspect (Preferred Example F1 or F'1 of Formula Ib): Substituting Malea Amine linker (preferred example Ea of formula Ia) with two The peptide/tripeptide-PAB cytotoxic drug CTD was condensed to obtain F1 or F'1, respectively.
  • the reaction route is as follows:
  • R is as defined for formula Ia
  • Rx represents halogen, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, nitrile or amide
  • Ry represents H or alkyl
  • the inventors have conducted extensive and intensive research and found a class of linker structures which can cross-couple the light chain-heavy chain and heavy chain-heavy chain of the antibody in whole/part, and obtain the coupling method.
  • Antibody-drug conjugates have a narrower drug/antibody ratio (DAR) distribution compared to traditional antibody-drug conjugates. Based on the above findings, the inventors completed the present invention.
  • DAR drug/antibody ratio
  • C1-C4 alkyl refers to a straight or branched alkyl group having from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl. Base, isobutyl, sec-butyl, tert-butyl, or the like.
  • C1-C4 alkoxy refers to a straight or branched alkoxy group having from 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso Butoxy, sec-butoxy, tert-butoxy, or the like.
  • halogen refers to F, Cl, Br and I.
  • C6-C10 aryl refers to an aryl group having 6 to 10 carbon atoms, such as phenyl, naphthyl and the like, which may be substituted or unsubstituted.
  • C6-C10 aryl refers to an aryl group having 6 to 10 carbon atoms, such as phenyl, naphthyl and the like, which may be substituted or unsubstituted.
  • 5-12 membered heteroaryl means having 5 to 12 carbon atoms and one or more (preferably 1 to 3) selected from O, S and/or N.
  • a heteroatom heteroaryl or heteroarylene preferably a 5-8 membered heteroaryl or heteroarylene.
  • the heteroaryl or heteroarylene may be substituted or unsubstituted.
  • the term "pharmaceutically acceptable” ingredient means a substance which is suitable for use in humans and/or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reaction), that is, a reasonable benefit/risk ratio.
  • the term "effective amount" means an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect.
  • the precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the condition, and the combination of therapeutic and/or therapeutic agents selected for administration. Therefore, it is useless to specify an accurate effective amount in advance. However, for a given situation, routine experimentation can be used to determine the effective amount, and the clinician is able to judge Broken out.
  • each of the chiral carbon atoms may be optionally in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.
  • compound of the invention refers to a compound of formula I.
  • the term also encompasses various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula I.
  • the term "pharmaceutically acceptable salt” refers to a salt of the compound of the invention formed with an acid or base suitable for use as a medicament.
  • Pharmaceutically acceptable salts include inorganic and organic salts.
  • a preferred class of salts are the salts of the compounds of the invention with acids.
  • Suitable acids for forming salts include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Organic acids such as maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzoic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.
  • mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid,
  • Organic acids such as maleic acid, lactic acid, malic acid, tartaric acid,
  • amino acid is intended to include any conventional amino acid, such as aspartic acid, glutamic acid, cysteine, asparagine, phenylalanine, glutamine, tyrosine. , serine, methionine (methionine), tryptophan, glycine, valine, leucine, alanine, isoleucine, valine, threonine, histidine, lysine, Arginine.
  • the trade name is intended to include the trade name product formulation, its corresponding generic, and the active pharmaceutical component of the trade name product.
  • antibody as used herein is used in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, as long as they behave.
  • the desired biological activity is obtained (Miller et al. (2003) Journal of Immunology 170: 4854-4861).
  • the antibody can be a murine, human, humanized, chimeric antibody or derived from other species.
  • An antibody is a protein produced by an immune system capable of recognizing and binding to a specific antigen (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York).
  • Target antigens generally have a large number of binding sites, also referred to as epitopes, that are recognized by the CDRs of a variety of antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen can have more than one corresponding antibody.
  • Antibodies include all-long immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, ie, molecules containing antigens or portions thereof that specifically bind to a target of interest, such targets, but not limited to cancer cells or production A cell of an autoimmune antibody associated with an autoimmune disease.
  • the immunoglobulins disclosed herein can have any type of immunoglobulin molecule (eg, IgG, IgE, IgM, IgD, and IgA), classes (eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclasses.
  • Immunoglobulins can be derived from any species. However, in one aspect, the immunoglobulin is derived from a human, a mouse or a rabbit.
  • antibody fragment comprises a portion of a full length antibody, typically its antigen binding or variable region.
  • antibody fragments include: Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al. (2004) Protein Eng. Design & Sel.
  • the antibody constituting the antibody drug conjugate of the present invention preferably retains the antigen binding ability in its original wild state. Therefore, the antibody of the present invention can, preferably, specifically bind to an antigen.
  • Antigens involved include, for example, tumor associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (as known or predicted) Functionally, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules involved in angiogenesis (eg, antigens known to bind antibodies may be one or a subset of the above categories) Other subsets contain other molecules/antigens with specific properties (compared to the target antigen).
  • Antibodies for use in antibody drug conjugates include, but are not limited to, cell surface receptors and tumor associated antigens Antibodies.
  • tumor associated antigens are well known in the art and can be prepared by antibody preparation methods and information well known in the art.
  • To develop effective cell-level targets for cancer diagnosis and treatment researchers have sought to find transmembrane or other tumor-associated polypeptides. These targets are capable of being specifically expressed on the surface of one or more cancer cells with little or no expression on the surface of one or more non-cancer cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancer cells. Confirmation of such tumor-associated factors can greatly enhance the specific targeting characteristics of cancer-based treatment of cancer.
  • Tumor-associated antigens include, but are not limited to, tumor-associated antigens (1)-(53) listed below. For convenience, antigen-related information well known in the art is indicated below, including name, other name, and gene bank accession number. Nucleic acid and protein sequences corresponding to tumor associated antigens can be found in public databases such as Genbank. Antibody-targeting corresponding tumor-associated antigens include all amino acid sequence variants and homologs, having at least 70%, 80%, 85%, 90%, or 95% homology to the sequences identified in the references, or The tumor-associated antigen sequences cited in the literature have completely identical biological properties and characteristics.
  • HER2 Human epidermal growth factor receptor 2 (English: human epidermal growth factor receptor 2, abbreviated as HER2, also known as Neu, ErbB-2, CD340 (differentiation group 340) or p185) A protein encoded by the ERBB2 gene.
  • HER2 is a member of the epidermal growth factor receptor (EGFR/ErbB) family;
  • HER3 Gene ID: 2065, epidermal factor receptor 3 (ErbB3/HER3) is A member of the epidermal growth factor transmembrane receptor family.
  • ErbB3/HER3 has been shown to be closely related to the efficacy of breast cancer, recurrence and metastasis, chemotherapy and endocrine therapy, and has become a promising candidate for therapeutic treatment); 3) CD19 (Gene ID: 930); (4) CD20 (Gene ID: 931); (5) CD22 (Gene ID: 933); (6) CD30 (Gene ID: 943); (7) CD33 (Gene ID : 945); (8) CD37 (Gene ID: 951); (9) CD45 (Gene ID: 5788); (10) CD56 (Gene ID: 4684); (11) CD66e (Gene ID: 1048); CD70 (Gene ID: 970); (13) CD74 (Gene ID: 972); (14) CD79b (Gene ID: 974); (15) CD138 (Gene ID: 6382); (16) CD147 (Gene ID: 682); (17) CD223 (Gene ID: 3902); (18) EpCAM (Gene ID: 4072); (19) Mucin 1
  • drug refers broadly to any compound having the desired biological activity and having reactive functional groups to prepare the conjugates of the invention. Desirable biological activities include, diagnosing, curing, ameliorating, treating, and preventing diseases in humans or other animals. Thus, as long as the necessary reactive functional groups are present, the term “drug” refers to compounds including the official National Pharmacopoeia, as well as, for example, the US Official Homeopathic Pharmacopoeia, the official National Formulary, or any of its supplements. Typical drugs are listed in the physician's desk medication reference (PDR) and the US Food and Drug Administration (FDA) Orange Book. As new drugs are constantly being discovered and developed, this patent states that these drugs should also be included in the "drugs" of the coupled drugs of the present invention.
  • PDR physician's desk medication reference
  • FDA US Food and Drug Administration
  • the drug refers to: a cytotoxic drug for cancer treatment, or a protein or polypeptide having a desired biological activity, such as a toxin such as acacia toxin, ricin A, and pseudomonas Toxins, and diphtheria toxins; others Suitable proteins include tumor necrosis factor, alpha interferon, beta interferon, neurogenic growth factor, platelet derived growth factor, tissue fibrinolytic growth factor, and biological response modulation agents such as lymphokines, interleukins -1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor, Or other growth factors.
  • a cytotoxic drug for cancer treatment or a protein or polypeptide having a desired biological activity
  • a toxin such as acacia toxin, ricin A, and pseudomonas Toxins, and diphtheria toxins
  • a preferred agent of the invention is maytansine or maytansinoid.
  • Maytansin compounds inhibit cell proliferation by inhibiting microtubule formation by tubulin.
  • Maytansin is a derivative of maytansine. Both maytansine and maytansinoids are highly cytotoxic, but they have significant limitations in the clinical application of cancer therapy, mainly due to the low selectivity of such molecules for tumors. However, this high cytotoxicity has made them the drug of choice for antibody drug conjugates.
  • the structure of deacetylmaytansine is listed below.
  • the auristatin peptide drug is an analog of Dolastatin 10, which is a biologically active polypeptide isolated from marine mollusk sea rabbits. Dolon toxin 10 inhibits tubulin polymerization by binding to tubulin (the same binding region as vincristine).
  • the rabbit toxin 10, the auristatin peptide PE, and the auristatin peptide E are all linear polypeptides containing four amino acids (three of which are unique to the sea rabbit toxin compound) and a C-terminal amide group.
  • Two representative autin peptide compounds, monomethyl auratin peptide E (MMAE) and monomethyl auristatin peptide F (MMAF) are the preferred drugs for antibody drug conjugates.
  • MMAE Monomethyl Auristatin E
  • MMAF Monomethyl Auristatin F
  • MMAD Monomethyl Dolastatin 10
  • Microtubulin is first isolated from myxobacteria culture by the research team and is a very potent cytostatic agent that acts by inhibiting tubulin polymerization and thereby inducing apoptosis.
  • the microtubulin D in tubulysin is the most potent and has 10 to 100-fold more activity than most other tubulin modulators, including epothilone, vinblastine and paclitaxel. Paclitaxel and vinblastine are currently used in the treatment of a variety of cancers, and epothilone derivatives are being evaluated for activity in clinical trials.
  • Microtuberin D is a complex tetrapeptide that can be divided into four regions, Mep (DN-methylpiperidinecarboxylic acid), Ile (isoleucine), Tuv (tubular proline, tubuvaline) and Tup (tubular phenylalanine, tubuphenylalanine), as shown below:
  • Cryptophycin is a novel anti-tumor active substance that inhibits the formation of microtubules isolated from the culture of cyanobacteria and is active against a variety of tumors.
  • Cryptophycin is a fat-soluble compound containing two peptide bonds and two ester bonds, having five optically active centers and one epoxy group. The dipeptide diester bonds are all in one macrocyclic structure.
  • the structure of Cryptophycin derivatives CP1 and CP2 is as follows:
  • Taltobulin Another preferred agent of the invention is the novel anti-microtubule agent Taltobulin (HTI-286, SPA-110).
  • Taltobulin inhibits multimerization of purified microtubules, interferes with intracellular microtubule organization, induces mitotic blockade, and induces apoptosis.
  • Taltobulin is a potent inhibitor of cell proliferation with an average IC50 of 2.5 nM for 18 human tumor cell lines.
  • Taltobulin is not a suitable substrate for p-glycoprotein compared to currently used anti-microtubule agents, and the Taltobulin structure is shown below.
  • the drug is the camptothecin derivative SN-38.
  • SN-38 is a biologically active metabolite of irinotecan hydrochloride (CPT-11) and is a class of topoisomerase inhibitors. SN-38 caused the strongest inhibition of DNA topoisomerase I, inhibited DNA synthesis in a dose- and time-dependent manner, and caused frequent DNA single-strand breaks. The structure of SN-38 is shown in the figure below.
  • the drug is a camptothecin derivative, Exatecan.
  • the Exatecan structure is shown below.
  • alpha-Amanitin is a mycotoxin derived from the poisonous mushroom Amanita phalloides, a bicyclic octapeptide that inhibits transcription of eukaryotic RNA polymerase II and RNA polymerase III.
  • Another preferred agent of the invention is a benzodipyridyl antibiotic (duocarmycins, CC-1065, etc.) and other cyclopropapyrroloind-4-one (CPI) derivatives.
  • Such compounds are potent DNA minor groove binding-alkylating agents.
  • the 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, which has weakened prodrug toxicity and enhanced water solubility (wherein the CBI-seco structure has the following general formula):
  • PBD pyrrolo[2,1-c][1,4]benzodi-azepines
  • PBD dimers PBD dimers
  • PBD is a natural product produced by Streptomyces, and its unique feature is the ability to form non-twisted covalent additions in the DNA minor groove, specifically at the ⁇ -guanine- ⁇ sequence.
  • the use of PBD as a partial small molecule strategy to target locked DNA sequences and as a new type of anticancer and antibacterial drugs has attracted increasing interest.
  • a flexible carbon chain is used to link the C8/C8' hydroxyl groups of the two PBD units, and the resulting dimer has enhanced biological activity.
  • PBD dimers are thought to be DNA damage that can produce sequence selectivity, such as reversed 5'-Pu-GATC-Py-3' interstrand crosslinks, resulting in their biological activity. These compounds have proven to be highly potent cytotoxic drugs and can be used as an alternative to antibody drug conjugates.
  • Another preferred drug of the invention is a derivative of PNU-159682, which is the major active metabolite of Nemorubicin in human liver microsomes, with a 3000-fold increase in activity compared to MMDX and doxorubicin.
  • the drug is not limited to the above-mentioned categories, but also includes all drugs that can be used for antibody drug conjugates. And especially those which are capable of coordination by an amide bond to a linker, such as by a cytotoxin having a basic amine group (primary amine or secondary amine), such as the cytotoxin D1-D12 shown above. structure.
  • a linker such as by a cytotoxin having a basic amine group (primary amine or secondary amine), such as the cytotoxin D1-D12 shown above. structure.
  • Linkers or “linkers of antibody drug conjugates” can be classified into two classes according to the mechanism of drug release in cells: non-cleavable linkers and cleavable linkers.
  • the drug release mechanism is: after the conjugate binds to the antigen and is endocytosed by the cell, the antibody is hydrolyzed in the lysosome, and the drug is released by the small molecule drug.
  • An active molecule composed of an amino acid residue of an antibody. The resulting change in the molecular structure of the drug does not diminish its cytotoxicity, but since the active molecule is charged (amino acid residues), it cannot penetrate into adjacent cells. Therefore, such active drugs cannot kill tumor cells (bystander effect) adjacent to the non-targeting antigen (antigen-negative cells).
  • a cleavable linker can cleave in the target cell and release the active drug (the small molecule drug itself).
  • Breakable linkers can be divided into two main classes: chemically labile linkers and enzyme labile linkers. Chemically labile linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties. Such properties include pH, glutathione concentration, and the like. pH sensitive linkers are often referred to as acid cleavage linkers. Such a linker is relatively stable in the neutral environment of blood (pH 7.3-7.5), but will be in the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). hydrolysis.
  • linkers such as hydrazine, carbonate, acetal, ketal.
  • Antibody drug conjugates based on such linkers typically have a shorter half-life (2-3 days) due to the limited plasma stability of the acid-cleaved linker. This shorter half-life limits the use of pH-sensitive linkers in a new generation of antibody drug conjugates to some extent.
  • disulfide bond For glutathione-sensitive linkers, it is also called disulfide bond. Drug release is based on a difference between the high concentration (in millimolar range) of intracellular glutathione and the relatively low concentration of glutathione (micromolar range) in the blood. This is especially true for tumor cells, where low oxygen levels result in enhanced reductase activity, resulting in higher glutathione concentrations. Disulfide bonds are thermodynamically stable and therefore have better stability in plasma.
  • Enzyme-labile linkers such as peptide linkers, provide better control of drug release.
  • Peptide linkers are capable of being efficiently cleaved by lysosome in vivo proteases such as cathepsin (Bathepsin B) or plasmin (increased levels of such enzymes in some tumor tissues). This peptide linkage is believed to be very stable in the plasma cycle because of the extracellular pH and serum protease inhibitors that cause proteases to be generally inactive.
  • enzyme-labile linkers are widely used as cleavable linkers for antibody drug conjugates.
  • Typical enzyme labile linkers include Val-Cit (VC), Phe-Lys, and the like.
  • Self-releasing linkers are typically chimeric between the cleavable linker and the active drug, or are themselves part of the cleavable linker.
  • the mechanism of action of the self-releasing linker is that when the cleavable linker is cleaved under suitable conditions, the self-releasing linker can spontaneously rearrange the structure, thereby releasing the active drug attached thereto.
  • Common suicide linkers include p-aminobenzyl alcohols (PAB) and beta-glucuronides.
  • a linker or coupling reagent of the invention comprising a diarylthiomaleamide unit and a coupling group.
  • the diarylthiomaleamide unit is used to crosslink the sulfhydryl group between the antibody chains (after reduction), while the coupling group is used to couple with the small molecule drug or drug-linker unit. Because of the bidentate binding of the diarylthiomaleamide unit to the two sulfur atoms of the open cysteine-cysteine disulfide bond in the antibody, these ADCs are homogeneous and comparable. ADCs with single-toothed joints are more stable. Thus they will have a increased in vivo half-life, reduce the amount of systemically released cytotoxin, and be safer than the ADC with a single-toothed linker.
  • the produced drug-linker unit is coupled to the antibody via the linker to form a partial interchain cross-linking conjugate.
  • the antibody/antibody ratio (DAR) distribution of the antibody drug conjugate prepared by the method of the present invention is narrower than that of the conventional antibody drug conjugate, thereby greatly improving product uniformity and pharmacological property uniformity.
  • the antibody drug conjugate can be used to target delivery of a drug to a target cell population, such as a tumor cell.
  • Antibody drug couple The conjugate can specifically bind to the cell surface protein, and the resulting conjugate is then endocytosed by the cell. Within the cell, the drug is released as an active drug to produce efficacy.
  • Antibodies include chimeric antibodies, humanized antibodies, human antibodies; antibody fragments that bind to an antigen; or antibody Fc fusion proteins; or proteins.
  • a "drug” is a highly active drug (see definitions), and in some cases the drug may be polyethylene glycol.
  • the antibody drug conjugates provided by the invention consist of an antibody, a linker, a linker and a drug, the linker being a cleavable linker combination or a non-cleavable linker.
  • Antibodies are globular proteins that contain a range of amino acid sites that can be used to couple drug-linkers. Due to its tertiary and quaternary structure, only solvent accessible amino acids are available for coupling. In fact, high yield couplings typically occur on the ⁇ -amino group of a lysine residue or the thiol group of a cysteine residue.
  • a large number of lysine side chains on the surface of the antibody protein result in a large number of sites for drug conjugation, resulting in a mixture of antibody drug conjugates that are produced, containing different drug coupling amounts (drug/antibody ratio, DAR) and Joint point.
  • DAR drug/antibody ratio
  • the coupled product provided by the present invention although still a mixture, has a narrow DAR distribution range compared to the antibody drug conjugate obtained by conventional coupling.
  • the average DAR value is close to 4, which is close to the range of optimal DAR values (2-4) for optimal antibody drug conjugates.
  • the preparation route of the antibody drug conjugate is as follows.
  • the antibody interchain disulfide bond is reduced to generate a total of 8 thiol groups, and the substituted maleic amide linker drug conjugate is cross-linked with the reduced antibody thiol to form a corresponding antibody drug conjugate, wherein the antibody drug
  • the conjugate is present in one or both of the forms as shown.
  • reaction buffer may be a buffer prepared in the following ratio: 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH) 2 PO 4 -NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 6-9; 50 mM disodium hydrogen phosphate - citric acid / 150 mM sodium chloride (NaCl) / 1 mM Diethyltriaminepentaacetic acid (DTPA), pH 6-9; 50 mM boric acid-borax/150 mM sodium chlor
  • the above reaction solution is cooled to 0-10 ° C. If DTT reduction is used, excess DTT is removed by desalting column or ultrafiltration after completion of the reduction reaction, and then substituted maleimide compound (previously 10 mg/ml dissolved in acetonitrile) ACN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF) or diethyl acetamide (DMA), and ensure the volume of organic solvent in the reaction solution The ratio is not more than 15%, and the coupling reaction is stirred at 0-37 ° C for 2-4 hours. If TCEP reduction is used, it is also possible to directly add a substituted maleimide compound for coupling without removing the remaining TCEP.
  • DTT reduction is used, excess DTT is removed by desalting column or ultrafiltration after completion of the reduction reaction, and then substituted maleimide compound (previously 10 mg/ml dissolved in acetonitrile) ACN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF
  • the coupling reaction mixture was purified by filtration using a sodium succinate/NaCl buffer or a histidine-acetic acid/sucrose gel using a desalting column, and a peak sample was collected based on the UV280 ultraviolet absorption value. Or ultrafiltration several times.
  • the bacteria were then sterilized by filtration and the resulting product was stored at a low temperature.
  • the temperature is from -100 to 60 ° C, and the pore size of the filtration device is preferably from 0.15 to 0.3 ⁇ m.
  • the drug antibody coupling ratio (DAR) of the obtained antibody drug conjugate is relatively uniform, and the antibody drug conjugate having different product homogeneity is also obtained by using different substituted maleimide connectors described in the patent, if necessary Samples with better homogeneity can be further purified, but not limited to the following methods: hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), ion exchange chromatography (IEC).
  • HIC hydrophobic interaction chromatography
  • SEC size exclusion chromatography
  • IEC ion exchange chromatography
  • the antibody-drug conjugate provided by the present invention can be targeted to a specific cell population and bind to a cell surface specific protein (antigen), the drug can be released into the cell in an active form by endocytosis or drug infiltration.
  • the antibody-drug conjugates of the invention can be used to treat a disease of interest, and the antibody-drug conjugates mentioned above can be administered to a subject (e.g., a human) by a suitable route in a therapeutically effective amount.
  • a subject in need of treatment can be a patient at risk or suspected of having a condition associated with the activity or amount of expression of a particular antigen. Such patients can be identified by routine physical examination.
  • compositions can be administered by other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or by implantation.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
  • it can be administered to the subject of injectable or biodegradable materials and methods by administration of an injectable depot route, for example using 1-, 3-, or 6 months.
  • the injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, Liquid polyethylene glycol, etc.).
  • the water-soluble antibody can be administered by infusion by a drip method, whereby a pharmaceutical preparation containing the antibody and a physiologically acceptable excipient.
  • Physiologically acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients.
  • An intramuscular preparation for example, a sterile preparation in the form of a suitable soluble salt of the antibody, a pharmaceutically acceptable excipient which can be dissolved and administered, such as a water exchange injection, 0.9% saline, or a 5% dextrose solution.
  • delivery can be by conventional methods in the art. For example, it can be introduced into cells by using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres.
  • the nucleic acid or vector can be delivered locally by direct injection or by using an infusion pump.
  • Other methods include the use of various transport and carrier systems through the use of conjugates and biodegradable polymers.
  • compositions of the present invention comprise a safe and effective amount of an antibody-drug conjugate of the invention and a pharmaceutically acceptable carrier.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared in the form of a solution, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the effective amount of the antibody-drug conjugate of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the bifunctional antibody conjugate such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, patient's weight, patient's immunity Status, route of administration, etc.
  • the antibody-drug conjugate of the present invention when administered at a dose of about 0.0001 mg to 50 mg/kg of animal body weight per day (preferably 0.001 mg to 10 mg/kg of animal body weight), a satisfactory effect can be obtained.
  • a separate dose can be administered several times per day, or the dose can be proportionally reduced.
  • Dosage forms for the compounds of the invention for topical administration include ointments, powders, patches, propellants and inhalants.
  • the active ingredient is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.
  • the compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.
  • a safe and effective amount of a compound of the invention is administered to a mammal (e.g., a human) in need of treatment wherein the dosage is a pharmaceutically effective effective dosage, for a 60 kg body weight
  • the dose to be administered is usually from 1 to 2000 mg, preferably from 5 to 500 mg.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the novel linker provided by the present invention can be coupled to an antibody by a simple chemical method, and the DAR value distribution of the conjugate obtained by using the linker is very narrow compared with the conventional antibody drug conjugate, thus generating The product uniformity is high, and the obtained component of the cross-linking product (DAR is 4) accounts for more than 80%.
  • the antibody-drug conjugate provided by the present invention has almost zero ratio of bare and low cross-linking ADC (mass detection does not detect components with DAR of 0 and 1).
  • the antibody-drug conjugate provided by the present invention has certain safety and effectiveness in treating tumors.
  • the hydrophilicity imparted by the ethylene glycol after coupling can be used to adjust the biomolecular properties; the in vitro tumor cell proliferation inhibitory activity of the cross-linker is more traditional than the traditional mcVC-PAB cross-linking biological activity, drug metabolism stability, safety and the like. The nature has improved or maintained.
  • the coupling method provided by the present invention is applicable to most antibodies, thereby avoiding cumbersome recombination and modification of each antibody to introduce a site-directed coupling site, and thus has broad application prospects.
  • the advantages of the maleimide-based disulfide bridge bridging crosslinking agent of the present invention include: faster crosslinking speed, crosslinking reaction time is usually The reaction can be completed within 2-4 hours. 6.
  • the maleimide-based disulfide bridge bridging has better stability, and the oxime ether exchange is less likely to occur in the body, and the introduction of a substituent at the Ar' site can greatly slow the maleic acid ratio compared with the unsubstituted phenyl group.
  • the cyclization secondary hydrolysis reaction after ring opening of the imine further enhances the stability of the antibody-drug conjugate in vitro and in vivo.
  • Figure 1 shows a comparative map of hydrophobic interaction chromatography (HIC) of patezumab and patomycin drug conjugates
  • Figure 2 shows a mass spectrometric comparison of the pertuzumab and panotex drug conjugates
  • Figure 3 shows the binding experiments of ADC-2, P-mcVC-MMAE, Pertuzumab mAb and NCI-N87 cell surface antigen Her2;
  • Figure 4 shows the proliferation inhibition assay of human breast cancer cell SK-BR-3 by ADC-2, P-mcVC-MMAE, Kadcyla and Pertuzumab;
  • Figure 5 shows proliferation inhibition experiments of human breast cancer cell line BT-474 by ADC-2, P-mcVC-MMAE, Kadcyla, and Pertuzumab;
  • Figure 6 shows the proliferation inhibition assay of human gastric cancer cell line N87 by ADC-2, P-mcVC-MMAE, Kadcyla and Pertuzumab;
  • Figure 7 shows the proliferation inhibition assay of ADC-2 on SKOV-3 human ovarian cancer cells, Du-145 human prostate cancer cells and Panc-1 human pancreatic cancer;
  • Figure 8 shows proliferation inhibition experiments of ADC-2 on human breast cancer MCF-7, MDA-MB-231 and MDA-MB-468 cells;
  • Figure 9-1 shows the proliferation inhibition assay of ADC-4 on human gastric cancer cell line N87;
  • Figure 9-2 shows inhibition of proliferation of Calu-3 human lung cancer cells by ADC-2, ADC-3 and ADC-4;
  • Figure 10 shows the activity of ADC-2, ADC-3, P-mcVC-MMAE, and Kadcyla inhibiting human gastric cancer NCI-N87 subcutaneous xenografts in nude mice.
  • FIG 11-1 Hydrophobic interaction chromatography (HIC) map of Pertuzumab
  • FIG. 11-2 Hydrophobic interaction chromatography (HIC) map of pertuzumab-drug conjugate ADC-I;
  • FIG 11-3 Hydrophobic interaction chromatography (HIC) map of pertuzumab-drug conjugate ADC-II;
  • FIG 11-4 Hydrophobic interaction chromatography (HIC) map of pertuzumab-drug conjugate ADC-III;
  • FIG 11-5 Hydrophobic interaction chromatography (HIC) map of pertuzumab-drug conjugate ADC-IV;
  • FIG 11-6 Hydrophobic interaction chromatography (HIC) map of pertuzumab-drug conjugate ADC-V;
  • FIG 11-7 Hydrophobic interaction chromatography (HIC) map of pertuzumab-drug conjugate ADC-VI;
  • FIG 11-8 Hydrophobic interaction chromatography (HIC) map of pertuzumab-drug conjugate ADC-VII;
  • FIG 12-1 Hydrophobic interaction chromatography (HIC) map of trastuzumab
  • FIG 12-2 Hydrophobic interaction chromatography (HIC) map of trastuzumab-drug conjugate ADC-VIII;
  • FIG. 13-1 Mass spectrum of pertuzumab
  • FIG. 13-2 Mass spectrum of pertuzumab-drug conjugate ADC-I;
  • FIG. 13-3 Mass spectrum of pertuzumab-drug conjugate ADC-II;
  • FIG. 13-4 Mass spectrum of pertuzumab-drug conjugate ADC-III;
  • FIG. 13-5 Mass spectrum of pertuzumab-drug conjugate ADC-IV;
  • FIG. 13-6 Mass spectrum of pertuzumab-drug conjugate ADC-V;
  • FIG. 13-7 Mass spectrum of pertuzumab-drug conjugate ADC-VI;
  • FIG. 13-8 Mass spectrum of pertuzumab-drug conjugate ADC-VII
  • FIG. 14-1 Mass spectrum of trastuzumab
  • Figure 14-2 Mass spectrum of trastuzumab-drug conjugate ADC-VIII;
  • Figure 15 shows the trend of the formation of secondary hydrolysate corresponding to each ADC control, ADC-I, ADC-II, and ADC-VII at room temperature of 0-7 days by LC-MS (Q-TOF);
  • Figure 16-1 shows the corresponding HIC profile of the control ADC at 0 day room temperature
  • Figure 16-2 shows the corresponding HIC profile of the control ADC at room temperature for 2 days
  • Figure 16-3 shows the corresponding HIC profile of the control ADC at room temperature for 4 days
  • Figure 16-4 shows the corresponding HIC profile of the control ADC at 7 days room temperature
  • Figure 17-1 shows the corresponding HIC spectrum of ADC-I at 0 day room temperature
  • Figure 17-2 shows the corresponding HIC spectrum of ADC-I at room temperature for 2 days
  • Figure 17-3 shows the corresponding HIC spectrum of ADC-I at room temperature for 4 days
  • Figure 17-4 shows the corresponding HIC spectrum of ADC-I at 7 days room temperature
  • Figure 18-1 shows the corresponding HIC spectrum of ADC-II at 0 day room temperature
  • Figure 18-2 shows the corresponding HIC spectrum of ADC-II at room temperature for 2 days;
  • Figure 18-3 shows the corresponding HIC spectrum of ADC-II at room temperature for 4 days
  • Figure 18-4 shows the corresponding HIC spectrum of ADC-II at 7 days room temperature
  • Figure 19-1 shows the corresponding HIC spectrum of ADC-VII at 0 day room temperature
  • Figure 19-2 shows the corresponding HIC spectrum of ADC-VII at room temperature for 2 days;
  • Figure 19-3 shows the corresponding HIC spectrum of ADC-VII at room temperature for 4 days
  • Figure 19-4 shows the corresponding HIC spectrum of ADC-VII at 7 days room temperature
  • Figure 20 Results of inhibition experiments on proliferation inhibition of human gastric cancer cell line NCI-N87 by ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII, and Pertuzumab (Perjeta);
  • Figure 21 Results of inhibition experiments on proliferation inhibition of human breast cancer cell line BT-474 by ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII, and Pertuzumab (Perjeta);
  • Figure 22 Results of inhibition experiments on proliferation inhibition of human gastric cancer cell line NCI-N87 by ADC-VIII and Trastuzumab (Herceptin);
  • Figure 23 A graph showing the results of inhibition of proliferation of human breast cancer cell line BT-474 by ADC-VIII and Trastuzumab (Herceptin);
  • Figure 24 P-mcVC-MMAE (1.0 mg/kg), control ADC (0.5, 1.0 mg/kg), ADC-I (1.0 mg/kg), ADC-IV (1.0 mg/kg), ADC-V ( 1.0 mg/kg), ADC-VI (1.0 mg/kg), ADC-VII (0.5, 1.0 mg/kg) inhibited the activity of human gastric cancer NCI-N87 subcutaneous xenografts in nude mice. .
  • the inventors have conducted extensive and intensive research and found a class of linker structures which can cross-couple the light chain-heavy chain and heavy chain-heavy chain of the antibody in whole/part, and obtain the coupling method.
  • the antibody drug conjugate has a narrower drug/antibody ratio (DAR) distribution compared to conventional antibody drug conjugates. Based on the above findings, the inventors completed the present invention.
  • DAR drug/antibody ratio
  • Specific design 1 Preparation of a substituted maleicamide linker fragment Ia and its use, wherein Ar' is selected from an unsubstituted C 6 -C 10 arylene group or an unsubstituted 5-12 membered heteroarylene group.
  • the substituted maleimide linker molecule represented by the formula Ia listed in the first aspect of the present invention can be synthesized by the method of the first scheme, and the intermediate B is obtained by substituting the n-glycol with the fluoronitrobenzene.
  • the amino compound C is obtained by reduction of the nitro group
  • the intermediate D is obtained by substituting the 2,3-dibromomaleimide with the arylthiophenol, and then reacting with methyl chloroformate to obtain the intermediate E, 2, 3-
  • the dibromomaleimide can also be directly reacted with methyl chloroformate to obtain the intermediate E', and the intermediate C is reacted with the intermediate E or the intermediate E' to obtain the linker molecule F. Examples of reaction routes and specific examples are as follows:
  • the intermediate compound B-1 (3.0 g, 9.52 mmol) was dissolved in acetone (30 mL), cooled in ice-water, and then freshly-prepared to the preparation of the reagent (15 mL). The reaction mixture was stirred at room temperature for 3 hours, cooled in an ice water bath, and slowly dripped. After adding isopropyl alcohol and stirring in an ice-water bath for 15 minutes, the organic solvent was evaporated to dryness, and the aqueous layer was evaporated to ethyl ether. Crude, this intermediate was used directly in the next step without purification.
  • the substituted maleimide linker molecule represented by formula Ia can also be synthesized by the method shown in the following figure, by substituting n-glycol with fluoronitrobenzene to obtain intermediate B, followed by tert-butyl bromoacetate.
  • the ester reaction can obtain the intermediate Z, and the intermediate Z can also be obtained by reacting n-glycol with t-butyl bromoacetate and then with t-butyl bromoacetate; the intermediate Z is reduced to obtain the intermediate Y; 2, 3 -Dibromomaleimide is substituted with arylthiophenol to obtain intermediate D, which is then reacted with methyl chloroformate to obtain intermediate E.
  • Intermediate E is reacted with intermediate Y to obtain intermediate X, intermediate X.
  • the tert-butyl ester in the middle is removed under acidic conditions to obtain a linker fragment molecule W.
  • An example of the reaction route is as follows:
  • the substituted maleic amide linker drug conjugate (Formula 13-Formula 19) represented by Formula Ib set forth in the second aspect of the present invention can be synthesized by the route outlined in Scheme 2, by Compound F and cytotoxicity.
  • the drug CTD (D1-D11, a toxic drug molecule can be obtained commercially) is condensed and coupled to obtain a series of molecules G. Examples of reaction routes and specific examples are as follows:
  • the substituted maleimide linker drug conjugate (Formula 22-Formula 41) represented by Formula Ib set forth in the second aspect of the present invention can be synthesized by the route outlined in Scheme 3, by Compound F and Dipeptide/
  • the amino group of the tripeptide-PAB linker (which is commercially available) is condensed, and the PAB group is condensed with the cytotoxic drug CTD (D1-D11) after activation with bis(p-nitrophenyl) carbonate. Together, a series of molecules K can be obtained.
  • reaction routes and specific examples are as follows:
  • the synthesis of the compound K-8 is the same as the synthesis of the compound K-1 in Example 3.1 except that the compound H-1 in the step f is replaced by H-4 (Ala-Ala-Asn-PAB), and the compound in the step h is used. D1 was changed to compound D2 (monomethyl auristatin E). A total of three steps of the reaction gave product K-8 as a yellow amorphous solid.
  • LC-MS (M +) Theoretical value: 1699.79, LC-MS (ESI , M + H +) Found: 1700.80.
  • the Patuxib antibody stock solution is treated with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 7.4 reaction buffer
  • KH 2 PO 4 -NaOH potassium dihydrogen phosphate-sodium hydroxide
  • NaCl sodium chloride
  • DTPA diethyltriamine pentaacetic acid
  • pH 7.4 reaction buffer The mixture was diluted to 5 mg/mL, and a 6.0-fold excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction solution was stirred at 35 ° C for 10 hours.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the above reaction solution was cooled to 8 ° C, and an appropriate amount of dimethyl sulfoxide (DMSO) was added without purification, and a compound 6 G in a molar excess ratio (10 mg/ml pre-dissolved in DMSO) was added to ensure the reaction system.
  • DMSO dimethyl sulfoxide
  • the volume of DMSO was not more than 15%, and the coupling was carried out by stirring at 37 ° C for 3 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a histidine-acetic acid/sucrose gel of pH 6.0, and a peak sample was collected based on the UV280 ultraviolet absorption value. It was then sterilized through a 0.15 micron pore size filter device and stored at -60 °C.
  • the Patuxib antibody stock solution is treated with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 7.4 reaction buffer
  • KH 2 PO 4 -NaOH potassium dihydrogen phosphate-sodium hydroxide
  • NaCl sodium chloride
  • DTPA diethyltriamine pentaacetic acid
  • pH 7.4 reaction buffer The mixture was diluted to 5 mg/mL, and a 10-fold excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction solution was stirred at 10 ° C for 4 hours.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the reaction solution was cooled to 5 ° C, and an appropriate amount of diethyl acetamide (DMA) was added without purification, and a compound K-2 (10 mg/ml pre-dissolved in DMA) was added in a 6-fold excess molar ratio to ensure the reaction system.
  • the volume of the medium DMA is not more than 10%, and the coupling is carried out by stirring at 25 ° C for 2.5 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a histidine-acetic acid/sucrose gel of pH 6.0, and a peak sample was collected based on the UV280 ultraviolet absorption value. It was then sterilized through a 0.22 micron pore size filter device and stored at -80 °C.
  • the Patuxib antibody stock solution is treated with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 7.4 reaction buffer
  • KH 2 PO 4 -NaOH potassium dihydrogen phosphate-sodium hydroxide
  • NaCl sodium chloride
  • DTPA diethyltriamine pentaacetic acid
  • pH 7.4 reaction buffer The mixture was diluted to 5 mg/mL, and a 20-fold excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction solution was stirred at 15 ° C for 2 hours.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the above reaction solution was cooled to 10 ° C, and an appropriate amount of acetonitrile (ACN) was added without purification, and a compound K-3 (10 mg/ml pre-dissolved in ACN) in a molar excess ratio was added to ensure the volume of ACN in the reaction system.
  • the ratio was not more than 10%, and the coupling was carried out by stirring at 10 ° C for 4 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a histidine-acetic acid/sucrose gel of pH 6.0, and a peak sample was collected according to the UV280 ultraviolet absorption value, followed by filtration sterilization, and the obtained product was cryopreserved; for example, via a 0.20 micron pore size.
  • the filter device was sterilized and stored at -90 °C.
  • the Patuxib antibody stock solution is treated with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 7.4 reaction buffer
  • KH 2 PO 4 -NaOH potassium dihydrogen phosphate-sodium hydroxide
  • NaCl sodium chloride
  • DTPA diethyltriamine pentaacetic acid
  • pH 7.4 reaction buffer The mixture was diluted to 5 mg/mL, and an 8-fold excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction solution was stirred at 25 ° C for 48 hours.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the reaction solution was cooled to 0 ° C, and an appropriate amount of dimethylformamide (DMF) was added without purification, and a compound K-4 (10 mg/ml pre-dissolved in DMF) was added in a 6-fold excess molar ratio to ensure the reaction system.
  • the volume of DMF in the medium did not exceed 8%, and the coupling was carried out by stirring at 0 ° C for 2 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a histidine-acetic acid/sucrose gel of pH 6.0, and a peak sample was collected based on the UV280 ultraviolet absorption value. It was then sterilized through a 0.3 m pore size filter device and stored at -100 °C.
  • Example 2 The sample obtained in Example 2 was subjected to hydrophobic interaction chromatography (HIC) comparison (Fig. 1) and mass spectrometry (Fig. 2) with pertuzumab, whereby the conjugate obtained by using the linker of the present invention was known.
  • HIC hydrophobic interaction chromatography
  • Fig. 2 mass spectrometry
  • the DAR value distribution is very narrow, the average DAR value is close to 4, and the obtained single component of the crosslinked product (DAR is 4) accounts for more than 80%, so the product produced is highly uniform; after identification, m is 3.0-4.2. .
  • Examples 2, 3, and 4 were each analyzed by the same method as described above, and DAR gave the same result, and m was in the range of 1 to 5.0.
  • the Biacore instrument's principle of detecting the intermolecular affinity of proteins is based on Surface Plasmon Resonance (SPR).
  • SPR is an optical physics phenomenon. When a bundle of P-polarized light is incident on the prism end face within a certain angle range, a surface plasma wave will be generated at the interface between the prism and the metal film (Au). When the propagation constant of the incident light wave When the propagation constants of the surface plasma waves match, the free electrons in the metal film resonate.
  • a biomolecular recognition film is first fixed on the surface of the sensor chip, and then the sample to be tested flows through the surface of the chip.
  • the surface of the gold film is caused.
  • the change in refractive index eventually leads to a change in the SPR angle.
  • information such as the concentration, affinity, kinetic constant and specificity of the analyte is obtained.
  • Binding experiments were performed using Biacore to detect binding activity of three batches of monoclonal samples of Pertuzumab, ADC-2, and ADC-4 to Human ErbB2.
  • the surface plasmon resonance technique was used to characterize the binding activity of three monoclonal antibody samples Pertuzumab, ADC-2, ADC-4 and Human ErbB2, and the results showed binding.
  • the experimental results showed that the above three monoclonal antibody samples had similar affinity to Human ErbB2, both in the range of 0.5-0.7 nM.
  • the experimental materials used in the following experiments were derived from: RPMI1640 medium, 0.25% trypsin-EDTA, fetal bovine serum, 100 x sodium pyruvate, 100 x streptomycin, purchased from Gibco, fluorescein isothiocyanate.
  • the isothiocyanate, FITC-labeled secondary antibody was purchased from Invitrogen, and the NCI-N87 gastric cancer cells were obtained from the Kunming Cell Bank of the Chinese Academy of Sciences. All other reagents were of analytical grade. FACSCalibur flow cytometer (BD).
  • Her2 is highly expressed in human gastric cancer NCI-N87 cells.
  • NCI-N87 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum in a 37 ° C, 5% CO 2 incubator, subcultured for 4 to 5 days, and cells were collected into 15 mL centrifuge tubes with cold PBS. The cells were washed twice, centrifuged at 1000 rpm for 5 min at 4 ° C, resuspended in PBS containing 5% fetal bovine serum, incubated at 37 ° C for 30 min, then centrifuged at 1000 ° C for 5 min at 4 ° C, supernatant, and resuspended in cold PBS.
  • ADC-2, P-mcVC-MMAE, and Pertuzumab mAb can bind to NCI-N87 cell surface antigen Her2, and with the increase of antibody concentration, ADC-2, P-mcVC-MMAE, Pertuzumab The binding to the Her2 receptor was also increased, and the affinity of the three for binding to Her2 in NCI-N87 cells was comparable, with no significant difference.
  • the experimental materials used in the following experiments were derived from: DMEM medium, DMEM/F12K medium, RPMI 1640 medium, 0.25% trypsin-EDTA, fetal bovine serum, 100 ⁇ sodium pyruvate, 100 ⁇ streptomycin. Gibco. Sulforhodamine B (SRB) was purchased from Sigma.
  • BT-474 human breast cancer cells SK-RB-3 human breast cancer cells, MDA-MB-231 human breast cancer cells, NCI-N87 human gastric cancer cells from Kunming cells of Chinese Academy of Sciences Library, Panc-1 human pancreatic cancer, MDA-MB-468 human breast cancer cells, MCF-7 human breast cancer cells from the Chinese Academy of Sciences Shanghai Institute of Life Sciences cell bank, SKOV-3 human ovarian cancer cells, Du-145 prostate Cancer cells are from the American Type Culture Collection (ATCC). All other reagents were of analytical grade. 96-well flat-bottom polystyrene (Corning, Cat. No. 3599). Synergy 2 microplate reader (Bio-Tek).
  • SRB sulforhodamine B
  • SRB is a pink anionic dye which is easily soluble in water and can specifically bind to basic amino acids of proteins in cells under acidic conditions. It produces an absorption peak at a wavelength of 510 nm. The absorbance is linearly positively correlated with the amount of cells. Therefore, it can be used for quantitative detection of the number of cells.
  • the cell lines selected in this example are: BT-474, SK-RB-3, MDA-MB-231, MDA-MB-468, MCF-7 human breast cancer cells, NCI-N87 human gastric cancer cells, SKOV-3 humans. Ovarian cancer cells, Du-145 human prostate cancer cells, Panc-1 human pancreatic cancer.
  • MDA-MB-468 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum in a logarithmic growth phase at 37 ° C, 5% CO 2 incubator.
  • the cells in the logarithmic growth phase were inoculated into a 96-well culture plate at a density of 2 ⁇ 103 to 9 ⁇ 103 cells per well, 100 ⁇ L per well, and cultured for 24 hours, and then added with different concentrations of the drug for 5 days.
  • Inhibition rate (%) (A control - A administration) / A control ⁇ 100%.
  • ADC-2, ADC-4, P-mcVC-MMAE, Kadcyla, and Pertuzumab were used to study the proliferation of a variety of Her2 high-expressing tumor cell lines in vitro, and ADC-2 was used for a variety of non- The tumor cell line with high expression of Her2 also studied the proliferation of cell culture in vitro.
  • ADC-2, P-mcVC-MMAE, and Kadcyla treated Her2 high-expressing SK-BR-3 and BT-474 human breast cancer cells and NCI-N87 human gastric cancer cells, all of which significantly inhibited tumors.
  • ADC-2 and P-mcVC-MMAE had significantly higher inhibitory activity against tumor cell proliferation than Kadcyla, and ADC-2 and P-mcVC-MMAE had comparable anti-tumor cell proliferation activities, of which ADC-2 was BT-474.
  • the tumor cell proliferation inhibitory activity was slightly higher than that of P-mcVC-MMAE; compared with Pertuzumab naked antibody, the antibody drug conjugates ADC-2, P-mcVC-MMAE, and Kadcyla significantly increased the tumor cell proliferation inhibitory activity.
  • ADC-2 also showed a good tumor cell proliferation inhibitory effect on SKOV-3 human ovarian cancer cells, Du-145 human prostate cancer cells and Panc-1 human pancreatic cancer which were not highly expressed by Her2.
  • ADC-2 anti-tumor cell proliferation activity was significantly reduced in human breast cancer MCF-7, MDA-MB-231 and MDA-MB-468 cells that did not express Her2 (Fig. 8), indicating antibody drug conjugate ADC-2 It has no effect on cells that do not express the target antigen, and thus, it indicates that the toxic and side effects will be significantly reduced.
  • ADC-4 showed potent tumor cell proliferation inhibition on Her2 high-expressing NCI-N87 human gastric cancer cells.
  • ADC-2, ADC-3 and ADC-4 showed potent tumor cell proliferation inhibition effects on Calu-3 human lung cancer cells expressed in Her2.
  • the efficacy of the combination of the invention can be measured in vivo by implanting an allograft or xenograft of cancer cells in a rodent and treating the tumor with the combination.
  • Test mice were treated with drugs or controls and monitored for weeks or longer to measure time to tumor doubling, log cell killing, and tumor suppression.
  • mice were subcutaneously inoculated with human gastric cancer NCI-N87 cells, and after the tumors were grown to 100-250 mm 3 , the animals were randomly grouped (D0). The dosage and administration schedule are shown in Table 1. The tumor volume was measured 2-3 times a week, the rats were weighed, and the data were recorded. The tumor volume (V) is calculated as:
  • V 1/2 ⁇ a ⁇ b 2
  • a and b represent length and width, respectively.
  • T/C(%) (T-T0)/(C-C0)x100
  • T and C are the tumor volumes at the end of the experiment
  • T 0 and C 0 are the tumor volumes at the beginning of the experiment.
  • Figure 10 shows that ADC2 (0.5, 1, 2 mg/kg, IV, once a week for 2 times) dose-dependently significantly inhibited the growth of human gastric cancer NCI-N87 subcutaneous xenografts in nude mice, and the tumor inhibition rates were 59%, 94%, and 200%, 3/6 tumors partially resolved in the 1 mg/kg group, and 6/6 tumors completely resolved in the 2 mg/kg group; ADC3 (0.5, 1, 2 mg/kg, IV, once a week, 2 times) the inhibition rate of NCI-N87 was 65%, 69% and 185%, respectively, 1/6 tumor partial regression and 5/6 tumor complete regression in 2mg/kg group; P-vcMMAE (1mg/kg, IV, once a week for 2 times) The tumor inhibition rate for NCI-N87 was 94%, and 2/6 tumors were partially resolved; the reference drug Kadcyla (2 mg/kg, IV, once a week for 2 times) The tumor inhibition rate of NCI-N87 was 77%.
  • Example group I compound synthesis and preparation method
  • Triethylene glycol (92 g, 613 mmol) was dissolved in tBuOH (200 mL).
  • tBuOH a solution of t-butyl bromoacetate (39.8 g, 204 mmol.
  • TLC detected the end of the reaction.
  • dichloromethane 400 ml
  • the organic phase was washed with 400 ml of water, and the aqueous phase was extracted once with 300 ml of dichloromethane.
  • the organic phase was combined and washed once with saturated brine and dried over anhydrous sodium sulfate Steamed and dried.
  • the crude product was purified by EtOAc EtOAc EtOAc (EtOAc)
  • step e The synthesis of compound E-4 is the same as the procedure for the synthesis of compound E-2 in Example 1.2 except that the 5-fluoro-2-nitrobenzotrifluoride in step b is replaced by 2-methoxy-4-fluoronitrobenzene.
  • the thiophenol in step e was changed to 4-(N-morpholinecarboxamide) thiophenol to give the product E-4 as an orange oil.
  • step e The synthesis of compound E-6 was carried out in the same manner as the synthesis of compound E-2 in Example 1.2 except that 5-fluoro-2-nitrobenzotrifluoride in step b was replaced with 5-fluoro-2-nitrobenzonitrile.
  • the thiophenol in step e was changed to 4-(N-morpholinecarboxamide) thiophenol to give the product E-6 as an orange oil.
  • step e The synthesis of compound E-7 was carried out in the same manner as the synthesis of compound E-2 in Example 1.2 except that the 5-fluoro-2-nitrobenzotrifluoride in step b was replaced by 2-fluoro-5-nitrobenzonitrile.
  • the thiophenol in step e was changed to 4-(N-morpholinecarboxamide) thiophenol to give the product E-7 as an orange oil.
  • the synthesis of the compound E-8 was the same as the synthesis of the compound E-2 in Example 1.2 except that the 5-fluoro-2-nitrobenzotrifluoride in the step b was changed to 5-fluoro-2-nitrobenzamide.
  • the thiophenol in step e was changed to 4-(N-morpholinecarboxamide) thiophenol to give the product E-8 as an orange oil.
  • reaction system was extracted with 100 ml of dichloromethane, washed once with 200 ml of 1N diluted hydrochloric acid, twice with 200 ml of water, once with 200 ml of saturated brine, dried over anhydrous sodium sulfate and evaporated to dryness.
  • the synthesis of the compound E-12 is the same as the synthesis of the compound E-11 in the step 1.11 except that the 2,6-difluoro-4-nitrophenol in the step g is replaced with 3-fluoro-4-nitrophenol.
  • the product E-12 was an orange oil.
  • the synthesis of the compound E-16 was carried out in the same manner as the procedure of the compound E-11 in Example 1.11 except that the triethylene glycol in the step a was changed to the pentaethylene glycol to give the product E-16 as an orange oil.
  • the synthesis of the compound E-17 was carried out in the same manner as the procedure of the compound E-11 in Example 1.11 except that the triethylene glycol in the step a was changed to hexaethylene glycol to give the product E-17 as an orange oil.
  • the Patuxib antibody stock solution is treated with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 7 reaction buffer
  • KH 2 PO 4 -NaOH potassium dihydrogen phosphate-sodium hydroxide
  • NaCl sodium chloride
  • DTPA diethyltriamine pentaacetic acid
  • pH 7 reaction buffer pH 7 reaction buffer
  • the mixture was diluted to 2 mg/mL, and a 6.0-fold excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction solution was stirred at 35 ° C for 2.5 hours.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the above reaction solution was cooled to 8 ° C, and an appropriate amount of dimethyl sulfoxide (DMSO) was added without purification, and a compound 6 F1-17 (10 mg/ml pre-dissolved in DMSO) was added to ensure a reaction system.
  • DMSO dimethyl sulfoxide
  • the volume of DMSO was not more than 15%, and the coupling was carried out by stirring at 37 ° C for 3 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a histidine-acetic acid/sucrose gel of pH 6.0, according to UV280. Peak samples were collected for UV absorbance. It was then sterilized through a 0.15 micron pore size filter device and stored at -60 °C.
  • the Patuxib antibody stock solution is treated with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 6 reaction buffer
  • KH 2 PO 4 -NaOH potassium dihydrogen phosphate-sodium hydroxide
  • NaCl sodium chloride
  • DTPA diethyltriamine pentaacetic acid
  • pH 6 reaction buffer pH 6 reaction buffer
  • the mixture was diluted to 5 mg/mL, and a 10-fold excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction solution was stirred at 10 ° C for 40 hours.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the reaction solution was cooled to 5 ° C, and an appropriate amount of diethyl acetamide (DMA) was added without purification, and a compound 6 F1 (10 mg/ml pre-dissolved in DMA) was added in a molar excess ratio to ensure the reaction system.
  • the volume of the medium DMA is not more than 10%, and the coupling is carried out by stirring at 25 ° C for 2.5 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a histidine-acetic acid/sucrose gel of pH 6.0, and a peak sample was collected based on the UV280 ultraviolet absorption value. It was then sterilized through a 0.22 micron pore size filter device and stored at -80 °C.
  • Patuxib antibody stock solution was diluted to 5 mg/mL with PBS//1 mM diethyltriaminepentaacetic acid (DTPA), pH 7.4 reaction buffer, and a 20-fold excess molar ratio of tris(2-carboxyethyl)phosphine salt was added.
  • DTPA diethyltriaminepentaacetic acid
  • TCEP acid salt
  • the above reaction solution was cooled to 10 ° C, and an appropriate amount of acetonitrile (ACN) was added without purification, and a compound 6 F1-20 (10 mg/ml pre-dissolved in ACN) was added to ensure the volume of ACN in the reaction system.
  • ACN acetonitrile
  • the ratio was not more than 10%, and the coupling was carried out by stirring at 10 ° C for 4 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a histidine-acetic acid/sucrose gel of pH 8.0, and a peak sample was collected according to the UV280 ultraviolet absorption value, followed by filtration sterilization, and the obtained product was cryopreserved; for example, via a 0.20 micron pore size.
  • the filter device was sterilized and stored at -90 °C.
  • the Patuxib antibody stock solution is treated with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 7 reaction buffer
  • KH 2 PO 4 -NaOH potassium dihydrogen phosphate-sodium hydroxide
  • NaCl sodium chloride
  • DTPA diethyltriamine pentaacetic acid
  • pH 7 reaction buffer pH 7 reaction buffer
  • the mixture was diluted to 8 mg/mL, and an 8-fold excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction solution was stirred at 25 ° C for 25 hours.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the reaction solution was cooled to 5 ° C, and an appropriate amount of dimethylformamide (DMF) was added without purification, and a compound 6 F1-19 (10 mg/ml pre-dissolved in DMF) was added to ensure the reaction system.
  • the volume of DMF in the medium did not exceed 8%, and the coupling was carried out by stirring at 0 ° C for 2 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a histidine-acetic acid/sucrose gel of pH 6.0, and a peak sample was collected based on the UV280 ultraviolet absorption value. It was then sterilized through a 0.3 micron pore size filter device and stored at -80 °C.
  • the Patuxib antibody stock solution was diluted to 6 mg/mL with 50 mM histidine-sodium hydroxide/150 mM sodium chloride (NaCl)/1 mM diethyltriaminepentaacetic acid (DTPA), pH 7.4, and added 8 times. Excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and the reaction was stirred at 35 ° C for 15 hours.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the reaction solution was cooled to 10 ° C, and an appropriate amount of dimethylformamide (DMF) was added without purification, and a compound 6-2 (10 mg/ml pre-dissolved in DMF) was added in a 6-fold excess molar ratio to ensure the reaction system.
  • the volume of DMF in the medium did not exceed 8%, and the coupling was carried out by stirring at 0 ° C for 5 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a histidine-acetic acid/sucrose gel of pH 6.0, and a peak sample was collected based on the UV280 ultraviolet absorption value. It was then sterilized through a 0.15 micron pore size filtration device and stored at -100 °C.
  • the Patuxib antibody stock solution was diluted to 10 mg/mL with 50 mM boric acid-borax/150 mM sodium chloride (NaCl)/1 mM diethyltriaminepentaacetic acid (DTPA), pH 9 reaction buffer, and added in an 8-fold excess molar ratio.
  • Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and the reaction solution was stirred at 25 ° C for 10 hours.
  • the above reaction solution was cooled to 10 ° C, and an appropriate amount of dimethylformamide (DMF) was added without purification, and a compound 6-2 (10 mg/ml pre-dissolved in DMF) was added in a 6-fold excess molar ratio to ensure the reaction system.
  • the volume of the medium DMF is not more than 8%, and the coupling is carried out by stirring at 0 ° C for 4 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a histidine-acetic acid/sucrose gel of pH 6.0, and a peak sample was collected based on the UV280 ultraviolet absorption value. It was then sterilized through a 0.2 micron pore size filter device and stored at -60 °C.
  • the Patuxib antibody stock solution was diluted with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriamine pentaacetic acid (DTPA), pH 8 reaction buffer To 3 mg/mL, an 8-fold excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction solution was stirred at 15 ° C for 48 hours.
  • KH 2 PO 4 -NaOH potassium dihydrogen phosphate-sodium hydroxide
  • NaCl sodium chloride
  • DTPA diethyltriamine pentaacetic acid
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the reaction solution was cooled to 0 ° C, and an appropriate amount of dimethylformamide (DMF) was added without purification, and a compound 6 F1-18 (10 mg/ml pre-dissolved in DMF) was added to ensure a reaction system.
  • the volume of the medium DMF was not more than 8%, and the coupling was carried out by stirring at 0 ° C for 3 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a histidine-acetic acid/sucrose gel of pH 6.0, and a peak sample was collected based on the UV280 ultraviolet absorption value. It was then sterilized through a 0.3 m pore size filter device and stored at -70 °C.
  • the trastuzocyte antibody stock solution was diluted to 5 mg/mL with 50 mM disodium hydrogen phosphate-citric acid/150 mM sodium chloride (NaCl)/1 mM diethyltriaminepentaacetic acid (DTPA), pH 7.4, and added 8 times. Excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), the reaction was stirred at 25 ° C for 5 hours.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • the reaction solution was cooled to 0 ° C, and an appropriate amount of dimethylformamide (DMF) was added without purification, and a compound 6 F1 (10 mg/ml pre-dissolved in DMF) was added in a molar excess ratio to ensure the reaction system.
  • the volume of DMF in the medium did not exceed 8%, and the coupling was carried out by stirring at 0 ° C for 2 hours.
  • the coupling reaction mixture was purified by filtration using a desalting column with a histidine-acetic acid/sucrose gel of pH 6.0, and a peak sample was collected based on the UV280 ultraviolet absorption value. It was then sterilized through a 0.3 m pore size filter device and stored at -80 °C.
  • Hydrophobic interaction chromatography HIC analysis of antibody-conjugated drugs can obtain important information such as the number and location of coupling sites and the drug to antibody ratio (DAR).
  • DAR drug to antibody ratio
  • the present invention is based on the disulfide bridge bridging of maleimide, which has better stability and is less prone to oxime ether exchange in the body, in order to further confirm that the introduction of a substituent at the Ar' site can greatly slow the ring opening of maleimide.
  • the subsequent cyclization of the second hydrolysis reaction enhances the stability of the antibody-drug conjugate.
  • ADC-I, ADC-II, and ADC-VII were selected and compared with the control ADC.
  • the ADC samples of the same protein concentration (10 mg/mL) were stored in the preparation buffer and placed at 25 ° C, respectively, at 0, 2. 4, 7 days sampling and determination.
  • ADC-I, ADC-II, secondary hydrolysate in the ADC-VII sample is ADC-I, ADC-II, secondary hydrolysate in the ADC-VII sample.
  • the HIC method was used to determine the change of 0, 2, 4, and 7 days for each ADC sample.
  • the control ADC sample appeared at the retention time of 6.094 at 7 days.
  • the impurity peaks were not significantly changed from 0 to 7 days in ADC-I, ADC-II, and ADC-VII samples. See Figures 17-1 to 17-4 and Figures 18-1 to 18-4, respectively. Figure 19-1 to 19-4.
  • the experimental materials used in the following experiments were derived from: DMEM medium, DMEM/F12K medium, RPMI 1640 medium, 0.25% trypsin-EDTA, fetal bovine serum, 100 ⁇ sodium pyruvate, 100 ⁇ streptomycin. Gibco. Sulforhodamine B (SRB) was purchased from Sigma. NCI-N87 human gastric cancer cells and BT-474 human breast cancer cells were obtained from the Kunming cell bank of the Chinese Academy of Sciences. All other reagents were of analytical grade. 96-well flat-bottom polystyrene (Corning, Cat. No. 3599). Synergy 2 microplate reader (Bio-Tek).
  • SRB sulforhodamine B
  • SRB is a pink anionic dye which is easily soluble in water and can specifically bind to basic amino acids of proteins in cells under acidic conditions. It produces an absorption peak at 510 nm. The absorbance is linear with the amount of cells. Related, it can be used as a quantitative test for the number of cells.
  • the cell lines selected in this example were: BT-474 human breast cancer cells and NCI-N87 human gastric cancer cells.
  • BT-474 and NCI-N87 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in a logarithmic growth phase at 37 ° C in a 5% CO 2 incubator, and the above cells in the logarithmic growth phase were respectively 2 ⁇ 103 ⁇ 9 ⁇ 103 cells were inoculated into the 96-well culture plate at a density of 100 ⁇ L per well. After 24 hours of culture, different concentrations of the drug were added for 5 days, and diluted by 3, 4 or 5-fold, respectively. Concentrations were set at each concentration, and the corresponding concentrations of vehicle control and cell-free medium wells were set.
  • the culture solution was decanted, and 100 ⁇ l of a pre-cooled trichloroacetic acid solution (30%, w/v) at 4 ° C was added, and fixed at 4 ° C for 1 hour, followed by washing 5 times with deionized water, and dried at room temperature.
  • a pre-cooled trichloroacetic acid solution (30%, w/v) at 4 ° C was added, and fixed at 4 ° C for 1 hour, followed by washing 5 times with deionized water, and dried at room temperature.
  • Add 100 ⁇ L of 0.4% (w/v) SRB stain (Sigma, 1% glacial acetic acid) to each well, incubate for 30 min at room temperature, rinse 4 times with 1% glacial acetic acid, remove unbound dye, and dry at room temperature. .
  • Inhibition rate (%) (A control - A administration) / A control ⁇ 100%.
  • ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII, and ADC-VIII were used to promote the proliferation of Her2 high-expressing tumor cell lines in vitro. Research. As shown in the table below, higher expression of Her2 was observed in ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII and ADC-VIII compared to naked anti-Perjeta and Herceptin. NCI-N87 human gastric cancer cells and BT-474 human breast cancer cells can significantly inhibit tumor cell proliferation. The corresponding proliferation inhibition curve is shown in Figure 20-23.
  • the efficacy of the combination of the invention can be measured in vivo by implanting an allograft or xenograft of cancer cells in a rodent and treating the tumor with the combination.
  • Test mice were treated with drugs or controls and monitored for weeks or longer to measure time to tumor doubling, log cell killing, and tumor suppression.
  • mice were subcutaneously inoculated with 6 106 human gastric cancer NCI-N87 cells, and after the tumors were grown to 100-200 mm3, the animals were grouped according to tumor volume (D0). Mice were injected intravenously (IV); volume was 10 mL/kg; solvent group was given the same volume of "solvent" (0.1% BSA normal saline); the specific administration dose and administration schedule are shown in the following table. Tumor volume was measured twice a week, the body weight of the mice was weighed, and data were recorded.
  • the experimental index is to investigate the effect of drugs on tumor growth, and the specific indicators are T/C% or tumor inhibition rate TGI (%).
  • the tumor diameter was measured twice a week with a vernier caliper.
  • the tumor volume (V) was calculated as:
  • V 1/2 ⁇ a ⁇ b2
  • a and b represent length and width, respectively.
  • T/C(%) (T-T0)/(C-C0)x100 where T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.
  • TGI Tumor inhibition rate
  • TGI tumor inhibition rate
  • tumor partial regression PR
  • CR complete tumor regression
  • the end point of the experiment was reached, or the tumor volume reached 1500 mm 3 , the animals were sacrificed by CO 2 anesthesia, and then the tumor was dissected and photographed.

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Abstract

本发明提供了一种与抗体偶联的双取代马来酰胺类连接子及其制备方法和用途，具体地，本发明通过一类新的连接子将强细胞毒活性物质和生物大分子进行偶联。该类连接子可选择性与二硫链同时作用，从而大大提高偶联物的物质均一性。本发明的连接子所制备的偶联物对于表达相应抗原的细胞株具有高抑制活性。本发明还提供了上述偶联物的制备方法和用途。

Description

用于抗体-药物偶联的双取代马来酰胺类连接子及其制备方法和用途 技术领域
本发明涉及一类新型二硫链桥接交联试剂、大分子、治疗用偶联物及其合成方法。更具体而言，本发明涉及通过基于取代马来酰胺的二硫链桥接交联试剂将细胞毒类药物和大分子进行交联而得到的偶联物及其制备方法和用途。
背景技术
多年来，大量研究集中在提高药物或其它制剂向靶细胞、组织或肿瘤递送效率以实现最大疗效和最小毒性。虽然为开发在体内和体外将生物活性分子输入细胞的有效方法已作出许多努力，但是没有一种证明是令人完全满意的。优化药物与其胞内靶分子的结合，同时最大程度减少药物的胞内再分布(如再分布到邻近细胞中)常常是困难或无效的。
目前经胃肠道外给予患者的大部分药物不是靶向的，这导致这种药物全身递送给不需要它们的机体细胞和组织，常常引起不良反应。这可导致药物的不良副作用，常常限制可给予的药物剂量(如化疗(抗癌)，细胞毒，酶抑制剂和抗病毒或抗微生物药物)。比较而言，虽然口服给药被认为是方便和经济的给药模式，但一旦该药物被吸收进入全身循环系统中时，也可能对未受疾病影响的细胞产生相同的非特异性毒性问题。其它复杂情况包括口服生物利用度问题和药物在内脏中滞留问题，这使内脏与药物接触过久，从而有产生内脏毒性的风险。因此，开发(药物递送)方法的主要目标是使药物特异性靶向细胞和组织。这种治疗的益处包括避免将这种药物不合适地递送给其它细胞和组织，如未受感染的细胞而产生的全身生理效应。可通过使生物活性物质即活性代谢物在细胞内累积或保留的方法、化合物和制剂实现胞内靶向。
抗体-药物偶联物(ADC)即免疫偶联物在局部递送细胞毒剂或细胞抑制剂即在癌症治疗中杀死或抑制肿瘤细胞的药物中的应用容许将药物模块靶向递送至肿瘤，并在其中发生胞内积蓄，其中系统施用这些未偶联的药剂在对试图消除的肿瘤细胞之外也对正常细胞产生了不可接受水平的毒性。改进ADC的治疗指数(即最高的功效与最低的毒性)的努力已经聚集于多克隆和单克隆抗体(mAb)的选择性以及药物连接和药效释放特性。
作为新型的靶向治疗药物，抗体-药物偶联物(Antibody Drug Conjguate,ADC)开创了肿瘤治疗方法的新纪元，其基本设计思想最早源于保罗·埃尔利希(Paul Ehrlich)于1913年首次提出的“魔术子弹”(Magic bullet)及药物靶向输送(Drug targeting)概念，即通过适当的载体将药物靶向输送到疾患部位。然而，受制于抗体及高活性细胞毒性药物技术的制约，直到2000年第一个用于治疗急性髓样白血病(AML)的抗体-药物偶联物(MylotargTM)才被FDA批准上市。近期西雅图基因公司(Seattle Genetics)研制的用于治疗霍奇金淋巴瘤(HL)/复发性间变性大细胞淋巴瘤(ALCL)的新药AdcetrisTM(2011)及健泰科生物技术公司(Genentech)研制的用于治疗乳腺癌的新药KadcylaTM(2013)相继通过FDA批准上市，则标志着抗体-药物偶联物在肿瘤治疗领域的应用进入了快速发展阶段。
抗体药物偶联物一般由三部分组成：抗体或抗体类配体，小分子药物，和将配体与药物偶联起来的连接子。目前进入临床试验的抗体药物偶联物结构中，高活性的细胞毒性药物通常是通过连接子连接在配体表面的赖氨酸残基，或者抗体铰链区域的半胱氨酸残基(由链间二硫键部分还原得到)上,最佳的药物/配体比值(DAR)为2-4。抗体表面大量的赖氨酸残基(超过80个)以及偶联反应的非选择性，导致偶联数目和位点的不确定性，进而导致生成的抗体药物偶联物的不均一性。例如，T-DM1(平均DAR值为3.5)的DAR值分布为0-8。同样，尽管抗 体铰链区的链间二硫键只有四对，但为达到最佳平均DAR值(2-4)的要求，需要部分还原链间二硫键。由于现有的还原剂(DTT,TCEP等)无法选择性地还原链间二硫键，因此生成的偶联物也不是均一的产物，由多种组分组成，其主要组分的DAR值为0,2,4,6,8，而且对应每一种特定DAR值的组分都存在由于连接位点不同而形成的异构体。抗体药物偶联物产品的不均一性可以导致各成员组分间药物动力学性质，效价以及毒性的不均一性。例如，具有较高DAR值的组分在体内被清除得更快，并导致更高的毒性。
为了解决抗体药物偶联物均一性问题，定点偶联技术近来得到了更多的青睐，这一技术从位点和数量两方面来控制抗体药物之间的偶联。尽管这些技术都能够实现偶联药物位点和数量的可控，所应用的抗体/蛋白都是通过基因重组的方式得到的。基因重组技术需要大量的工作和精巧的设计，以寻找合宜的位点供药物偶联或聚乙二醇修饰，而现有基因重组技术所获得定点修饰的抗体/蛋白的表达量均较低，因此在大规模制备生产上非常耗时而且研发和最终产业化费用成本非常高。而通过改造后的抗体/蛋白也还需要进一步验证其自身的体内药效与安全性等因素。
针对以上偶联技术存在的问题，通过简单的化学方法对现有抗体实现定点偶联目的，会节省大量的人力物力财力，因此更加富有吸引力。其中已有相关的研究包括：波利泰里克斯有限公司报道的一种偶联技术CN200480019814.4；Igenica Biotherapeutics公司申请的WO2014197871A2；索伦托医疗有限公司申请的CN201380025774.3；上海新理念生物医药科技有限公司申请的CN201310025021.4等专利文献。然而上述技术存在偶联试剂合成路线较长、偶联试剂化学稳定性不佳、抗体偶联物电泳图较为杂乱，提示偶联过程中可能存在副反应、现有方案并未解决体内循环过程中的巯基交换(逆迈克尔加成反应)等问题。
因此，本领域迫切需要提供高效、简单、实用的化学偶联方法，而且达到定点偶联的目的，同时还需要兼顾提高抗体-药物偶联物的稳定性、安全性等性质。
发明内容
本发明的目的是提供一种能够与大多数抗体简单地进行偶联的连接子。
本发明的第一方面，提供了一种取代马来酰胺类连接子片断，结构为式Ia所示：
Figure PCTCN2017112958-appb-000001
其中，R为X或ArS-，
X选自下组：卤素，优选为溴或碘；
Ar选自下组：取代或未取代的C6-C10芳基，取代或未取代的5-12元杂芳基；
优选地，Ar选自苯基、卤代苯、C1-C4烷基苯基、C1-C4烷氧基苯基、2-吡啶基、2-嘧啶基、1-甲基咪唑-2-基、
Figure PCTCN2017112958-appb-000002
其中W为与羰基连接的胺基R1，R1选自-NH2、
Figure PCTCN2017112958-appb-000003
Figure PCTCN2017112958-appb-000004
等；其中：C1-C4烷基苯基进一步优选为4-甲基苯基；C1-C4烷氧基苯基进一步优选为4-甲氧基苯基。
Ar’选自下组：取代或未取代的C6-C10亚芳基，取代或未取代的5-12元亚杂芳基；优选地， Ar’选自取代或未取代的亚苯基或吡啶基，所述的取代指基团上的氢原子被选自下组的一个或多个取代基所取代：卤素、C1-C4烷基、C1-C4烷氧基、三氟甲基、腈基、酰胺基等。
L1为连接于Ar’基团上的-O(CH2CH2O)n-，其中n选自1-20中任一整数，优选为1-10中任一整数。
在另一优选例中，所述连接子片段具有选自下组的结构：
Figure PCTCN2017112958-appb-000005
Figure PCTCN2017112958-appb-000006
Figure PCTCN2017112958-appb-000007
Figure PCTCN2017112958-appb-000008
本发明的第二方面，提供了一种含有如本发明第一方面所述的式Ia连接子片断的取代马来酰胺类连接子-药物缀合物，及其药学上可接受的盐或溶剂物，结构如通式Ib所示：
Figure PCTCN2017112958-appb-000009
其中，R、Ar’、L1的定义同上；
L2为化学键或AA-PAB结构；其中，AA为二肽或三肽片断(即2-3个氨基酸通过肽键连接形成的片段)，优选包括Val-Cit(缬氨酸-瓜氨酸)、Val-Ala(缬氨酸-甘氨酸)、Phe-Lys(苯丙氨酸-赖氨酸)、Ala-Ala-Asn(甘氨酸-甘氨酸-天冬酰胺)、D-Ala-Phe-Lys(D型甘氨酸-苯丙氨酸-赖氨酸)等，PAB为对-氨基苄基氨甲酰基；
CTD为通过酰胺键键合于L2的细胞毒性类小分子药物和/或治疗自身免疫疾病和抗炎症的药物；优选CTD选自下组：微管蛋白抑制剂、拓扑异构酶抑制剂、DNA结合剂；更优选所述的微管蛋白抑制剂选自下组：美登素衍生物、Monomethyl auristatin E、Monomethylauristatin F、Monomethyl Dolastatin 10、Tubulysin类衍生物、Cryptophycin类衍生物、Taltobulin。优选的，所述的DNA结合剂选自下组：PBD类衍生物、duocarmycin类衍生物。优选的，所述的拓扑异构酶抑制剂选自下组：阿霉素代谢产物PNU-159682衍生物、伊立替康代谢产物SN38衍生物、依沙替康。
更具体的，所述的CTD具有选自D1-D13及D13’的分子结构，选自以下组：
Figure PCTCN2017112958-appb-000010
Figure PCTCN2017112958-appb-000011
Figure PCTCN2017112958-appb-000012
在另一优选例中，所述的式Ib化合物选自下组：
Figure PCTCN2017112958-appb-000013
Figure PCTCN2017112958-appb-000014
Figure PCTCN2017112958-appb-000015
Figure PCTCN2017112958-appb-000016
Figure PCTCN2017112958-appb-000017
Figure PCTCN2017112958-appb-000018
Figure PCTCN2017112958-appb-000019
Figure PCTCN2017112958-appb-000020
Figure PCTCN2017112958-appb-000021
Figure PCTCN2017112958-appb-000022
Figure PCTCN2017112958-appb-000023
本发明的第三方面，提供了一种抗体-药物偶联物，所述的偶联物是用如本发明第二方面所述的式Ib取代马来酰胺类连接子药物缀合物与抗体进行偶联形成的。
在另一优选例中，所述的偶联物共价连接有一个或多个药物组分。
在另一优选例中，所述的偶联物中，抗体和药物是通过共价方式(如通过分别共价连接于连接子上)进行偶联。
本发明的另一方面，提供了一种抗体-药物偶联物，所述的偶联物具有通式Ic和/或Id的结构；
Figure PCTCN2017112958-appb-000024
Figure PCTCN2017112958-appb-000025
其中，Ar’、L1、L2、CTD的定义同上；
m＝1.0～5.0，优选为3.0-4.2；
Ab选自下组：为蛋白质、酶、抗体、抗体片段、多肽。
在另一优选例中，所述的通式Id为通式Ic中N-苯基马来酰胺开环后产物。
在另一优选例中，所述的抗体或Ab选自下组：单克隆抗体、双特异性抗体、嵌合抗体、人源化抗体、抗体片段(优选为抗体Fab片段)。
在另一优选例中，所述的抗体-药物偶联物中，通过所述抗体或抗体片断铰链区的二硫链还原生成一对半胱氨酸残基，并通过所述半胱氨酸残基中巯基与所述通式Ib中的芳基硫醚发生取代反应，从而将所述通式Ib化合物连接于所述抗体或抗体片段上。
在另一优选例中，所述的CTD为细胞毒性类小分子药物，优选为微管蛋白抑制剂、拓扑异构酶抑制剂或DNA结合剂。
在另一进一步优选例中，所述的微管蛋白抑制剂选自美登素(maytansine)衍生物，Monomethyl auristatin E(MMAE)，Monomethylauristatin F(MMAF)，Monomethyl Dolastatin 10，Tubulysin类衍生物，Cryptophycin类衍生物，Taltobulin。
在另一进一步优选例中，所述的DNA结合剂选自PBD类衍生物，duocarmycin类衍生物。
在另一进一步优选例中，所述的拓扑异构酶抑制剂选自阿霉素(Doxorubicin)代谢产物PNU-159682衍生物，伊立替康(irinotecan,CPT-11)代谢产物SN38衍生物。
在另一优选例中，所述抗体是能够与选自下组的肿瘤相关抗原结合的抗体：
Figure PCTCN2017112958-appb-000026
在另一优选例中，所述的抗体为HER2抗体，进一步优选为曲妥珠单抗(Trastuzumab)或帕妥珠单抗(Pertuzumab)。
在另一优选例中，所述的抗体为EGFR抗体，进一步优选为Erbitux或Vectibix。
在另一优选例中，所述的抗体为Tissue factor(TF)抗体。
本发明的第四方面，提供了一种药物组合物，上述药物组合物包括：(a)如本发明第三方面所述的抗体-药物偶联物；和(b)药学上可接受的稀释剂，载剂或赋形剂。
本发明的第五方面，提供了一种如本发明第三方面中任一项所述的抗体-药物偶联物在用于制备治疗肿瘤的药物的用途
在另一优选例中，所述的肿瘤选自下组：乳腺癌，卵巢癌，非霍奇金淋巴瘤，霍奇金淋巴瘤，急性淋巴细胞性白血病，间变性大细胞淋巴瘤，多发性骨髓瘤，前列腺癌，非小细胞肺癌，小细胞肺癌，恶性黑色素瘤，鳞状细胞癌，胶质母细胞瘤，肾细胞癌，胃肠道肿瘤，胰腺癌，前列腺癌，直结肠，胃癌，神经胶质瘤，间皮瘤。
本发明的另一方面，提供了一种治疗肿瘤的方法，所述的方法包括步骤：对需要的对象施用治疗有效量的如本发明第三方面所述的抗体-药物偶联物。
在另一优选例中，所述的对象是哺乳动物，优选为人。
本发明的第六方面，提供了一种如本发明第五方面所述的抗体-药物偶联物的制备方法，所述方法包括步骤：
(1)用抗体与还原试剂在缓冲液中反应，得到经还原后的抗体；
(2)用连接子-药物缀合物与步骤(1)得到的经还原后的抗体在缓冲液与一定量有机溶剂混合液中进行交联，得到抗体-药物偶联物。
步骤(1)中所述抗体经还原试剂还原，从而使抗体链间二硫键被还原，产生巯基基团。
在另一优选例中，所述的还原试剂为三(2-羧乙基)膦盐酸盐(TCEP)、beta-巯基乙醇、beta-巯基乙胺盐酸盐、或二硫苏糖醇(DTT)。
在另一优选例中，所述的缓冲液选自下组：磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/氯化钠(NaCl)/二乙基三胺五乙酸(DTPA)缓冲液、磷酸氢二钠-柠檬酸/氯化钠(NaCl)/二乙基三胺五乙酸(DTPA)、硼酸-硼砂/氯化钠(NaCl)/二乙基三胺五乙酸(DTPA)、组氨酸-氢氧化钠/氯化钠(NaCl)/二乙基三胺五乙酸(DTPA)，和PBS/二乙基三胺五乙酸(DTPA)。
在另一优选例中，所述的步骤(2)中，有机溶剂在反应液中的体积占比不超过15％。
在另一优选例中，所述的步骤(2)中的有机溶剂选自下组：乙腈(ACN)、二甲基甲酰胺(DMF)、二甲基乙酰胺(DMA)、二甲基亚砜(DMSO)。
在另一优选例中，所述的步骤(2)中，所述的偶联反应在0-37℃下进行。
在另一优选例中，若所述的步骤(1)采用beta-巯基乙醇、beta-巯基乙胺盐酸盐或DTT还原，在所述的步骤(1)和步骤(2)之间还包括：在还原反应完成后，对产物进行过脱盐柱或超滤以去除还原剂。
在另一优选例中，所述方法的反应路线如下：
Figure PCTCN2017112958-appb-000027
其中R、Ab、Ar’、L1、L2、CTD、m的定义同上。
在另一优选例中，所述方法包括以下步骤：
1)还原：将抗体原液用反应缓冲液稀释至2-10mg/mL，加入140-200倍过量摩尔比的二硫苏糖醇(DTT)，或加入6.0-20倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于10-35℃搅动2-48小时；
2)偶联：将上述反应液冷至0-10℃，再加入取代马来酰亚胺类化合物，在0-37℃搅拌2-5小时。
在另一进一步优选例中，还包括该步骤：当步骤1)采用DTT还原时，在步骤1)还原反应完成后将反应液过脱盐柱或超滤除去过量的DTT；
在另一进一步优选例中，所述取代马来酰亚胺类化合物可预先溶于有机溶剂中，其有机溶剂优先选自：乙腈(ACN)、二甲亚砜(DMSO)、二甲基甲酰胺(DMF)或二乙基乙酰胺(DMA)；进一步优选，取代马来酰亚胺类化合物和有机溶剂按10mg/ml溶解，并保证有机溶剂的体积占比不超过反应液的15％。
在另一进一步优选例中，还包括该步骤：在步骤2)偶联反应完成后将反应混合物用琥珀酸钠/NaCl缓冲液或组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。
在另一进一步优选例中，还包括该步骤：在步骤2)偶联反应完成后将反应混合物超滤，然后过滤除菌，所得产物低温保存；进一步优选保存温度为-100～60℃；进一步优选，所述超滤采用的装置孔径为0.15～0.3微米。
在另一进一步优选例中，所述步骤1)采用TCEP进行还原。可不去除过量的TCEP。
在另一进一步优选例中，步骤1)所述的反应缓冲液可以是：50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM磷酸氢二钠-柠檬酸/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM硼酸-硼砂/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM组氨酸-氢氧化钠/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9和PBS//1mM二乙基三胺五乙酸(DTPA)，pH 6-9。
所得抗体-药物偶联物的药物抗体偶联比(DAR)较为均一，采用本发明中所述的不同取代马来酰亚胺连接子也可将产物均一性有一定差别的抗体-药物偶联物，如需要获得均一性更好的样品，可进一步利用但不限于以下方法进行分离纯化：疏水作用层析方法(HIC)、分子排阻色谱法(SEC)、离子交换层析(IEC)。
本发明的第七方面，提供了第一方面所述的取代马来酰亚胺类连接子(式Ia的优选例E)的制备方法：
通过中间体C与二卤代马来酸酐环合反应得中间体D，再与芳基硫酚进行取代反应后获 得连接子片断分子E，反应式如下：
Figure PCTCN2017112958-appb-000028
其中R、n同上所述，X代表卤素，优选Br、Cl；U、V各自独立代表N或C。
在另一优选例中，所述C可通过B还原而得，反应式如下：
Figure PCTCN2017112958-appb-000029
其中R、n、U、V与上述相同。
在另一进一步优选例中，所述B可通过A与氟代硝基苯取代反应而得，反应式如下：
Figure PCTCN2017112958-appb-000030
其中R、n、U、V与上述相同。
在另一进一步优选例中，所述B可通过下式制备：
Figure PCTCN2017112958-appb-000031
其中R、n、U、V与上述相同。
在另一更进一步优选例中，A由n甘醇与卤代乙酸叔丁酯反应而得。反应式如下：
Figure PCTCN2017112958-appb-000032
其中n、X与上述相同。
本发明的第八方面，提供了第二方面所述的取代马来酰亚胺类连接子-药物缀合物(式Ib的优选例F1或F’1)的制备方法：取代马来酰亚胺类连接子(式Ia的优选例Ea)与带有二 肽/三肽-PAB细胞毒药物CTD进行缩合，分别得到F1或F’1。
反应路线如下：
Figure PCTCN2017112958-appb-000033
其中R同式Ia的定义，Rx代表卤素、C1-C4烷基、C1-C4烷氧基、三氟甲基、腈基或酰胺基，Ry代表H或烷基。
本发明人经过广泛而深入的研究，发现了一类连接子结构，该连接子可以全部/部分交叉偶联抗体的轻链-重链及重链-重链，且应用此种偶联方法得到的抗体-药物偶联物，与传统抗体-药物偶联物相比，具有更窄的药物/抗体比值(DAR)分布。基于上述发现，发明人完成了本发明。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
术语
在本文中，除特别说明之处，术语“C1-C4烷基”指具有1-4个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、或类似基团。
术语“C1-C4烷氧基”指具有1-4个碳原子的直链或支链烷氧基，例如甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、仲丁氧基、叔丁氧基、或类似基团。
术语“卤素”指F、Cl、Br和I。
术语“C6-C10芳基”指具有6-10个碳原子的芳基，例如苯基、萘基等，所述的芳基可以是取代或未取代的。
术语“C6-C10芳基”指具有6-10个碳原子的芳基，例如苯基、萘基等，所述的芳基可以是取代或未取代的。
术语“5-12元杂芳基”、“5-12元亚杂芳基”指具有5-12个碳原子和一个或多个(优选1-3个)选自O、S和/或N的杂原子的杂芳基或亚杂芳基，优选5-8元杂芳基或亚杂芳基。所述的杂芳基或亚杂芳基可以是取代或未取代的。
本发明中，术语“药学上可接受的”成分是指适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)，即有合理的效益/风险比的物质。
本发明中，术语“有效量”指治疗剂治疗、缓解或预防目标疾病或状况的量，或是表现出可检测的治疗或预防效果的量。对于某一对象的精确有效量取决于该对象的体型和健康状况、病症的性质和程度、以及选择给予的治疗剂和/或治疗剂的组合。因此，预先指定准确的有效量是没用的。然而，对于某给定的状况而言，可以用常规实验来确定该有效量，临床医师是能够判 断出来的。
除非特别说明，本发明中，所有出现的化合物均意在包括所有可能的光学异构体，如单一手性的化合物，或各种不同手性化合物的混合物(即外消旋体)。本发明的所有化合物之中，各手性碳原子可以任选地为R构型或S构型，或R构型和S构型的混合物。
如本文所用，术语“本发明化合物”指式I所示的化合物。该术语还包括及式I化合物的各种晶型形式、药学上可接受的盐、水合物或溶剂合物。
如本文所用，术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐。适合形成盐的酸包括但并不限于：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。
除非特别说明，本文中所使用的“氨基酸”意在包括任何常规氨基酸，如天冬氨酸、谷氨酸、半胱氨酸、天冬酰胺、苯丙氨酸、谷氨酰胺、酪氨酸、丝氨酸、甲硫氨酸(蛋氨酸)、色氨酸、甘氨酸、缬氨酸、亮氨酸、丙氨酸、异亮氨酸、脯氨酸、苏氨酸、组氨酸、赖氨酸、精氨酸。
当本文中使用商品名时，该商品名意在包括商品名产品制剂、其相应的仿制药，以及商品名产品的活性药物组分。
本文的术语“抗体”以其最广泛的含义使用并且特别覆盖单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体(例如双特异性抗体)和抗体片段，只要它们表现出所需的生物活性(Miller等(2003)Journal of Immunology 170:4854-4861)。抗体可以为鼠、人、人源化、嵌合的抗体或来源于其它物种。抗体为由能够识别和结合特异性抗原的免疫系统产生的蛋白质(Janeway,C.,Travers,P.,Walport,M.,Shlomchik(2001)ImmunoBiology,5thEd.,Garland Publishing,NewYork)。靶抗原一般具有由多种抗体的CDRs识别的大量结合位点，也称作表位。特异性结合不同表位的各抗体具有不同的结构。因此，一种抗原可以具有一种以上相应的抗体。抗体包括全-长免疫球蛋白分子或全-长免疫球蛋白分子的免疫活性部分，即含有特异性结合所关注靶标的抗原或其部分的分子，这类靶标包括，但不限于癌细胞或产生与自身免疫性疾病相关的自身免疫抗体的细胞。本文披露的免疫球蛋白可以具有免疫球蛋白分子的任意类型(例如IgG、IgE、IgM、IgD和IgA)、类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类。免疫球蛋白可以来源于任意的物种。然而，在一个方面中，免疫球蛋白来源于人、鼠或兔。
“抗体片段”包含全长抗体的一部分，一般为其抗原结合区或可变区。抗体片段的实例包括：Fab、Fab’、F(ab’)2和Fv片段；双抗体；线性抗体；微抗体(minibody)(Olafsen等(2004)Protein Eng.Design&Sel.17(4):315-323)；Fab表达文库制备的片段；抗-独特型(抗-Id)抗体；CDR(互补决定区)；和以免疫特异性方式结合癌细胞抗原、病毒抗原或微生物抗原的上述任意的表位-结合片段；单-链抗体分子；和由抗体片段形成的多特异性抗体。
本发明中组成抗体药物偶联物的抗体最好保持其原有野生状态时的抗原结合能力。因此，本发明中的抗体能够，最好专一性地，与抗原结合。涉及的抗原包括，例如，肿瘤相关抗原(TAA)，细胞表面受体蛋白和其他细胞表面分子，细胞存活调节因子，细胞增殖调节因子，与组织生长与分化相关的分子(如已知或预知的具有功能性的)，淋巴因子，细胞因子，参与细胞循环调节的分子，参与血管生成的分子，以及与血管生成有关的分子(如已知抗体结合的抗原可以是上述分类中一个或一个子集，而其它的子集则包含其它的具有特殊性质的分子/抗原(与目标抗原相比)。
应用在抗体药物偶联物中的抗体包括，但不局限于，针对细胞表面受体和肿瘤相关抗原 的抗体。这样的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)-(53)。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，与参考文献中确认的序列具有至少70％，80％，85％，90％，或95％的同源性，或者具备与引用文献中的肿瘤相关抗原序列具有完全一致的生物性质和特征。
(1)HER2(Gene ID:2064，人类表皮生长因子受体2(英语：human epidermal growth factor receptor 2，缩写为HER2，亦称为Neu、ErbB-2、CD340(分化群340)或p185)是一种由ERBB2基因编码的蛋白质。HER2是表皮生长因子受体(EGFR/ErbB)家族中的成员之一)；(2)HER3(Gene ID:2065,表皮因子受体3(ErbB3/HER3)是表皮生长因子跨膜受体家族的成员之一。近年来证实ErbB3/HER3与乳腺癌的发病、复发转移、化疗以及内分泌治疗的疗效密切相关，已成为非常有前景的治疗候选靶点)；(3)CD19(Gene ID:930)；(4)CD20(Gene ID:931)；(5)CD22(Gene ID:933)；(6)CD30(Gene ID:943)；(7)CD33(Gene ID:945)；(8)CD37(Gene ID:951)；(9)CD45(Gene ID:5788)；(10)CD56(Gene ID:4684)；(11)CD66e(Gene ID:1048)；(12)CD70(Gene ID:970)；(13)CD74(Gene ID:972)；(14)CD79b(Gene ID:974)；(15)CD138(Gene ID:6382)；(16)CD147(Gene ID:682)；(17)CD223(Gene ID:3902)；(18)EpCAM(Gene ID:4072)；(19)Mucin 1(Gene ID:4582)；(20)STEAP1(Gene ID:26872)；(21)GPNMB(Gene ID:10457)；(22)FGF2(Gene ID:2247)；(23)FOLR1(Gene ID:2348)；(24)EGFR(Gene ID:1956)；(25)EGFRvIII(GenBank:GM832119.1)；(26)Tissue factor(TF)(Gene ID:2152)；(27)c-MET(Gene ID:4233)；(28)Nectin 4(Gene ID:81607)；(29)AGS-16；(30)Guanylyl cyclase C(Gene ID:2984)；(31)Mesothelin(Gene ID:10232)；(32)SLC44A4(Gene ID:80736)；(33)PSMA(Gene ID:2346)；(34)EphA2(Gene ID:1969)；(35)AGS-5；(36)GPC-3(Gene ID:2719)；(37)c-KIT(Gene ID:3815)；(38)RoR1(Gene ID:4919)；(39)PD-L1(Gene ID:29126)；(40)CD27L(Gene ID:970)；(41)5T4(Gene ID:7162)；(42)Mucin 16(Gene ID:94025)；(43)NaPi2b(Gene ID:10568)；(44)STEAP(Gene ID:26872)；(45)SLITRK6(Gene ID:84189)；(46)ETBR(Gene ID:1910)；(47)BCMA(Gene ID:608)；(48)Trop-2(Gene ID:4070)；(49)CEACAM5(Gene ID:1048)；(50)SC-16；(51)SLC39A6(Gene ID:25800)；(52)Delta-like protein3(DLL3)(Gene ID:10683)；(53)Claudin 18.2(Gene ID:51208)。
如本文所用，“药物”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。因此，只要具有必需的反应性官能团，术语“药物”涉及的化合物包括正式国家药典，以及例如美国正式同种疗法药典，正式全国处方集，或者其任何增补本等确认的药物。典型的药物列于医师案头用药参考(PDR)和美国食品药品监督管理局(FDA)的橙皮书。随着新型药物不断被发现和发展，本专利规定这些药物也应纳入本发明所述偶联药物的中的“药物”。
较佳地，所述的药物是指：用于癌症治疗的细胞毒性药物，或具有期望生物活性的蛋白或多肽，例如一种毒素，如相思子毒素，蓖麻毒素A，假单胞菌外毒素，和白喉毒素；其他 合适的蛋白包括肿瘤坏死因子，α-干扰素，β-干扰素，神经原生长因子，血小板衍生生长因子，组织型纤酶溶原生长因子，以及生物反应调节制剂，例如淋巴因子，白细胞介素-1(IL-1),白细胞介素-2(IL-2),白细胞介素-6(IL-6),粒细胞巨噬细胞集落刺激因子(GM-CSF),粒细胞集落刺激因子，或其它生长因子。
一种优选的本发明药物是美登素或类美登素。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。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。以下列出了去乙酰基美登素的结构。
Figure PCTCN2017112958-appb-000034
另一种优选的本发明药物是耳抑素肽类药物。耳抑素肽类药物是海兔毒素10(Dolastatin10)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。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。
Figure PCTCN2017112958-appb-000035
Monomethyl Auristatin E(MMAE)
Figure PCTCN2017112958-appb-000036
Monomethyl Auristatin F(MMAF)
Figure PCTCN2017112958-appb-000037
Monomethyl Dolastatin 10(MMAD)
另一种优选的本发明药物是微管溶素(Tubulysin)。微管溶素首先由研究小组从粘细菌培养物中分离，是非常有效的细胞生长抑制剂，其通过抑制微管蛋白聚合并从而诱导细胞凋亡而起作用。微管溶素中的微管溶素D是最有效的，具有超过大多数其他微管蛋白调节剂(包括埃博霉素、长春碱和紫杉醇)10至100倍的活性。紫杉醇和长春碱目前用于多种癌症的治疗，而埃博霉素衍生物正在临床试验中进行活性评价。微管溶素D的合成衍生物将提供与抑制和关键结合相互作用有关的必要信息，并且作为抗癌剂可以具有优越的性质，所述抗癌剂作为分离的实体或作为靶向抗体或配体上的化学弹头。微管溶素D是复杂的四肽，其可以分为四个区域，Mep(D-N-甲基哌啶甲酸)、Ile(异亮氨酸)、Tuv(管式缬氨酸，tubuvaline)和Tup(管式苯丙氨酸，tubuphenylalanine)，如下式所示：
Figure PCTCN2017112958-appb-000038
另一种优选的本发明药物是可抑制微管聚合的微生物来源的cryptophycin衍生物。Cryptophycin是从蓝藻的培养物中分离得到的一种能抑制微管生成的新型抗肿瘤活性物质，对多种肿瘤有活性。Cryptophycin是一个脂溶性化合物，含有2个肽键及2个酯键，有5个光学活性中心及1个环氧基团。双肽双酯键都在一个大环结构中。Cryptophycin衍生物CP1和CP2结构如下式所示：
Figure PCTCN2017112958-appb-000039
另一种优选的本发明药物是新型抗微管剂Taltobulin(HTI-286、SPA-110)。Taltobulin抑制纯化微管的多聚化，干扰细胞内微管组织、诱导有丝分裂阻断以及诱导细胞凋亡。Taltobulin是细胞增殖强效抑制剂，对18种人肿瘤细胞系的平均IC50为2.5nM。和目前应用的抗微管剂相比，Taltobulin并不是p-糖蛋白的合适的底物，其中Taltobulin结构如下图所示。
Figure PCTCN2017112958-appb-000040
一方面，药物是喜树碱类药物衍生物SN-38。SN-38是伊立替康盐酸盐(CPT-11)的生物活性代谢物，为一类拓扑异构酶抑制剂。SN-38引起DNA拓扑异构酶I的抑制最强，剂量依赖性和时间依赖性地抑制DNA的合成，并造成频繁的DNA单链断裂。其中SN-38结构如下图所示。
Figure PCTCN2017112958-appb-000041
一方面，药物是喜树碱类药物衍生物Exatecan。是拓扑异构酶I抑制剂喜树碱的合成类似物，活性较SN-38更强，可引起DNA拓扑异构酶I的抑制最强，剂量依赖性和时间依赖性地抑制DNA的合成，并造成频繁的DNA单链断裂。其中Exatecan结构如下式所示。
Figure PCTCN2017112958-appb-000042
另一种优选的本发明药物是鹅膏覃碱类药物(alpha-Amanitin)，结构如下图所示。alpha-Amanitin是一种来自毒蘑菇鬼笔鹅蕈(Amanita phalloides)的真菌毒素，二环八肽，能抑制真核RNA聚合酶Ⅱ与RNA聚合酶Ⅲ转录。
Figure PCTCN2017112958-appb-000043
另一种优选的本发明药物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，具有弱化的前药毒性和增强的水溶性(其中CBI-seco类结构通式如下图所示)：
Figure PCTCN2017112958-appb-000044
另一种优选的本发明药物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。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。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 PCTCN2017112958-appb-000045
另一种优选的本发明药物是PNU-159682衍生物，PNU-159682是Nemorubicin在人肝微粒体中的主要活性代谢产物，与MMDX和阿霉素相比，活性提高3000倍。
Figure PCTCN2017112958-appb-000046
另一方面，药物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。并且尤其是那些能够通过与接头的酰胺键来配位，如通过具有碱性胺基(一级胺或二级胺)来配位的细胞毒素，例如上文中所示的细胞毒素D1-D12的结构。
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：不可断裂连接子和可断裂连接子。
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)。
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(pH7.3-7.5)，但是在弱酸性的内涵体(pH5.0-6.5)和溶酶体(pH4.5-5.0)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(CathepsinB)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包括Val-Cit(VC),Phe-Lys等。
自释放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。自释放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，自释放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包括对氨基苄醇类(PAB)和β-葡萄糖醛酸苷类(β-Glucuronide)等。
连接子
本发明的连接子或偶联试剂，包含二芳硫基马来酰胺单元和一个偶联基团。二芳硫基马来酰胺单元用于交联抗体链间的巯基基团(还原后)，而偶联基团用于与小分子药物或药物-连接子单元偶联。由于该二芳硫基马来酰胺单元与抗体中的开放半胱氨酸-半胱氨酸二硫键的两个硫原子的二齿结合(bidentate binding)，因此这些ADC是均质的并比含有单齿接头的ADC具有更强的稳定性。因此它们将具有增长的体内半衰期，减少全身性释放的细胞毒素的量，并且比具有单齿接头的ADC更加安全的药物性质。
在另一个方面，所产生的药物-连接子单元通过该连接子与抗体偶联，生成部分链间交联的偶联物。与传统的抗体药物偶联物相比，应用本发明方法制备的抗体药物偶联物的药物/抗体比值(DAR)分布更窄，从而大幅提升了产品均一性及药理学特性均一性。
该抗体药物偶联物可用于靶向输送药物到达目标细胞群体，例如肿瘤细胞。抗体药物偶 联物可以特异性的与细胞表面蛋白结合，所产生的结合物随即被细胞内吞。在细胞内，药物以活性药物的方式释放出来产生功效。抗体包括嵌合抗体，人源化抗体，人抗体；可与抗原结合的抗体片段；或者抗体Fc融合蛋白；或者蛋白。“药物”是高活性药物(见定义部分)，在某种情况下，药物可以是聚乙二醇。
抗体-药物偶联物
本发明提供的抗体药物偶联物由抗体、连接子、连接子和药物组成，所述连接子是可断裂连接子组合或不可断裂连接子。
抗体是球状蛋白，含有一系列氨基酸位点可用于偶联药物-连接子。由于其三级和四级结构，只有溶剂可及的氨基酸可供偶联。事实上，高收率的偶联通常发生在赖氨酸残基的ε-氨基基团或半胱氨酸残基的巯基基团上。
抗体蛋白表面的大量赖氨酸侧链导致大量的位点可供药物偶联，从而导致生成的抗体药物偶联物是混合物，含有不同的药物偶联数量(药物/抗体比值，DAR)和偶联位点。
本发明提供的偶联产品，尽管仍然是混合物，但与传统方式偶联得到的抗体药物偶联物相比，其DAR分布范围很窄。其平均DAR值接近4，接近最佳抗体药物偶联物平均DAR值(2-4)范围。此外，偶联产品极少不含有裸抗(DAR＝0)，这一组分对细胞毒杀不起作用。同时，偶联产品也不含有重度偶联产品(DAR＝8),这一组分在体内的清除速度很快，相对于低DAR的组分而言。因此，本发明提供的抗体药物偶联物产品非均一性得到很大的改善。
抗体-药物偶联物的制备方法
抗体药物偶联物制备路线如下所示。抗体链间二硫键被还原产生共8个巯基基团骤，取代马来酰胺类连接子药物缀合物与还原后的抗体巯基交联，生成相应的抗体药物偶联物，其中该抗体药物偶联物存在如图所示中的一种或二种形式。
Figure PCTCN2017112958-appb-000047
将抗体原液用反应缓冲液稀释至2-10mg/mL，加入140-200倍过量摩尔比的二硫苏糖醇(DTT)，或加入6.0-20倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于10-35℃搅动2-48小时；在此所述反应缓冲液可以是按以下比例制备的缓冲液：50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM磷酸氢二钠-柠檬酸/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM硼酸-硼砂/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9；50mM组氨酸-氢氧化钠/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6-9和PBS//1mM二乙基三胺五乙酸(DTPA)，pH 6-9。
将上述反应液冷至0-10℃，若采用DTT还原，需在还原反应完成后过脱盐柱或超滤除去过量的DTT，再加入取代马来酰胺类化合物(预先10mg/ml溶在乙腈(ACN)、二甲亚砜(DMSO)、二甲基甲酰胺(DMF)或二乙基乙酰胺(DMA)中)，并保证反应液中有机溶剂的体积 占比不超过15％，偶联反应于0-37℃搅动2-4小时。若采用TCEP还原，也可不需除去剩余TCEP，直接加入取代马来酰胺类化合物进行偶联。
采用脱盐柱将偶联反应混合物用琥珀酸钠/NaCl缓冲液或组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。或超滤数遍。然后过滤除菌，所得产物低温保存。优选温度为-100—60℃，过滤装置的孔径优选0.15-0.3微米。
所得抗体药物偶联物的药物抗体偶联比(DAR)较为均一，采用本专利中所述的不同取代马来酰胺连接头也将产物均一性有一定差别的抗体药物偶联物，如需要获得均一性更好的样品，可进一步利用但不限于以下方法进行分离纯化：疏水作用层析方法(HIC)、分子排阻色谱法(SEC)、离子交换层析(IEC)。
药物组合物和施用方法
由于本发明提供的抗体-药物偶联物，可以靶向瞄准特殊的细胞群体，与细胞表面特异蛋白(抗原)结合，从而通过结合物内吞或药物渗入使得药物以活性形式释放到细胞内，因此，本发明的抗体-药物偶联物可以用于治疗目标疾病，上面提到的抗体-药物偶联物可以以治疗有效量，通过合适的途径给予受试者(例如人)。需要治疗的受试者可以是有风险，或怀疑患有与特定抗原的活性或表达量有关病症的患者。这样的患者可以通过常规体检来鉴定。
常规方法，已知的医学领域的普通技术人员，可以用于施用药物组合物给受试者，这取决于疾病的要治疗的类型或疾病的部位。此组合物还可以通过其它常规途径，例如，口服、肠胃外给药、通过吸入喷雾、局部、直肠、经鼻、口腔、阴道或通过植入进行给药。本文所用的术语“肠胃外”包括皮下、皮内、静脉内、肌内、关节内、动脉内、滑膜内、胸骨内、鞘内、病灶内和颅内注射或输注技术。此外，它可以施用到通过施用可注射的贮库途径，例如使用1-，3-，或6个月的贮库可注射或可生物降解的材料和方法的主题。
注射组合物可以含有各种载体如植物油，二甲基乙酰胺(dimethylactamide)，二甲基甲酰胺，乳酸乙酯，碳酸乙酯，肉豆蔻酸异丙酯，乙醇，多元醇(甘油，丙二醇，液体聚乙二醇，等等)。对于静脉内注射，水溶性抗体可以通过点滴方法，由此含有抗体和生理上可接受的赋形剂的药物制剂输注给药。生理上可接受的赋形剂可以包括，例如，5％葡萄糖，0.9％盐水，林格溶液或其它合适的赋形剂。肌内制剂，例如，抗体的一个合适的可溶盐形式的无菌制剂，可以溶解和施用的药用赋形剂诸如水换注射液，0.9％盐水，或5％葡萄糖溶液。
当用本发明的抗体-药物偶联物治疗时，可以通过本领域常规的方法进行递送。例如，它可以通过使用脂质体、水凝胶、环糊精、生物可降解的纳米胶囊、或生物粘附性微球被引入到细胞中。或者，所述核酸或载体可在本地通过直接注射或通过使用输注泵递送。其它方法包括通过使用缀合物和生物可降解的聚合物的使用各种运输和载体系统。
本发明的药物组合物含有安全有效量的本发明的抗体-药物偶联物以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物可以被制成溶液剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。
本发明所述的抗体-药物偶联物的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的双功能抗体偶联物的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本发明的抗体-药物偶联物每天以约0.0001mg-50mg/kg动物体重(较佳的0.001mg-10mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫 切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药，或者与其他药学上可接受的治疗剂联合给药。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选5～500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
实验表明，本发明的主要优点在于：
1、本发明提供的新型连接子，可通过简单的化学方法与抗体偶联，与传统抗体药物偶联物相比，应用这种连接子得到的偶联物DAR值分布非常窄，因此生成的产品均一性高，获得的交联物单一分布的组份(DAR为4)占比80％以上。
2、本发明提供的抗体-药物偶联物，裸抗和低交联度的ADC占比几乎为零(质谱检测不出DAR为0和1的组份)。
3、申请人通过大量的实验证明，本发明提供的抗体-药物偶联物，在治疗肿瘤方面具有一定安全性和有效性。偶联后乙二醇所赋予的亲水性可以用来调节生物分子特性；交联物的体外肿瘤细胞增殖抑制活性较传统mcVC-PAB交联生物学活性、药物代谢稳定性、安全性等成药性质方面有所提高或保持。
4、本发明提供的偶联方法，适用于大部分抗体，从而可以避免对每一种抗体进行繁琐的重组改造以引入定点偶联位点，因此具有广泛的应用前景。
5、本发明提供的偶联方法与现有偶联方法相比，本发明的基于马来酰胺的二硫链桥接交联试剂的优点包括：具有较快的交联速度，交联反应时间通常在2-4小时以内便可反应完毕。6、本发明基于马来酰亚胺的二硫链桥接具有更好的稳定性，在体内不易发生巯醚交换，同时在Ar’部位引入取代基较未取代的苯基可以大大减缓马来酰亚胺开环后的环合二次水解反应，进一步加强了抗体-药物偶联物在体外和体内的稳定性。
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，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
附图说明
图1：显示了帕妥珠单抗与帕妥珠药物偶联物疏水作用层析(HIC)对比图谱；
图2：显示了帕妥珠单抗与帕妥珠药物偶联物质谱对比图谱；
图3：显示了ADC-2、P-mcVC-MMAE、Pertuzumab单抗与NCI-N87细胞表面抗原Her2结合实验；
图4：显示了ADC-2、P-mcVC-MMAE、Kadcyla、Pertuzumab对人乳腺癌细胞SK-BR-3的增殖抑制实验；
图5：显示了ADC-2、P-mcVC-MMAE、Kadcyla、Pertuzumab对人乳腺癌细胞BT-474的增殖抑制实验；
图6：显示了ADC-2、P-mcVC-MMAE、Kadcyla、Pertuzumab对人胃癌细胞N87的增殖抑制实验；
图7：显示了ADC-2对SKOV-3人卵巢癌细胞、Du-145人前列腺癌细胞和Panc-1人胰腺癌的增殖抑制实验；
图8：显示了ADC-2对人乳腺癌MCF-7、MDA-MB-231和MDA-MB-468细胞的增殖抑制实验；
图9-1：显示了ADC-4对人胃癌细胞N87的增殖抑制实验；
图9-2：显示了ADC-2、ADC-3与ADC-4对Calu-3人肺癌细胞的增殖抑制实验；
图10：显示了ADC-2、ADC-3、P-mcVC-MMAE、Kadcyla抑制人胃癌NCI-N87裸小鼠皮下移植瘤的活性研究。
图11-1：帕妥珠单抗(Pertuzumab)的疏水作用层析(HIC)图谱；
图11-2：帕妥珠单抗-药物偶联物ADC-I的疏水作用层析(HIC)图谱；
图11-3：帕妥珠单抗-药物偶联物ADC-II的疏水作用层析(HIC)图谱；
图11-4：帕妥珠单抗-药物偶联物ADC-III的疏水作用层析(HIC)图谱；
图11-5：帕妥珠单抗-药物偶联物ADC-IV的疏水作用层析(HIC)图谱；
图11-6：帕妥珠单抗-药物偶联物ADC-V的疏水作用层析(HIC)图谱；
图11-7：帕妥珠单抗-药物偶联物ADC-VI的疏水作用层析(HIC)图谱；
图11-8：帕妥珠单抗-药物偶联物ADC-VII的疏水作用层析(HIC)图谱；
图12-1：曲妥珠单抗(Trastuzumab)的疏水作用层析(HIC)图谱；
图12-2：曲妥珠单抗-药物偶联物ADC-VIII的疏水作用层析(HIC)图谱；
图13-1：帕妥珠单抗(Pertuzumab)的质谱图谱；
图13-2：帕妥珠单抗-药物偶联物ADC-I的质谱图谱；
图13-3：帕妥珠单抗-药物偶联物ADC-II的质谱图谱；
图13-4：帕妥珠单抗-药物偶联物ADC-III的质谱图谱；
图13-5：帕妥珠单抗-药物偶联物ADC-IV的质谱图谱；
图13-6：帕妥珠单抗-药物偶联物ADC-V的质谱图谱；
图13-7：帕妥珠单抗-药物偶联物ADC-VI的质谱图谱；
图13-8：帕妥珠单抗-药物偶联物ADC-VII的质谱图谱；
图14-1：曲妥珠单抗(Trastuzumab)的质谱图谱；
图14-2：曲妥珠单抗-药物偶联物ADC-VIII的质谱图谱；
图15：显示了采用LC-MS(Q-TOF)测定各个ADC对照、ADC-I、ADC-II、ADC-VII在0-7天室温下对应的二次水解产物生成变化趋势图；
图16-1：显示了对照ADC在0天室温下对应的HIC图谱；
图16-2：显示了对照ADC在2天室温下对应的HIC图谱；
图16-3：显示了对照ADC在4天室温下对应的HIC图谱；
图16-4：显示了对照ADC在7天室温下对应的HIC图谱；
图17-1：显示了ADC-I在0天室温下对应的HIC图谱；
图17-2：显示了ADC-I在2天室温下对应的HIC图谱；
图17-3：显示了ADC-I在4天室温下对应的HIC图谱；
图17-4：显示了ADC-I在7天室温下对应的HIC图谱；
图18-1：显示了ADC-II在0天室温下对应的HIC图谱；
图18-2：显示了ADC-II在2天室温下对应的HIC图谱；
图18-3：显示了ADC-II在4天室温下对应的HIC图谱；
图18-4：显示了ADC-II在7天室温下对应的HIC图谱；
图19-1：显示了ADC-VII在0天室温下对应的HIC图谱；
图19-2：显示了ADC-VII在2天室温下对应的HIC图谱；
图19-3：显示了ADC-VII在4天室温下对应的HIC图谱；
图19-4：显示了ADC-VII在7天室温下对应的HIC图谱；
图20：ADC-I、ADC-II、ADC-III、ADC-IV、ADC-V、ADC-VI、ADC-VII、Pertuzumab(Perjeta)对人胃癌细胞NCI-N87的增殖抑制实验结果图；
图21：ADC-I、ADC-II、ADC-III、ADC-IV、ADC-V、ADC-VI、ADC-VII、Pertuzumab(Perjeta)对人乳腺癌细胞BT-474的增殖抑制实验结果图；
图22：ADC-VIII、Trastuzumab(Herceptin)对人胃癌细胞NCI-N87的增殖抑制实验结果图；
图23：ADC-VIII、Trastuzumab(Herceptin)对人乳腺癌细胞BT-474的增殖抑制实验结果图；
图24：P-mcVC-MMAE(1.0mg/kg)、对照ADC(0.5，1.0mg/kg)、ADC-I(1.0mg/kg)、ADC-IV(1.0mg/kg)、ADC-V(1.0mg/kg)、ADC-VI(1.0mg/kg)、ADC-VII(0.5,1.0mg/kg)抑制人胃癌NCI-N87裸小鼠皮下移植瘤的活性研究图。。
具体实施方式
本发明人经过广泛而深入的研究，发现了一类连接子结构，该连接子可以全部/部分交叉偶联抗体的轻链-重链及重链-重链，且应用此种偶联方法得到的抗体药物偶联物，与传统抗体药物偶联物相比，具有更窄的药物/抗体比值(DAR)分布。基于上述发现，发明人完成了本发明。
具体设计一：取代马来酰胺类连接子片断Ia制备及其应用，其中Ar’选自未取代的C6-C10亚芳基，或未取代的5-12元亚杂芳基。
Figure PCTCN2017112958-appb-000048
一、化合物合成与制备方法
方案一：
本发明第一方面中列出的式Ia所代表的取代马来酰胺类连接子片断分子可通过方案一中的方法合成，通过n甘醇与氟代硝基苯取代反应得到中间体B，后经还原硝基获得氨基化合物C，2,3-二溴马来酰亚胺与芳基硫酚进行取代反应后获得中间体D，后与氯甲酸甲酯反应获得中间体E，2,3-二溴马来酰亚胺也可直接与氯甲酸甲酯反应获得中间体E’，中间体C与中间体E或中间体E’反应获得连接子片断分子F。反应路线与具体实例举例如下：
Figure PCTCN2017112958-appb-000049
实施例1(式1-12化合物的合成与制备)
1.1化合物F-1(式1)的合成
1.1.1中间体B-1的合成(步骤a)
Figure PCTCN2017112958-appb-000050
称取4-氟硝基苯(10.0g，0.071mol)，二甘醇(75.2g，0.71mol)和碳酸钾(14.7g，0.11mol)置于250mL圆底瓶，氮气保护下在80℃搅拌22小时。慢慢降温至室温，二氯甲烷萃取，依次用1mol/L稀盐酸、水和饱和食盐水洗涤，无水硫酸钠干燥，旋干溶剂。柱层析(硅胶，200-300目，PE/EtOAC10:1)得到产物，淡黄色透明液体(15.1g，产率94％)。LC-MS(M+)理论值:227.08，LC-MS(ESI,M+H+)实测值:228.12。
1.1.2中间体C-1的合成(步骤b)
Figure PCTCN2017112958-appb-000051
中间体化合物B-1(3.0g，9.52mmol)溶在丙酮(30mL)，冰水浴冷却，慢慢滴加新鲜制备琼斯试剂(15mL)，反应混合物室温搅拌3小时，冰水浴冷却，慢慢滴加异丙醇，冰水浴中继续搅拌15分钟后，旋蒸除去有机溶剂，乙醚萃取水相3次，合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，旋蒸除去溶剂，得黄色油状粗品，该中间体不经纯化分离直接用于下步反应。上述中间体溶于四氢呋喃(30mL)中，加入10％钯炭(300mg)，在30℃氢化反应6小时。抽滤除去催化剂后，旋蒸除去溶剂，得棕黄色油状粗品。不需进一步纯化用于下步反应。LC-MS(M+)理论值:211.08，LC-MS(ESI,M+H+)实测值:212.05。
1.1.3中间体D-1的合成(步骤c)
Figure PCTCN2017112958-appb-000052
2,3-二溴马来酰亚胺(7.0g，27.69mmol)溶于甲醇(80mL)中，依次加入乙酸钠(4.5g，55.4mmol)和苯硫酚(11.3mL，110.7mmol)。反应混合物室温搅拌30分钟，旋蒸除去有机溶剂，二氯甲烷萃取，依次用水和饱和食盐水洗涤，无水硫酸钠干燥，旋干溶剂。粗品用石油醚/乙酸乙酯重结晶，抽滤干燥，得亮黄色固体D-1(6.5g，产率75％)。
1.1.4中间体E-1的合成(步骤d)
Figure PCTCN2017112958-appb-000053
中间体D-1(3.0g，9.6mmol)和N-甲基吗啡啉(1.37mL，12.5mmol)溶于乙酸乙酯(40mL)中，冰水浴冷却，慢慢滴加入氯甲酸甲酯(1.11mL，14.4mmol)，反应混合物室温搅拌30分钟。反应液用乙酸乙酯稀释后，加水萃取，依次用水和饱和食盐水洗涤，无水硫酸钠干燥，旋干溶剂。粗品用石油醚/乙酸乙酯重结晶，抽滤干燥，得亮黄色固体E-1(3.5g，产率98％)。
1.1.5化合物F-1的合成(步骤e)
Figure PCTCN2017112958-appb-000054
中间体C-1(2.0g，9.5mmol)溶于无水二氯甲烷(40ml)中，加入中间体E-1(3.5g，9.5mmol)，氮气保护下反应混合物室温搅拌24小时，称取硅胶(12g，200-300目)加入，反应混合物继续室温搅拌过夜。旋干溶剂，干法柱层析(硅胶，200-300目，二氯甲烷/甲醇10:1)得到产物F-1为橘黄色油状(4.1g，产率85％)。LC-MS(M+)理论值:507.08，LC-MS(ESI,M+H+)实测值:508.11。
1.2化合物F-2(式2)的合成
Figure PCTCN2017112958-appb-000055
化合物F-2的合成与例1.1中化合物F-1的合成步骤相同，只是将步骤a中的二甘醇换为三甘醇。经5步反应得到产物F-2为橘黄色油状。LC-MS(M+)理论值:551.11，LC-MS(ESI,M+H+)实测值:552.13。
1.3化合物F-3(式3)的合成
Figure PCTCN2017112958-appb-000056
化合物F-3的合成与例1.1中化合物F-1的合成步骤相同，只是将步骤a中的二甘醇换为四甘醇。经5步反应得到产物F-3为橘黄色油状。LC-MS(M+)理论值:595.13，LC-MS(ESI,M+H+)实测值:596.14。
1.4化合物F-4(式4)的合成
Figure PCTCN2017112958-appb-000057
化合物F-3的合成与例1.1中化合物F-1的合成步骤相同，只是将步骤a中的二甘醇换为五甘醇。经5步反应得到产物F-3为橘黄色油状。LC-MS(M+)理论值:639.16，LC-MS(ESI,M+H+)实测值:640.18。
1.5化合物F-5(式5)的合成
Figure PCTCN2017112958-appb-000058
化合物F-3的合成与例1.1中化合物F-1的合成步骤相同，只是将步骤a中的二甘醇换为六甘醇。经5步反应得到产物F-3为橘黄色油状。LC-MS(M+)理论值:683.19，LC-MS(ESI,M+H+)实测值:684.21。
1.6化合物F-6(式6)的合成
Figure PCTCN2017112958-appb-000059
化合物F-3的合成与例1.1中化合物F-1的合成步骤相同，只是将步骤a中的二甘醇换为八甘醇。经5步反应得到产物F-3为橘黄色油状。LC-MS(M+)理论值:771.24，LC-MS(ESI,M+H+)实测值:772.26。
1.7化合物F-7(式7)的合成
Figure PCTCN2017112958-appb-000060
化合物F-3的合成与例1.1中化合物F-1的合成步骤相同，只是将步骤a中的二甘醇换为十甘醇。经5步反应得到产物F-3为橘黄色油状。LC-MS(M+)理论值:895.29，LC-MS(ESI,M+H+)实测值:896.31。
1.8化合物F-8(式8)的合成
Figure PCTCN2017112958-appb-000061
化合物F-8的合成与例1.3中化合物F-3的合成步骤相同，只是将步骤c中的苯硫酚换为对甲 基苯硫酚。共经5步反应得到产物F-8为橘黄色油状。LC-MS(M+)理论值:623.16，LC-MS(ESI,M+H+)实测值:624.18。
1.9化合物F-9(式9)的合成
Figure PCTCN2017112958-appb-000062
化合物F-9的合成与例1.3中化合物F-3的合成步骤相同，只是将步骤c中的苯硫酚换为对甲氧基苯硫酚。共经5步反应得到产物F-9为橘黄色油状。LC-MS(M+)理论值:655.15，LC-MS(ESI,M+H+)实测值:656.17。
1.10化合物F-10(式10)的合成
Figure PCTCN2017112958-appb-000063
化合物F-9的合成与例1.3中化合物F-3的合成步骤相同，只是将步骤c中的苯硫酚换为4-(N-甲基甲酰胺)苯硫酚。共经5步反应得到产物F-10为橘黄色油状。LC-MS(M+)理论值:709.18，LC-MS(ESI,M+H+)实测值:710.23。
1.11化合物F-11(式11)的合成
Figure PCTCN2017112958-appb-000064
化合物F-10的合成与例1.3中化合物F-3的合成步骤相同，只是将步骤c中的苯硫酚换为2-巯基吡啶。共经5步反应得到产物F-10为橘黄色油状。LC-MS(M+)理论值:597.12，LC-MS(ESI,M+H+)实测值:598.13。
1.12化合物F-12(式12)的合成
Figure PCTCN2017112958-appb-000065
化合物F-11的合成与例1.3中化合物F-3的合成步骤相同，只是将步骤c中的苯硫酚换为2-巯基嘧啶。共经5步反应得到产物F-11为橘黄色油状。LC-MS(M+)理论值:599.11，LC-MS(ESI,M+H+)实测值:600.13。
1.13化合物F-13(式13)的合成
Figure PCTCN2017112958-appb-000066
化合物F-12的合成与例1.3中化合物F-3的合成步骤相同，只是将步骤a中的对氟硝基苯换为间氟硝基苯。共经5步反应得到产物F-12为橘黄色油状。LC-MS(M+)理论值:595.13，LC-MS(ESI,M+H+)实测值:596.15。
1.14化合物F-14(式14)的合成
Figure PCTCN2017112958-appb-000067
化合物F-12的合成与例1.3中化合物F-3的合成步骤相同，只是将步骤e中的中间体E换为溴代的中间体E’。共经4步反应得到产物F-13为无色油状。LC-MS(M+)理论值:534.95，LC-MS(ESI,M+H+)实测值:536.01。
1.15化合物F-15(式15)的合成
Figure PCTCN2017112958-appb-000068
化合物F-15的合成与例1.3中化合物F-3的合成步骤相同，只是将步骤c中的苯硫酚换为2-巯基-1-甲基咪唑。共经5步反应得到产物F-15为橘黄色油状。LC-MS(M+)理论值:603.15，LC-MS(ESI,M+H+)实测值:604.14。
1.16化合物F-16(式16)的合成
Figure PCTCN2017112958-appb-000069
化合物F-16的合成与例1.3中化合物F-3的合成步骤相同，只是将步骤c中的苯硫酚换为4-(N-吗啉甲酰胺)苯硫酚。共经5步反应得到产物F-16为橘黄色油状。LC-MS(M+)理论值:821.23，LC-MS(ESI,M+H+)实测值:822.21。
1.17化合物F-17(式17)的合成
Figure PCTCN2017112958-appb-000070
化合物F-17的合成与例1.3中化合物F-3的合成步骤相同，只是将步骤c中的苯硫酚换为4-(N-2-甲氧基乙基甲酰胺)苯硫酚。共经5步反应得到产物F-17为橘黄色油状。LC-MS(M+)理论值:797.23，LC-MS(ESI,M+H+)实测值:798.31。
1.18化合物F-18(式18)的合成
Figure PCTCN2017112958-appb-000071
化合物F-18的合成与例1.3中化合物F-3的合成步骤相同，只是将步骤c中的苯硫酚换为4-(N-甲氧基-N-甲基甲酰胺)苯硫酚。共经5步反应得到产物F-18为橘黄色油状。LC-MS(M+)理论值:769.20，LC-MS(ESI,M+H+)实测值:770.28。
式Ia所代表的取代马来酰胺类连接子片断分子还可通过以下图中所示的方法合成，通过n甘醇与氟代硝基苯取代反应得到中间体B，后经与溴乙酸叔丁酯反应获得中间体Z，中间体Z还可通过n甘醇与与溴乙酸叔丁酯反应后再与溴乙酸叔丁酯进行取代反应获得；中间体Z经还原得到中间体Y；2,3-二溴马来酰亚胺与芳基硫酚进行取代反应后获得中间体D，后与氯甲酸甲酯反应获得中间体E，中间体E与中间体Y反应获得中间体X，中间体X中的叔丁酯在酸性条件下脱除获得连接子片断分子W。反应路线举例如下：
Figure PCTCN2017112958-appb-000072
方案二：
本发明第二方面中列出的式Ib所代表的取代马来酰胺类连接子药物缀合物(式13-式19)可通过方案二中所列的路线进行合成，通过化合物F与细胞毒药物CTD(D1-D11，毒性药物分子可通过商业渠道获得)进行缩合偶联，可获得系列分子G。反应路线与具体实例举例如下：
Figure PCTCN2017112958-appb-000073
实施例2(式19-25化合物的合成与制备)
2.1化合物G-1(式19)的合成
Figure PCTCN2017112958-appb-000074
化合物F-3(200mg，0.34mmol)与化合物D1(220mg,0.34mmol)溶于N,N-二甲基甲酰胺(10 mL)中，室温下加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI)(77mg，0.40mmol)与1-羟基苯并三唑(HOBT)(54mg,0.40mmol)，反应混合物室温搅拌过夜。反应液用乙酸乙酯稀释后，加水萃取，依次用水和饱和食盐水洗涤，无水硫酸钠干燥，旋干溶剂。粗品通过硅胶层析柱分离纯化(二氯甲烷/甲醇)，抽滤干燥，得黄色无定形固体G-1(3.5g，产率98％)。LC-MS(M+)理论值:1226.40，LC-MS(ESI,M+H+)实测值:1227.42。
2.2化合物G-2(式20)的合成
Figure PCTCN2017112958-appb-000075
化合物G-2的合成与例2.1中化合物G-1的合成步骤相同，只是将其中的化合物D1换为化合物D2。得到产物G-2为黄色无定形固体。LC-MS(M+)理论值:1294.63，LC-MS(ESI,M+H+)实测值:1295.64。
2.3化合物G-3(式21)的合成
Figure PCTCN2017112958-appb-000076
化合物G-3的合成与例2.1中化合物G-1的合成步骤相同，只是将其中的化合物D1换为化合物D3。得到产物G-3为黄色无定形固体。LC-MS(M+)理论值:1308.61，LC-MS(ESI,M+H+)实测值:1309.63。
2.4化合物G-4(式22)的合成
Figure PCTCN2017112958-appb-000077
化合物G-4的合成与例2.1中化合物G-1的合成步骤相同，只是将其中的化合物D1换为化合物D9。得到产物G-4为黄色无定形固体。LC-MS(M+)理论值:1302.41，LC-MS(ESI,M+H+)实测值:1303.43。
2.5化合物G-5(式23)的合成
Figure PCTCN2017112958-appb-000078
化合物G-5的合成与例2.1中化合物G-1的合成步骤相同，只是将其中的化合物D1换为化合物D6。得到产物G-5为黄色无定形固体。LC-MS(M+)理论值:1290.51，LC-MS(ESI,M+H+)实测值:1291.53。
2.6化合物G-6(式24)的合成
Figure PCTCN2017112958-appb-000079
化合物G-6的合成与例2.1中化合物G-1的合成步骤相同，只是将其中的化合物D1换为化合物D7。得到产物G-6为黄色无定形固体。LC-MS(M+)理论值:1274.44，LC-MS(ESI,M+H+)实测值1275.45。
2.7化合物G-7(式25)的合成
Figure PCTCN2017112958-appb-000080
化合物G-7的合成与例2.1中化合物G-1的合成步骤相同，只是将其中的化合物D1换为化合物D11。得到产物G-7为黄色无定形固体。LC-MS(M+)理论值:1291.38，LC-MS(ESI,M+H+)实测值:1292.40。
方案三：
本发明第二方面列出的式Ib所代表的取代马来酰胺类连接子药物缀合物(式22-式41)可通过方案三中所列的路线进行合成，通过化合物F与二肽/三肽-PAB连接子的氨基(该连接子可通过商业渠道获得)进行缩合，PAB基团再与二(对硝基苯)碳酸酯活化后与细胞毒药物CTD(D1-D11)进行缩合偶联，可获得系列分子K。反应路线与具体实例举例如下：
Figure PCTCN2017112958-appb-000081
3.1化合物K-1(式26)的合成
3.1.1中间体I-1的合成(步骤f)
Figure PCTCN2017112958-appb-000082
化合物F-3(2.0g，3.36mmol)溶于无水N,N二甲基甲酰胺(25ml)中，氮气保护下依次加入EDCI(963mg，5.04mmol)，HOBt(681mg，5.04mmol)和N-甲基吗啡啉(1.11ml，10.08mmol)，反应混合物氮气保护下室温搅拌20分钟，最后加入化合物H-1(Val-Cit-PAB,1.91g，5.04mmol)。反应混合物氮气保护下室温搅拌过夜。旋干溶剂，干法柱层析(硅胶，200-300目，DCM/MeOH10:1)得到产物I-1为橘黄色油状(2.0g，产率62.2％)。LC-MS(M+)理论值:1631.60，LC-MS(ESI,M+H+)实测值:1632.62。
3.1.2中间体J-1的合成(步骤g)
Figure PCTCN2017112958-appb-000083
化合物I-1(1.5g，1.57mmol)溶于无水N,N二甲基甲酰胺(10mL)中，氮气保护下依次加入N,N-二异丙基乙胺(0.52mL，3.14mmol)和二(对硝基苯)碳酸酯(717mg，2.335mmol)，室温搅 拌15小时。旋干溶剂，干法柱层析(硅胶，200-300目，DCM/MeOH 20:1)得到产物J-1为橘黄色油状(1.4g，产率79.9％)。LC-MS(M+)理论值:1121.35，LC-MS(ESI,M+H+)实测值:1122.37。
3.1.3化合物K-1的合成(步骤h)
Figure PCTCN2017112958-appb-000084
化合物J-1(0.5g，0.52mmol)与溶于无水N,N二甲基甲酰胺(5mL)中，氮气保护下依次加入N,N-二异丙基乙胺(0.172mL，1.04mmol)，HOBt(70mg，0.52mmol)和化合物D1(340mg,0.52mmol)，反应混合物氮气保护下室温搅拌过夜。旋干溶剂，干法柱层析(硅胶，200-300目，DCM/MeOH10:1)得到产物K-1为黄色无定形固体(310mg，产率36.33％)。LC-MS(M+)理论值:1631.60，LC-MS(ESI,M+H+)实测值:1632.62。
3.2化合物K-2(式27)的合成
Figure PCTCN2017112958-appb-000085
化合物K-2的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤h中的化合物D1换为化合物D2(一甲基澳瑞他汀E)。共经3步反应得到产物K-2为黄色无定形固体。LC-MS(M+)理论值:1699.83，LC-MS(ESI,M+H+)实测值:1610.85。
3.3化合物K-3(式28)的合成
Figure PCTCN2017112958-appb-000086
化合物K-3的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤f中的化合物F-3换为F-5，将步骤h中的化合物D1换为化合物D2(一甲基澳瑞他汀E)。共经3步反应得到产物K-3为黄色无定形固体。LC-MS(M+)理论值:1787.88，LC-MS(ESI,M+H+)实测值:1788.90。
3.4化合物K-4(式29)的合成
Figure PCTCN2017112958-appb-000087
化合物K-4的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤f中的化合物F-3换为F-6，将步骤h中的化合物D1换为化合物D2(一甲基澳瑞他汀E)。共经3步反应得到产物K-4为黄色无定形固体。LC-MS(M+)理论值:1875.93，LC-MS(ESI,M+H+)实测值:1876.95。
3.5化合物K-5(式30)的合成
Figure PCTCN2017112958-appb-000088
化合物K-5的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤f中的化合物F-3换为F-7，将步骤h中的化合物D1换为化合物D2(一甲基澳瑞他汀E)。共经3步反应得到产物K-5为黄色无定形固体。LC-MS(M+)理论值:1963.99，LC-MS(ESI,M+H+)实测值:1965.01。
3.6化合物K-6(式31)的合成
Figure PCTCN2017112958-appb-000089
化合物K-6的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤f中的化合物H-1换为H-2(Val-Ala-PAB)，将步骤h中的化合物D1换为化合物D2(一甲基澳瑞他汀E)。共经3步反应得到产物K-6为黄色无定形固体。LC-MS(M+)理论值:1613.78，LC-MS(ESI,M+H+)实测值:1614.80。
3.7化合物K-7(式32)的合成
Figure PCTCN2017112958-appb-000090
化合物K-7的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤f中的化合物H-1换为H-3(Phe-Lys-PAB)，将步骤h中的化合物D1换为化合物D2(一甲基澳瑞他汀E)。共经3步反应得到产物K-7为黄色无定形固体。LC-MS(M+)理论值:1718.84，LC-MS(ESI,M+H+)实测值:1719.86。
3.8化合物K-8(式33)的合成
Figure PCTCN2017112958-appb-000091
化合物K-8的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤f中的化合物H-1换为H-4(Ala-Ala-Asn-PAB)，将步骤h中的化合物D1换为化合物D2(一甲基澳瑞他汀E)。共经3步反应得到产物K-8为黄色无定形固体。LC-MS(M+)理论值:1699.79，LC-MS(ESI,M+H+)实测值:1700.80。
3.9化合物K-9(式34)的合成
Figure PCTCN2017112958-appb-000092
化合物K-8的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤f中的化合物H-1 换为H-5(D-Ala-Phe-Lys-PAB)，将步骤h中的化合物D1换为化合物D2(一甲基澳瑞他汀E)。共经3步反应得到产物K-9为黄色无定形固体。LC-MS(M+)理论值:1789.88，LC-MS(ESI,M+H+)实测值:1790.90。
3.10化合物K-10(式35)的合成
Figure PCTCN2017112958-appb-000093
化合物K-10的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤h中的化合物D1换为化合物D3(一甲基澳瑞他汀F)。共经3步反应得到产物K-10为黄色无定形固体。LC-MS(M+)理论值:1713.81，LC-MS(ESI,M+H+)实测值:1714.83。
3.11化合物K-11(式36)的合成
Figure PCTCN2017112958-appb-000094
化合物K-11的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤h中的化合物D1换为化合物D4(Monomethyl Dolastatin 10,MMAD)。共经3步反应得到产物K-11为黄色无定形固体。LC-MS(M+)理论值:1752.80，LC-MS(ESI,M+H+)实测值:1753.82。
3.12化合物K-12(式37)的合成
Figure PCTCN2017112958-appb-000095
化合物K-12的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤h中的化合物D1换为化合物D5(Tubulysin衍生物1)。共经3步反应得到产物K-12为黄色无定形固体。LC-MS(M+)理论值:1506.59，LC-MS(ESI,M+H+)实测值:1507.61。
3.13化合物K-13(式38)的合成
Figure PCTCN2017112958-appb-000096
化合物K-13的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤h中的化合物D1换为化合物D6(Tubulysin衍生物2)。共经3步反应得到产物K-13为黄色无定形固体。LC-MS(M+)理论值:1695.71，LC-MS(ESI,M+H+)实测值:1696.73。
3.14化合物K-14(式39)的合成
Figure PCTCN2017112958-appb-000097
化合物K-14的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤h中的化合物D1换为化合物D7(Cryptophycin衍生物)。共经3步反应得到产物K-14为黄色无定形固体。LC-MS(M+)理论值:1679.64，LC-MS(ESI,M+H+)实测值:1680.66。
3.15化合物K-15(式40)的合成
Figure PCTCN2017112958-appb-000098
化合物K-15的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤h中的化合物D1换为化合物D8(Taltobulin)。共经3步反应得到产物K-15为棕黄色无定形固体。LC-MS(M+)理论值:1455.65，LC-MS(ESI,M+H+)实测值:1456.67。
3.16化合物K-16(式41)的合成
Figure PCTCN2017112958-appb-000099
化合物K-16的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤h中的化合物D1换为化合物D9(PBD dimer)。共经3步反应得到产物K-16为棕黄色无定形固体。LC-MS(M+)理论值:1707.61，LC-MS(ESI,M+H+)实测值:1708.63。
3.17化合物K-17(式42)的合成
Figure PCTCN2017112958-appb-000100
化合物K-17的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤h中的化合物D1换为化合物D10(Duocarmycin衍生物1)。共经3步反应得到产物K-17为棕黄色无定形固体。LC-MS(M+)理论值:1618.55，LC-MS(ESI,M+H+)实测值:1619.57。
3.18化合物K-18(式43)的合成
Figure PCTCN2017112958-appb-000101
化合物K-18的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤h中的化合物D1换为化合物D11(Duocarmycin衍生物2)。共经3步反应得到产物K-18为棕黄色无定形固体。LC-MS(M+)理论值:1696.58，LC-MS(ESI,M+H+)实测值:1697.60。
3.19化合物K-19(式44)的合成
Figure PCTCN2017112958-appb-000102
化合物K-19的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤h中的化合物D1换为化合物D12(PNU-159682衍生物)。共经3步反应得到产物K-19为棕黄色无定形固体。LC-MS(M+)理论值:1737.61，LC-MS(ESI,M+H+)实测值:1738.68。
3.20化合物K-20(式45)的合成
Figure PCTCN2017112958-appb-000103
化合物K-20的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤f中的中间体F-3换为中间体F-14。共经3步反应得到产物K-20为黄色无定形固体。LC-MS(M+)理论值:1639.64，LC-MS(ESI,M+H+)实测值:1640.61。
3.21化合物K-21(式46)的合成
Figure PCTCN2017112958-appb-000104
化合物K-21的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤f中的中间体F-3换为中间体F-11。共经3步反应得到产物K-21为黄色无定形固体。LC-MS(M+)理论值:1701.82，LC-MS(ESI,M+H+)实测值:1702.86。
3.22化合物K-22(式47)的合成
Figure PCTCN2017112958-appb-000105
化合物K-22的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤f中的中间体F-3换为中间体F-16。共经3步反应得到产物K-22为黄色无定形固体。LC-MS(M+)理论值:1925.92，LC-MS(ESI,M+H+)实测值:1926.88。
3.23化合物K-23(式48)的合成
Figure PCTCN2017112958-appb-000106
化合物K-24的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤f中的中间体F-3换为中间体F-16,将步骤h中的化合物D1换为化合物D11(Duocarmycin衍生物2)。共经3步反应得到产物K-24为黄色无定形固体。LC-MS(M+)理论值:1848.64，LC-MS(ESI,M+H+)实测值:1849.58。
3.24化合物K-24(式49)的合成
Figure PCTCN2017112958-appb-000107
化合物K-25的合成与例3.1中化合物K-1的合成步骤相同，只是将步骤f中的中间体F-3换为中间体F-16,将步骤h中的化合物D1换为化合物D13(SN38衍生物)。共经3步反应得到产物K-25为黄色无定形固体。LC-MS(M+)理论值:1714.64，LC-MS(ESI,M+H+)实测值:1715.61。
二、抗体偶联物制备方法与检测
实施例一：ADC-1的制备
将帕妥珠抗体原液用50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 7.4反应缓冲液稀释至5mg/mL，加入6.0倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于35℃搅动10小时。
将上述反应液冷至8℃，未经纯化加入适量的二甲亚砜(DMSO)，再加入6倍过量摩尔比的化合物G-2(10mg/ml预先溶在DMSO中)，保证反应体系中DMSO的体积占比不超过15％，于37℃搅动3小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.15微米孔径的过滤装置除菌，-60℃保存。
实施例二：ADC-2的制备
将帕妥珠抗体原液用50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 7.4反应缓冲液稀释至5mg/mL，加入10倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于10℃搅动4小时。
将上述反应液冷至5℃，未经纯化加入适量的二乙基乙酰胺(DMA)，再加入6倍过量摩尔比的化合物K-2(10mg/ml预先溶在DMA中)，保证反应体系中DMA的体积占比不超过10％，于25℃搅动2.5小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.22微米孔径的过滤装置除菌，-80℃保存。
实施例三：ADC-3的制备
将帕妥珠抗体原液用50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 7.4反应缓冲液稀释至5mg/mL，加入20倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于15℃搅动2小时。
将上述反应液冷至10℃，未经纯化加入适量的乙腈(ACN)，再加入6倍过量摩尔比的化合物K-3(10mg/ml预先溶在ACN中)，保证反应体系中ACN的体积占比不超过10％，于10℃搅动4小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品，然后过滤除菌，所得产物低温保存；如经由0.20微米孔径的过滤装置除菌，-90℃保存。
实施例四：ADC-4的制备
将帕妥珠抗体原液用50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 7.4反应缓冲液稀释至5mg/mL，加入8倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于25℃搅动48小时。
将上述反应液冷至0℃，未经纯化加入适量的二甲基甲酰胺(DMF)，再加入6倍过量摩尔比的化合物K-4(10mg/ml预先溶在DMF中)，保证反应体系中DMF的体积占比不超过8％，于0℃搅动2小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.3米孔径的过滤装置除菌，-100℃保存。
将实施例二所得样品与帕妥珠单抗进行疏水作用层析(HIC)对比(图1)和、质谱对比(图2)，由此可知，应用本发明所述连接子得到的偶联物DAR值分布非常窄，其平均DAR值接近4，获得的交联物单一分布的组份(DAR为4)占比80％以上，因此生成的产品均一性高；经鉴定，m为3.0-4.2。将实施例二、三、四分别采用上述相同的方法进行分析，DAR可得到相同的结果，m均在1-5.0范围内。
三、抗体偶联物的生物学检测
1.分子结合实验检测
Biacore仪检测蛋白分子间亲和力的工作原理是基于表面等离子共振(Surface Plasmon Resonance，SPR)技术。SPR是一种光学物理现象，当一束P偏振光在一定的角度范围内入射到棱镜端面，在棱镜与金属薄膜(Au)的界面将产生表面等离子波。当入射光波的传播常数与 表面等离子波的传播常数相匹配时，引起金属膜内自由电子产生共振。分析时，先在传感芯片表面固定一层生物分子识别膜，然后将待测样品流过芯片表面，若样品中有能够与芯片表面的生物分子识别膜相互作用的分子，会引起金膜表面折射率变化，最终导致SPR角变化，通过检测SPR角度变化，获得被分析物的浓度、亲和力、动力学常数和特异性等信息。
使用Biacore进行结合实验来检测Pertuzumab，ADC-2，ADC-4共3个批次单克隆样品与Human ErbB2的结合活性。
表1 3个单克隆抗体样品与Human ErbB2的亲和力和动力学参数
样品 ka(1/Ms) kd(1/s) KD(M)
Pertuzumab 1.902E+05 1.239E-03 6.517E-10
ADC-2 2.232E+05 1.294E-04 5.799E-10
ADC-4 1.956E+05 1.310E-04 6.969E-10
本实验用表面等离子共振技术表征3个单克隆抗体样品Pertuzumab,ADC-2，ADC-4与Human ErbB2的的结合活性，结果显示均有结合。实验结果表明，上述3个单克隆抗体样品与Human ErbB2的亲和力相似，均在0.5-0.7nM范围。
2.细胞水平ADC-2与Her2的亲和力测定实验
以下实验中所用的实验材料来源于：RPMI1640培养基、0.25％胰蛋白酶-EDTA、胎牛血清、100×丙酮酸钠、100×青链霉素购自Gibco公司，异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的二抗购自Invitrogen公司,NCI-N87胃癌细胞来自中国科学院昆明细胞库。其他试剂均是分析纯。FACSCalibur流式细胞仪(BD)。
在本实施例中，研究了ADC-2，P-mcVC-MMAE，Pertuzumab与Her2高表达细胞结合的亲和力。
本实施例选择Her2高表达人胃癌NCI-N87细胞。NCI-N87细胞在含10％胎牛血清的RPMI1640培养基中于37℃，5％CO2培养箱中培养，取传代培养4～5天的细胞计数，收集细胞至15mL离心管中，用冷PBS洗涤细胞2次，4℃条件，1000rpm离心5min，用含5％胎牛血清的PBS重悬细胞，37℃条件孵育30min，然后4℃，1000rpm离心5min，去上清，用冷PBS重悬细胞，按1×106细胞/1.5mL分装至EP管中，4℃，1000rpm离心5min，去上清，加入0.5mL不同浓度的ADC-2、P-mcVC-MMAE、Pertuzumab、人IgG，冰上放置40min,4℃，1000rpm离心5min，用1mL冷PBS洗涤2次，加入200μL荧光标记(FITC)标记的二抗，冰上避光放置40min后，4℃，1000rpm离心5min，去上清，1mL冷PBS洗涤2次，加入0.5mL冷PBS重悬细胞，冰上避光放置，采用FACSCalibur流式细胞仪检测测定不同浓度的ADC-2、P-mcVC-MMAE、Pertuzumab与细胞结合的平均荧光强度(mean fluorescence intensity，MFI)，人IgG结合的荧光强度为非特异性结合。
如图3所示，ADC-2、P-mcVC-MMAE、Pertuzumab单抗均能与NCI-N87细胞表面抗原Her2结合，且随着抗体浓度的增加，ADC-2、P-mcVC-MMAE、Pertuzumab与Her2受体的结合也增加，三者与NCI-N87细胞Her2结合的亲和力相当，没有明显差别。
3.体外细胞增殖测定生物活性实验
以下实验中所用的实验材料来源于：DMEM培养基、DMEM/F12K培养基、RPMI 1640培养基、0.25％胰蛋白酶-EDTA、胎牛血清、100×丙酮酸钠、100×青链霉素购自Gibco公司。磺酰罗丹明B(Sulforhodamine B，SRB)购自Sigma公司。BT-474人乳腺癌细胞、SK-RB-3人乳腺癌细胞、MDA-MB-231人乳腺癌细胞、NCI-N87人胃癌细胞来自中国科学院昆明细胞 库，Panc-1人胰腺癌、MDA-MB-468人乳腺癌细胞、MCF-7人乳腺癌细胞来自中国科学院上海生命科学研究院细胞库，SKOV-3人卵巢癌细胞、Du-145人前列腺癌细胞来自美国典型培养物保藏中心(ATCC)。其它试剂均为分析纯。96孔平底聚苯乙烯(Corning，产品目录号3599)。Synergy 2酶标仪(Bio-Tek)。
在本实施例中，研究了ADC-2，ADC-4，P-mcVC-MMAE,Kadcyla，Pertuzumab对肿瘤细胞系增殖的作用。
本实施例使用磺酰罗丹明B(SRB)比色法来评价药物组合的抗增殖作用。SRB是一种粉红色阴离子染料，易溶于水，在酸性条件下可特异性地与细胞内组成蛋白质的碱性氨基酸结合；在510nm波长下产生吸收峰，吸光值与细胞量成线性正相关，故可用作细胞数的定量检测。本实施例选择的细胞系有：BT-474、SK-RB-3、MDA-MB-231、MDA-MB-468、MCF-7人乳腺癌细胞、NCI-N87人胃癌细胞、SKOV-3人卵巢癌细胞、Du-145人前列腺癌细胞、Panc-1人胰腺癌。
BT-474、SK-BR-3、NCI-N87细胞在含10％胎牛血清的RPMI 1640培养基中,SKOV-3、Du-145、Panc-1、MCF-7、MDA-MB-231细胞在含10％胎牛血清的DMEM培养基中,MDA-MB-468细胞在含10％胎牛血清的DMEM/F12培养基中，于37℃，5％CO2培养箱中培养至对数生长期，将处于对数生长期的上述细胞分别以2×103～9×103个细胞每孔的密度接种至96孔培养板，每孔100μL，培养24小时后，加入不同浓度的药物作用5天，分别以3、4或5-倍稀释制备9个浓度，每个浓度设复孔，并设相应浓度的溶媒对照及无细胞培养基孔。药物作用结束后，倾去培养液，加入4℃预冷的三氯乙酸溶液(30％，w/v)100μl，于4℃固定1小时，随后用去离子水冲洗5遍，室温干燥后，每孔加入0.4％(w/v)的SRB染液(Sigma，1％冰乙酸配制)100μL，室温下孵育染色30min后，用1％冰乙酸冲洗4次，去除未结合的染料，室温晾干。每孔加入10mM Tris溶液100μL，室温下孵育染色15min后，用1％冰乙酸冲洗五次洗去未结合的SRB，室温干燥后，每孔加入10mM Tris缓冲液(pH＝10.5)溶解与细胞蛋白结合的染料，采用Synergy 2酶标仪(Bio-Tek)波长510nm和690nm处测定光吸收值(OD值)，并得到A＝OD510-OD690。
抑制率(％)＝(A对照-A给药)/A对照×100％。
本实验使用了ADC-2，ADC-4，P-mcVC-MMAE,Kadcyla，Pertuzumab对多种Her2高表达的肿瘤细胞系进行了体外细胞培养增殖作用的研究，同时使用ADC-2对多种非Her2高表达的肿瘤细胞系也进行了体外细胞培养增殖作用的研究。如图4-6所示，ADC-2、P-mcVC-MMAE、Kadcyla处理Her2高表达的SK-BR-3和BT-474人乳腺癌细胞、NCI-N87人胃癌细胞，均能明显抑制肿瘤细胞增殖，ADC-2和P-mcVC-MMAE对肿瘤细胞增殖抑制活性要明显高于Kadcyla，ADC-2与P-mcVC-MMAE的抗肿瘤细胞增殖活性基本相当，其中ADC-2对BT-474肿瘤细胞增殖抑制活性略高于P-mcVC-MMAE；与Pertuzumab裸抗比较，抗体药物偶联物ADC-2、P-mcVC-MMAE、Kadcyla对肿瘤细胞增殖抑制活性都显著提高。如图7所示，ADC-2对非Her2高表达的SKOV-3人卵巢癌细胞、Du-145人前列腺癌细胞和Panc-1人胰腺癌，也显示出良好的肿瘤细胞增殖抑制作用。对不表达Her2的人乳腺癌MCF-7、MDA-MB-231和MDA-MB-468细胞，ADC-2抗肿瘤细胞增殖活性则显著降低(图8)，表明抗体药物偶联物ADC-2对不表达靶标抗原的细胞基本无作用，因而，预示其毒副作用将明显降低。如图9-1示，ADC-4对Her2高表达的NCI-N87人胃癌细胞显示强效肿瘤细胞增殖抑制作用。此外，如图9-2所示，ADC-2、ADC-3与ADC-4对于Her2中表达的Calu-3人肺癌细胞显示强效肿瘤细胞增殖抑制作用。
4.体内抗肿瘤功效测定实验
可以在体内测量本发明的组合的功效，即在啮齿类动物中植入癌细胞的同种异体移植物或异种移植物，并用所述组合处理肿瘤。将受试小鼠用药物或对照处理，并监测数周或更长时间以测量到达肿瘤倍增的时间，对数细胞杀伤，和肿瘤抑制。
1)实验动物
BALB/cA-nude裸小鼠，6-7周，♀，购自上海灵畅生物科技有限公司。生产许可证号：SCXK(沪)2013-0018；动物合格证号2013001815683。饲养环境：SPF级。
2)实验步骤
裸小鼠皮下接种人胃癌NCI-N87细胞，待肿瘤生长至100-250mm3后，将动物随机分组(D0)。给药剂量和给药方案见表1。每周测2-3次瘤体积，称鼠重，记录数据。肿瘤体积(V)计算公式为：
V＝1/2×a×b2其中a、b分别表示长、宽。
T/C(％)＝(T-T0)/(C-C0)x100其中T、C为实验结束时的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
3)实验结果
图10显示了ADC2(0.5、1、2mg/kg，IV，每周1次，共2次)剂量依赖性地显著抑制人胃癌NCI-N87裸小鼠皮下移植瘤的生长，抑瘤率分别为59％、94％和200％，1mg/kg组有3/6肿瘤部分消退，2mg/kg组有6/6肿瘤完全消退；ADC3(0.5、1、2mg/kg，IV，每周1次，共2次)对NCI-N87的抑瘤率分别为65％、69％和185％，2mg/kg组有1/6肿瘤部分消退和5/6肿瘤完全消退；P-vcMMAE(1mg/kg，IV，每周1次，共2次)对NCI-N87的抑瘤率为94％，有2/6肿瘤部分消退；参比药物Kadcyla(2mg/kg，IV，每周1次，共2次)对NCI-N87的抑瘤率分别为77％。荷瘤小鼠对以上药物均能很好耐受，没有体重减轻等症状发生。
具体设计二：取代马来酰胺类连接子片断Ia制备及其应用，其中Ar’选自取代的C6-C10亚芳基，或取代的5-12元亚杂芳基；
实施例组一、化合物合成与制备方法
1.1化合物E-1(式Ia-1)的合成
1.1.1中间体A-1(步骤a)
Figure PCTCN2017112958-appb-000108
将三甘醇(92g,613mmol)溶于tBuOH(200ml)中。冰浴下加入KOtBu(22.91g,204mmol)搅拌半小时，氩气保护下，滴加溴乙酸叔丁酯(39.8g,204mmol)在tBuOH(40ml)的溶液，室温搅拌过夜。第二天，TLC检测反应结束。旋蒸除去叔丁醇后，剩余物加入400ml二氯甲烷，有机相用400ml水洗，该水相用300ml二氯甲烷萃取一次，合并有机相后用饱和食盐水洗一次，无水硫酸钠干燥，旋蒸蒸干。粗产物经石油醚：乙酸乙酯＝3:1-->1:1柱层析，得中间体A-1(24g,44.5％yield)，为黄色油状物。
1.1.2中间体B-1(步骤b)
Figure PCTCN2017112958-appb-000109
将中间体A-1(7.8g,29.5mmol)，5-氟-2-硝基三氟甲苯(9.26g,44.3mmol)，K2CO3(6.12 g,44.3mmol)粉末在250mL圆底反应瓶中，氮气保护下加热至80℃搅拌48小时，TLC监测，仅有少量原料剩余。：
降至室温用500二氯甲烷萃取，400ml 1N稀盐酸洗一次，400ml水洗一次，400ml饱和食盐水洗一次，无水硫酸钠干燥，旋蒸蒸干。柱层析(200目～300目硅胶)纯化，石油醚：乙酸乙酯30:1-10:1淋洗，得中间体B-1(7.5g,56.1％yield)，为黄色油状物。
1.1.3中间体C-1
Figure PCTCN2017112958-appb-000110
将中间体B-1(6g,13.23mmol)溶于100毫升无水乙醇中并且将溶液，加入装有10％Pd-C1.2g的反应瓶中。加氢反应6小时(1atm,38℃)，TLC检测反应完全。硅藻土过滤反应液，滤饼用乙醇淋洗，滤液旋蒸蒸干，得中间体C-1(5g,89％yield)为黄色油状物。
1.1.4化合物E-1
Figure PCTCN2017112958-appb-000111
称取中间体C-1(0.8g,1.889mmol)于平行反应管中，氮气保护下加入AcOH(3ml)，搅拌溶解。后慢慢加入3,4-二溴马来酸酐(0.483g,1.889mmol)。氮气保护下加热到110℃搅拌过夜。TLC检测反应。将反应液冷却到室温后，旋蒸蒸干溶剂，并加入甲苯旋蒸蒸干两次，得棕色油状化合物E-1。无需纯化直接用于下步反应。
1.2化合物E-2(式Ia-2)的合成(步骤e)
Figure PCTCN2017112958-appb-000112
称取化合物E-1(2.0g,1.35mmol)于100毫升圆底瓶中,氮气保护下加入30ml无水二氯甲烷搅拌溶解。称取297mg苯硫酚氮气保护下加入反应液中,溶解后在冰浴下慢慢滴加DIPEA(0.44ml,2.70mmol)，完毕后搅拌5分钟，撤去冰浴。在氮气保护下室温搅拌2小时，TLC检测反应结束。
减压蒸干溶剂后，柱层析(200目～300目硅胶)分离纯化，二氯甲烷装柱和淋洗，然后慢慢加大极性从2％至10％甲醇淋洗，收集蒸干溶剂得橘黄色油状产物E-2(0.92g,79％yield)。LC-MS(M+)理论值:595.13，实测值:596.15(ESI,M+H+)。
1.3化合物E-3(式Ia-3)的合成
化合物E-3的合成与例1.2中化合物E-2的合成步骤相同，只是将步骤e中的苯硫酚换为2-巯基吡啶，得到产物E-3为橘黄色油状物。
1.4化合物E-4(式Ia-4)的合成
化合物E-4的合成与例1.2中化合物E-2的合成步骤相同，只是将步骤b中的5-氟-2-硝基三氟甲苯换为2-甲氧基-4-氟硝基苯，步骤e中的苯硫酚换为4-(N-吗啉甲酰胺)苯硫酚，得到产物E-4为橘黄色油状物。
1.5化合物E-5(式Ia-5)的合成
化合物E-5的合成与例1.2中化合物E-2的合成步骤相同，只是将步骤b中的5-氟-2-硝基三氟甲苯换为1-氟-2-甲氧基-4-硝基苯，步骤e中的苯硫酚换为4-(N-吗啉甲酰胺)苯硫酚，得到产物E-5为橘黄色油状物。
1.6化合物E-6(式Ia-6)的合成
化合物E-6的合成与例1.2中化合物E-2的合成步骤相同，只是将步骤b中的5-氟-2-硝基三氟甲苯换为5-氟-2-硝基苯甲腈，步骤e中的苯硫酚换为4-(N-吗啉甲酰胺)苯硫酚，得到产物E-6为橘黄色油状物。
1.7化合物E-7(式Ia-7)的合成
化合物E-7的合成与例1.2中化合物E-2的合成步骤相同，只是将步骤b中的5-氟-2-硝基三氟甲苯换为2-氟-5-硝基苯甲腈，步骤e中的苯硫酚换为4-(N-吗啉甲酰胺)苯硫酚，得到产物E-7为橘黄色油状物。
1.8化合物E-8(式Ia-8)的合成
化合物E-8的合成与例1.2中化合物E-2的合成步骤相同，只是将步骤b中的5-氟-2-硝基三氟甲苯换为5-氟-2-硝基苯甲酰胺，步骤e中的苯硫酚换为4-(N-吗啉甲酰胺)苯硫酚，得到产物E-8为橘黄色油状物。
1.9化合物E-9(式Ia-9)的合成
化合物E-9的合成与例1.2中化合物E-2的合成步骤相同，只是将步骤b中的5-氟-2-硝基三氟甲苯换为4-氟-1-硝基-2-三氟甲基苯，步骤e中的苯硫酚换为4-(N-吗啉甲酰胺)苯硫酚，得到产物E-9为橘黄色油状物。
1.10化合物E-10(式Ia-10)的合成
化合物E-10的合成与例1.2中化合物E-2的合成步骤相同，只是将步骤b中的5-氟-2-硝基三氟甲苯换为1-氟-4-硝基-2-三氟甲基苯，步骤e中的苯硫酚换为4-(N-吗啉甲酰胺)苯硫酚，得到产物E-10为橘黄色油状物。
1.11化合物E-11(式Ia-11)的合成
1.11.1中间体F-11(步骤f)
Figure PCTCN2017112958-appb-000113
在250ml圆底瓶中将中间体A-1(4g,15.13mmol),三乙胺(2.53ml,18.16mmol)和二甲胺基吡啶(0.370g,3.03mmol)溶于100毫升分子筛干燥二氯甲烷中搅拌，冰浴下分批加入对甲基苯磺酰氯(3.17g,16.65mmol)，移至室温氩气保护搅拌过夜。
将反应体系加入100ml二氯甲烷萃取，用200ml 1N稀盐酸洗一次，200ml水洗两次，200ml饱和盐水洗一次，无水硫酸钠干燥，旋蒸蒸干有机相。用200-300目硅胶装柱，PE:EA＝5:1-2:1洗脱进行柱层析分离。旋蒸蒸干得中间体F-11(2.8g,收率44.2％)。
1.11.2中间体B-11(步骤g)
Figure PCTCN2017112958-appb-000114
将中间体F-11(1g,2.389mmol)，2,6-二氟-4-硝基苯酚(0.315g,1.797mmol)，溶于20mlDMF中，加入K2CO3(0.497g,3.59mmol)，加热至100度搅拌5小时。旋蒸蒸干溶剂，加入200ml二氯甲烷溶解，萃取，分别用200ml 1N稀盐酸，200ml水和200ml饱和盐水洗各洗一次，无水硫酸钠干燥，旋蒸蒸干，用200-300目硅胶装柱，PE：EA＝5:1-3:1洗脱柱层析纯化，旋蒸蒸干得中间体B-11(600mg,收率79％)
1.11.3中间体C-11
Figure PCTCN2017112958-appb-000115
将中间体B-11(600mg,1.42mmol)溶于100毫升无水乙醇中并且将溶液，加入装有
10％Pd-C 120mg的反应瓶中。加氢反应6小时(1atm,38℃)，TLC检测反应完全。硅藻土过滤反应液，滤饼用乙醇淋洗，滤液旋蒸蒸干，得中间体C-11(450mg,收率81％)为黄色油状物。
1.11.4中间体D-11
Figure PCTCN2017112958-appb-000116
称取中间体C-11(0.40g,1.02mmol)于平行反应管中，氮气保护下加入AcOH(3ml)，搅拌溶解。后慢慢加入3,4-二溴马来酸酐(0.261g,1.02mmol)。氮气保护下加热到110℃搅拌过夜。TLC检测反应。将反应液冷却到室温后，旋蒸蒸干溶剂，并加入甲苯旋蒸蒸干两次，得棕色油状化合物D-11。无需纯化直接用于下步反应。
1.11.5中间体E-11
Figure PCTCN2017112958-appb-000117
称取化合物D-11(600mg,0.95mmol)于100毫升圆底瓶中,氮气保护下加入30ml无水二氯甲烷搅拌溶解。称取425mg(1.91mmol)4-(N-吗啉甲酰胺)苯硫酚氮气保护下加入反应液中,溶解后在冰浴下慢慢滴加DIPEA(0.36ml,1.91mmol)，完毕后搅拌5分钟，撤去冰浴。在氮气保护下室温搅拌2小时，TLC检测反应结束。
减压蒸干溶剂后，柱层析(200目～300目硅胶)分离纯化，二氯甲烷装柱和淋洗，然后慢慢加大极性从2％至10％甲醇淋洗，收集蒸干溶剂得橘黄色油状产物E-11(0.62g,76％yield)。LC-MS(M+)理论值:857.21，实测值:858.23(ESI,M+H+)。
1.12化合物E-12(式Ia-12)的合成
化合物E-12的合成与例1.11中化合物E-11的合成步骤相同，只是将步骤g中的2,6-二氟-4-硝基苯酚换为3-氟-4-硝基苯酚，得到产物E-12为橘黄色油状物。
1.13化合物E-13(式Ia-13)的合成
化合物E-13的合成与例1.11中化合物E-11的合成步骤相同，只是将步骤g中的2,6-二氟-4-硝基苯酚换为2,5-二氟-4-硝基苯酚，得到产物E-13为橘黄色油状物。
1.14化合物E-14(式Ia-14)的合成
化合物E-14的合成与例1.11中化合物E-11的合成步骤相同，只是将步骤a中的三甘醇换为二甘醇，得到产物E-14为橘黄色油状物。
1.15化合物E-15(式Ia-15)的合成
化合物E-15的合成与例1.11中化合物E-11的合成步骤相同，只是将步骤a中的三甘醇换为四甘醇，得到产物E-15为橘黄色油状物。
1.16化合物E-16(式Ia-16)的合成
化合物E-16的合成与例1.11中化合物E-11的合成步骤相同，只是将步骤a中的三甘醇换为五甘醇，得到产物E-16为橘黄色油状物。
1.17化合物E-17(式Ia-17)的合成
化合物E-17的合成与例1.11中化合物E-11的合成步骤相同，只是将步骤a中的三甘醇换为六甘醇，得到产物E-17为橘黄色油状物。
1.18化合物E-18(式Ia-18)的合成
化合物E-18的合成与例1.11中化合物E-11的合成步骤相同，只是将步骤a中的三甘醇换为十二甘醇，得到产物E-18为橘黄色油状物。
1.19化合物E-19(式Ia-19)的合成
化合物E-19的合成与例1.11中化合物E-11的合成步骤相同，只是将步骤e中的4-(N-吗啉甲酰胺)苯硫酚换为1,1-二氧化硫代吗啉，得到产物E-19为橘黄色油状物。
1.20化合物E-20(式Ia-20)的合成
化合物E-20的合成与例1.11中化合物E-11的合成步骤相同，只是将步骤e中的4-(N-吗啉甲酰胺)苯硫酚换为4-(N-甲基甲酰胺)苯硫酚，得到产物E-20为橘黄色油状物。
1.21化合物E-21(式Ia-21)的合成
化合物E-21的合成与例1.2中化合物E-2的合成步骤相同，只是将步骤b中的5-氟-2-硝基三氟甲苯换为2-硝基-5-氟吡啶，步骤e中的苯硫酚换为4-(N-吗啉甲酰胺)苯硫酚，得到产物E-21为橘黄色油状物。
1.22化合物E-22(式Ia-22)的合成
化合物E-21的合成与例1.2中化合物E-2的合成步骤相同，只是将步骤b中的5-氟-2-硝基三氟甲苯换为2-氟-5-硝基吡啶，步骤e中的苯硫酚换为4-(N-吗啉甲酰胺)苯硫酚，得到产物E-22为橘黄色油状物。
实施例组2：式Ib-1～Ib-24的合成与制备
2.1化合物F1-1(式Ib-1)的合成
Figure PCTCN2017112958-appb-000118
向100毫升圆底瓶中称入化合物E1-9(300mg,0.337mmol)，氮气保护下加入无水DMF(20mL)使其完全溶解后,依次称取HATU(154mg,0.404mmol)和DIEA(0.11ml,0.674mmol)加入瓶中。室温搅拌15分钟后加入化合物D1-1(219mg,0.337mmol),氮气保护下室温搅拌过夜。TLC与HPLC跟踪反应过夜，原料E9消失。减压蒸干溶剂，做定量分析，后经反相HPLC纯化，得产物为黄色无定形粉末F1-1(0.350g,0.230mmol,68.2％yield)。LC-MS(M+)理论值:1520.48，实测值:1521.51(ESI,M+H+)。
2.2化合物F1-2(式Ib-2)的合成
Figure PCTCN2017112958-appb-000119
化合物F1-2的合成与例2.1中化合物F1-1的合成步骤相同，只是其中D1-1换为化合物D1-2，得到产物F1-2为黄色无定形粉末。LC-MS(M+)理论值:1993.91，实测值:1994.93(ESI,M+H+)。
2.3化合物F1-3(式Ib-3)的合成
Figure PCTCN2017112958-appb-000120
化合物F1-3的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中D1-1换为化合物D1-3，得到产物F1-3为黄色无定形粉末。LC-MS(M+)理论值:2007.89，实测值:2008.91(ESI,M+H+)。
2.4化合物F1-4(式Ib-4)的合成
Figure PCTCN2017112958-appb-000121
化合物F1-4的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中D1-1换为化合物D1-4，得到产物F1-4为黄色无定形粉末。LC-MS(M+)理论值:2046.88，实测值:2047.86(ESI,M+H+)。
2.5化合物F1-5(式Ib-5)的合成
Figure PCTCN2017112958-appb-000122
化合物F1-5的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中D1-1换为化合物D1-5，得到产物F1-5为黄色无定形粉末。LC-MS(M+)理论值:1800.67，实测值:1801.65(ESI,M+H+)。
2.6化合物F1-6(式Ib-6)的合成
Figure PCTCN2017112958-appb-000123
化合物F1-6的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中D1-1换为化合物D1-6，得到产物F1-6为黄色无定形粉末。LC-MS(M+)理论值:1989.79，实测值:1990.80(ESI,M+H+)。
2.7化合物F1-7(式Ib-7)的合成
Figure PCTCN2017112958-appb-000124
化合物F1-7的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中化合物D1-1换为化合物D1-7，得到产物F1-7为黄色无定形粉末。LC-MS(M+)理论值:1973.72，实测值:1974.72(ESI,M+H+)。
2.8化合物F1-8(式Ib-8)的合成
Figure PCTCN2017112958-appb-000125
化合物F1-8的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中D1-1换为化合物D1-8，得到产物F1-8为黄色无定形粉末。LC-MS(M+)理论值:1973.72，实测值:1974.72(ESI,M+H+)。
2.9化合物F1-9(式Ib-9)的合成
Figure PCTCN2017112958-appb-000126
化合物F1-9的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中E1-9与D1-1分别换为化合物E1-17与D1-9，得到产物F1-9为黄色无定形粉末。LC-MS(M+)理论值:2015.72，实测值:2016.73(ESI,M+H+)。
2.10化合物F1-10(式Ib-10)的合成
Figure PCTCN2017112958-appb-000127
化合物F1-10的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中D1-1换为化合物D1-10，得到产物F1-10为黄色无定形粉末。LC-MS(M+)理论值:1912.63，实测值:1913.65(ESI,M+H+)。
2.11化合物F1-11(式Ib-11)的合成
Figure PCTCN2017112958-appb-000128
化合物F1-11的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中D1-1换为化合物D1-11，得到产物F1-11为黄色无定形粉末。LC-MS(M+)理论值:1916.63，实测值:1917.61(ESI,M+H+)。
2.12化合物F1-12(式Ib-12)的合成
Figure PCTCN2017112958-appb-000129
化合物F1-12的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中D1-1换为化合物D1-12，得到产物F1-12为黄色无定形粉末。LC-MS(M+)理论值:2031.70，实测值:2032.71(ESI,M+H+)。
2.13化合物F1-13(式Ib-13)的合成
Figure PCTCN2017112958-appb-000130
化合物F1-13的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中D1-1换为化合物D1-13，得到产物F1-13为黄色无定形粉末。LC-MS(M+)理论值:1711.57，实测值:1712.55(ESI,M+H+)。
2.14化合物F1-14(式Ib-14)的合成
Figure PCTCN2017112958-appb-000131
化合物F1-14的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中D1-1换为化合物D1-2，得到产物F1-14为黄色无定形粉末。LC-MS(M+)理论值:1767.82，实测值:1768.83(ESI,M+H+)。
2.15化合物F1-15(式Ib-15)的合成
Figure PCTCN2017112958-appb-000132
化合物F1-15的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中E1-9与D1-1分别换为化合物E-19与D1-2，得到产物F1-15为黄色无定形粉末。LC-MS(M+)理论值:2057.84，实测值:2058.87(ESI,M+H+)。
2.16化合物F1-16(式Ib-16)的合成
Figure PCTCN2017112958-appb-000133
化合物F1-16的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中E1-9与D1-1分别换为化合物E1-20与D1-2，得到产物F1-16为黄色无定形粉末。LC-MS(M+)理论值:1849.85，实测值:1850.83(ESI,M+H+)。
2.17化合物F1-17(式Ib-17)的合成
Figure PCTCN2017112958-appb-000134
化合物F1-17的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中E1-9与D1-1分别换为化合物E1-10与D1-2，得到产物F1-17为黄色无定形粉末。LC-MS(M+)理论值:1993.91，实测值:1994.90(ESI,M+H+)。
2.18化合物F1-18(式Ib-18)的合成
Figure PCTCN2017112958-appb-000135
化合物F1-18的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中E1-9与D1-1分别换为化合物E1-11与D1-2，得到产物F1-18为黄色无定形粉末。LC-MS(M+)理论值:1961.90，实测值:1962.91(ESI,M+H+)。
2.19化合物F1-19(式Ib-19)的合成
Figure PCTCN2017112958-appb-000136
Figure PCTCN2017112958-appb-000137
化合物F1-19的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中E1-9与D1-1分别换为化合物E1-5与D1-2，得到产物F1-19为黄色无定形粉末。LC-MS(M+)理论值:1955.93，实测值:1956.95(ESI,M+H+)。
2.20化合物F1-20(式Ib-20)的合成
Figure PCTCN2017112958-appb-000138
化合物F1-20的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中E1-9与D1-1分别换为化合物E1-4与D1-2，得到产物F1-20为黄色无定形粉末。LC-MS(M+)理论值:1955.93，实测值:1956.95(ESI,M+H+)。
2.21化合物F1-21(式Ib-21)的合成
Figure PCTCN2017112958-appb-000139
Figure PCTCN2017112958-appb-000140
化合物F1-21的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中E1-9与D1-1分别换为化合物E1-12与D1-2，得到产物F1-21为黄色无定形粉末。LC-MS(M+)理论值:1943.91，实测值:1944.90(ESI,M+H+)。
2.22化合物F1-22(式Ib-22)的合成
Figure PCTCN2017112958-appb-000141
化合物F1-22的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中E1-9与D1-1分别换为化合物E1-6与D1-2，得到产物F1-22为黄色无定形粉末。LC-MS(M+)理论值:1950.92，实测值:1951.93(ESI,M+H+)。
2.23化合物F1-23(式Ib-23)的合成
Figure PCTCN2017112958-appb-000142
Figure PCTCN2017112958-appb-000143
化合物F1-23的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中E1-9与D1-1分别换为化合物E1-21与D1-2，得到产物F1-23为黄色无定形粉末。LC-MS(M+)理论值:1926.92，实测值:1927.93(ESI,M+H+)。
2.24化合物F1-24(式Ib-24)的合成
Figure PCTCN2017112958-appb-000144
化合物F1-24的合成与例2.1中化合物F1-1的合成步骤相同，只是将其中E1-9与D1-1分别换为化合物E1-22与D1-2，得到产物F1-24为黄色无定形粉末。LC-MS(M+)理论值:1926.92，实测值:1927.93(ESI,M+H+)。
实施例组二、抗体偶联物的制备
1、ADC-I的制备
将帕妥珠抗体原液用50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 7反应缓冲液稀释至2mg/mL，加入6.0倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于35℃搅动2.5小时。
将上述反应液冷至8℃，未经纯化加入适量的二甲亚砜(DMSO)，再加入6倍过量摩尔比的化合物F1-17(10mg/ml预先溶在DMSO中)，保证反应体系中DMSO的体积占比不超过15％，于37℃搅动3小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280 紫外吸收值收集出峰样品。然后经由0.15微米孔径的过滤装置除菌，-60℃保存。
2、ADC-II的制备
将帕妥珠抗体原液用50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 6反应缓冲液稀释至5mg/mL，加入10倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于10℃搅动40小时。
将上述反应液冷至5℃，未经纯化加入适量的二乙基乙酰胺(DMA)，再加入6倍过量摩尔比的化合物F1-2(10mg/ml预先溶在DMA中)，保证反应体系中DMA的体积占比不超过10％，于25℃搅动2.5小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.22微米孔径的过滤装置除菌，-80℃保存。
3、ADC-III的制备
将帕妥珠抗体原液用PBS//1mM二乙基三胺五乙酸(DTPA)，pH 7.4反应缓冲液稀释至5mg/mL，加入20倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于15℃搅动2小时。
将上述反应液冷至10℃，未经纯化加入适量的乙腈(ACN)，再加入6倍过量摩尔比的化合物F1-20(10mg/ml预先溶在ACN中)，保证反应体系中ACN的体积占比不超过10％，于10℃搅动4小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 8.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品，然后过滤除菌，所得产物低温保存；如经由0.20微米孔径的过滤装置除菌，-90℃保存。
4、ADC-IV的制备
将帕妥珠抗体原液用50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 7反应缓冲液稀释至8mg/mL，加入8倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于25℃搅动25小时。
将上述反应液冷至5℃，未经纯化加入适量的二甲基甲酰胺(DMF)，再加入6倍过量摩尔比的化合物F1-19(10mg/ml预先溶在DMF中)，保证反应体系中DMF的体积占比不超过8％，于0℃搅动2小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.3微米孔径的过滤装置除菌，-80℃保存。
5、ADC-V的制备
将帕妥珠抗体原液用50mM组氨酸-氢氧化钠/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 7.4反应缓冲液稀释至6mg/mL，加入8倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于35℃搅动15小时。
将上述反应液冷至10℃，未经纯化加入适量的二甲基甲酰胺(DMF)，再加入6倍过量摩尔比的化合物F1-22(10mg/ml预先溶在DMF中)，保证反应体系中DMF的体积占比不超过8％，于0℃搅动5小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.15微米孔径的过滤装置除菌，-100℃保存。
6、ADC-VI的制备
将帕妥珠抗体原液用50mM硼酸-硼砂/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 9反应缓冲液稀释至10mg/mL，加入8倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于25℃搅动10小时。
将上述反应液冷至10℃，未经纯化加入适量的二甲基甲酰胺(DMF)，再加入6倍过量摩尔比的化合物F1-21(10mg/ml预先溶在DMF中)，保证反应体系中DMF的体积占比不超过8％，于0℃搅动4小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.2微米孔径的过滤装置除菌，-60℃保存。
7、ADC-VII的制备
将帕妥珠抗体原液用50mM磷酸二氢钾-氢氧化钠(KH2PO4-NaOH)/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH8反应缓冲液稀释至3mg/mL，加入8倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于15℃搅动48小时。
将上述反应液冷至0℃，未经纯化加入适量的二甲基甲酰胺(DMF)，再加入6倍过量摩尔比的化合物F1-18(10mg/ml预先溶在DMF中)，保证反应体系中DMF的体积占比不超过8％，于0℃搅动3小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.3米孔径的过滤装置除菌，-70℃保存。
8、ADC-VIII的制备
将曲妥珠抗体原液用50mM磷酸氢二钠-柠檬酸/150mM氯化钠(NaCl)/1mM二乙基三胺五乙酸(DTPA)，pH 7.4反应缓冲液稀释至5mg/mL，加入8倍过量摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于25℃搅动5小时。
将上述反应液冷至0℃，未经纯化加入适量的二甲基甲酰胺(DMF)，再加入6倍过量摩尔比的化合物F1-2(10mg/ml预先溶在DMF中)，保证反应体系中DMF的体积占比不超过8％，于0℃搅动2小时进行偶联。
采用脱盐柱将偶联反应混合物用pH 6.0的组氨酸-醋酸/蔗糖凝胶过滤纯化，根据UV280紫外吸收值收集出峰样品。然后经由0.3米孔径的过滤装置除菌，-80℃保存。
实施例组三、抗体偶联物的检测及稳定性研究
针对抗体偶联药物进行疏水相互作用色谱HIC分析能够获得偶联位点数目和位置和药物抗体偶联比(drug to antibody ratio，DAR)等重要信息。我们基于以下条件就上述ADC产物进行HIC分析，分析图谱见图11-1至11-8与图12-1至12-2。
Agilent 1290Infinity
色谱柱:Waters Protein-Pak Hi Res HIC(4.6*100mm，2.5μm)
流动相：2.5M硫酸铵(含125mM磷酸缓冲液)：125mM磷酸缓冲液：异丙醇
流速：0.7mL/min，柱温：25℃
此外，液质联用技术，已用于ADC药物结构和组成分析，评价ADC药物接头的稳定性，分析测定不同DAR组分的相对比例等。我们基于以下条件就上述ADC产物进行LCMS分析。分析图谱见图13-1至13-8与图14-1至14-2。
仪器：Agilent 6520Q-TOF
色谱柱:Polyhydroxyethyl-A(PHEA)(PolyLC,Columbia,MD)2.1mm*200mm；5μm
particles with 300
Figure PCTCN2017112958-appb-000145
pores
流动相:200mM醋酸铵
流速:0.1mL/min；
柱温:25℃
本发明基于马来酰胺的二硫链桥接具有更好的稳定性，在体内不易发生巯醚交换，为了进一步证实在Ar’部位引入取代基较未取代的苯基可以大大减缓马来酰胺开环后的环合二次水解反应，同时可以加强了抗体-药物偶联物的稳定性。本实验中我们制备了对照ADC，由帕妥珠抗体与Ar’仅为1,4-取代的苯环化合物(如下式)，进行偶联，偶联方法同ADC-I的制备。
Figure PCTCN2017112958-appb-000146
并挑选了ADC-I,ADC-II,ADC-VII与对照ADC进行比较，分别将相同蛋白浓度(10mg/mL)储存于制剂buffer中的ADC样品，置于25℃，分别于0、2、4、7天取样测定。
采用LC-MS(Q-TOF)测定各个抗体-药物偶联物(ADC)中对应的二次水解产物，提取二次水解产物的质谱特征峰，得其峰面积。比较0-7天峰面积的变化情况得到ADC二次水解产物的趋势，详见以下数据与图15。从数据中可以看出对照ADC二次水解产物明显高于
ADC-I,ADC-II,ADC-VII样品中的二次水解产物。
Figure PCTCN2017112958-appb-000147
此外，采用各个ADC样品还用HIC方法测定0、2、4、7天的变化情况，从图16-1至16-4中可以看出，在对照ADC样品在7天时在保留时间6.904位置出现了杂质峰，而ADC-I,ADC-II,ADC-VII样品中从0至7天HIC图谱基本上变化不明显，分别参见图17-1至17-4、图18-1至18-4、图19-1至19-4。
实施例组四、抗体偶联物的生物学检测
1.体外细胞增殖测定生物活性实验
以下实验中所用的实验材料来源于：DMEM培养基、DMEM/F12K培养基、RPMI 1640培养基、0.25％胰蛋白酶-EDTA、胎牛血清、100×丙酮酸钠、100×青链霉素购自Gibco公司。磺酰罗丹明B(Sulforhodamine B，SRB)购自Sigma公司。NCI-N87人胃癌细胞、BT-474人乳腺癌细胞来自中国科学院昆明细胞库。其它试剂均为分析纯。96孔平底聚苯乙烯(Corning，产品目录号3599)。Synergy 2酶标仪(Bio-Tek)。
在本实施例中，研究了ADC-I，ADC-II，ADC-III，ADC-IV，ADC-V，ADC-VI，ADC-VII，ADC-VIII对肿瘤细胞系增殖的作用。
本实施例使用磺酰罗丹明B(SRB)比色法来评价药物组合的抗增殖作用。SRB是一种粉红色阴离子染料，易溶于水，在酸性条件下可特异性地与细胞内组成蛋白质的碱性氨基酸结合；在510 nm波长下产生吸收峰，吸光值与细胞量成线性正相关，故可用作细胞数的定量检测。 本实施例选择的细胞系有：BT-474人乳腺癌细胞、NCI-N87人胃癌细胞。
BT-474、NCI-N87细胞在含10％胎牛血清的RPMI 1640培养基中，于37℃，5％CO2培养箱中培养至对数生长期，将处于对数生长期的上述细胞分别以2×103～9×103个细胞每孔的密度接种至96孔培养板，每孔100μL，培养24小时后，加入不同浓度的药物作用5天，分别以3、4或5-倍稀释制备9个浓度，每个浓度设复孔，并设相应浓度的溶媒对照及无细胞培养基孔。药物作用结束后，倾去培养液，加入4℃预冷的三氯乙酸溶液(30％，w/v)100μl，于4℃固定1小时，随后用去离子水冲洗5遍，室温干燥后，每孔加入0.4％(w/v)的SRB染液(Sigma，1％冰乙酸配制)100μL，室温下孵育染色30min后，用1％冰乙酸冲洗4次，去除未结合的染料，室温晾干。每孔加入10mM Tris溶液100μL，室温下孵育染色15min后，用1％冰乙酸冲洗五次洗去未结合的SRB，室温干燥后，每孔加入10mM Tris缓冲液(pH＝10.5)溶解与细胞蛋白结合的染料，采用Synergy 2酶标仪(Bio-Tek)波长510nm和690nm处测定光吸收值(OD值)，并得到A＝OD510-OD690。
抑制率(％)＝(A对照-A给药)/A对照×100％。
本实验使用了ADC-I，ADC-II，ADC-III，ADC-IV，ADC-V，ADC-VI，ADC-VII，ADC-VIII对Her2高表达的肿瘤细胞系进行了体外细胞培养增殖作用的研究。如下表所示，相对于裸抗Perjeta与Herceptin，对应的ADC-I，ADC-II，ADC-III，ADC-IV，ADC-V，ADC-VI，ADC-VII与ADC-VIII处理Her2高表达的NCI-N87人胃癌细胞、BT-474人乳腺癌细胞，均能明显抑制肿瘤细胞增殖。对应的增殖抑制曲线见图20-23。
Figure PCTCN2017112958-appb-000148
2.体内抗肿瘤功效测定实验
可以在体内测量本发明的组合的功效，即在啮齿类动物中植入癌细胞的同种异体移植物或异种移植物，并用所述组合处理肿瘤。将受试小鼠用药物或对照处理，并监测数周或更长时间以测量到达肿瘤倍增的时间，对数细胞杀伤，和肿瘤抑制。
4)实验动物
BALB/cA-nude裸小鼠，6-7周，♀，购自上海灵畅生物科技有限公司。生产许可证号：SCXK(沪)2013-0018；动物合格证号2013001815683。饲养环境：SPF级。
5)实验步骤
裸小鼠皮下接种6 106人胃癌NCI-N87细胞，待肿瘤生长至100-200mm3后，根据肿瘤体积将动物分组(D0)。小鼠静脉注射(IV)；给药体积10mL/kg；溶剂组给予相同体积的“溶剂” (0.1％BSA生理盐水)；具体给药剂量和给药方案见下表。每周测2次肿瘤体积，称小鼠体重，记录数据。
实验指标为考察药物对肿瘤生长的影响，具体指标为T/C％或抑瘤率TGI(％)。
每周二次用游标卡尺测量肿瘤直径,肿瘤体积(V)计算公式为：
V＝1/2×a×b2 其中a、b分别表示长、宽。
T/C(％)＝(T-T0)/(C-C0)x100其中T、C为实验结束时的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
抑瘤率(TGI)(％)＝100-T/C(％)。
当肿瘤出现消退时，抑瘤率(TGI)(％)＝100-(T-T0)/T0x100
如果肿瘤比起始体积缩小，即T<T0或C<C0时，即定义为肿瘤部分消退(PR)；如果肿瘤完全消失，即定义为肿瘤完全消退(CR)。
实验结束(D21)、达到实验终点、或肿瘤体积达到1500mm3，CO2麻醉处死动物，随后解剖取瘤并拍照。
6)实验结果
药物对HER2阳性人胃癌NCI-N87裸小鼠皮下移植瘤的疗效见下表和图24；荷瘤小鼠对以上药物均能较好耐受，没有体重减轻等症状发生。
Figure PCTCN2017112958-appb-000149
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (27)

  1. 一种取代马来酰胺类连接子片断，结构为式Ia所示：
    Figure PCTCN2017112958-appb-100001
    其中，R为X或ArS-，
    X选自下组：卤素；
    Ar选自下组：取代或未取代的C6-C10芳基，取代或未取代的5-12元杂芳基；
    Ar’选自下组：取代或未取代的C6-C10亚芳基，取代或未取代的5-12元亚杂芳基；
    L1为连接于Ar’基团上的-O(CH2CH2O)n-，其中n选自1-20中任一整数。
  2. 如权利要求1所述的取代马来酰胺类连接子片断，其特征在于，Ar选自下组：苯基、卤代苯、C1-C4烷基苯基、C1-C4烷氧基苯基、4-甲基苯基、4-甲氧基苯基、2-吡啶基、2-嘧啶基、1-甲基咪唑-2-基、
    Figure PCTCN2017112958-appb-100002
    其中W为与羰基连接的胺基R1，R1选自-NH2、
    Figure PCTCN2017112958-appb-100003
    Figure PCTCN2017112958-appb-100004
  3. 如权利要求1所述的取代马来酰亚胺类连接子片断，其特征在于，Ar’选自取代的亚苯基或吡啶基，所述的取代指基团上的氢原子被选自下组的一个或多个取代基所取代：卤素、C1-C4烷基、C1-C4烷氧基、三氟甲基、腈基、酰胺基。
  4. 如权利要求1所述的取代马来酰胺类连接子片断，其特征在于，所述连接子片段具有选自下组的结构：
    Figure PCTCN2017112958-appb-100005
    Figure PCTCN2017112958-appb-100006
    Figure PCTCN2017112958-appb-100007
    Figure PCTCN2017112958-appb-100008
  5. 一种取代马来酰亚胺类连接子-药物缀合物，及其药学上可接受的盐或溶剂物，结构如通式Ib所示：
    Figure PCTCN2017112958-appb-100009
    其中，R、Ar’、L1如权利要求1的定义；
    L2为化学键，或AA-PAB结构；其中，AA为二肽或三肽片断，PAB为对-氨基苄基氨甲酰基；
    CTD为通过酰胺键键合于L2的细胞毒性类小分子药物和/或治疗自身免疫疾病和抗炎症的药物。
  6. 如权利要求5所述的取代马来酰胺类连接子-药物缀合物，及其药学上可接受的盐或溶剂物，其特征在于，所述的AA选自下组：Val-Cit、Val-Ala、Phe-Lys、Ala‐Ala‐Asn、D‐Ala‐Phe‐Lys。
  7. 如权利要求5所述的取代马来酰胺类连接子-药物缀合物，及其药学上可接受的盐或溶剂物，其特征在于，所述的CTD选自下组：微管蛋白抑制剂、拓扑异构酶抑制剂、DNA结合剂。
  8. 如权利要求7所述的取代马来酰胺类连接子-药物缀合物，及其药学上可接受的盐或溶剂 物，其特征在于，所述的微管蛋白抑制剂选自下组：美登素衍生物、Monomethyl auristatin E、Monomethylauristatin F、Monomethyl Dolastatin 10、Tubulysin类衍生物、Cryptophycin类衍生物、Taltobulin。
  9. 如权利要求7所述的取代马来酰胺类连接子-药物缀合物，及其药学上可接受的盐或溶剂物，其特征在于，所述的DNA结合剂选自下组：PBD类衍生物、duocarmycin类衍生物。
  10. 如权利要求7所述的取代马来酰胺类连接子-药物缀合物，及其药学上可接受的盐或溶剂物，其特征在于，所述的拓扑异构酶抑制剂选自下组：阿霉素代谢产物PNU-159682衍生物、伊立替康代谢产物SN38衍生物、依沙替康。
  11. 如权利要求5所述的取代马来酰胺类连接子-药物缀合物，及其药学上可接受的盐或溶剂物，其特征在于，所述的CTD具有选自D1-D13’的分子结构：
    Figure PCTCN2017112958-appb-100010
    Figure PCTCN2017112958-appb-100011
  12. 如权利要求5所述的取代马来酰胺类连接子药物缀合物，及其药学上可接受的盐或溶剂物，其特征在于，所述的式Ib化合物选自下组：
    Figure PCTCN2017112958-appb-100012
    Figure PCTCN2017112958-appb-100013
    Figure PCTCN2017112958-appb-100014
    Figure PCTCN2017112958-appb-100015
    Figure PCTCN2017112958-appb-100016
    Figure PCTCN2017112958-appb-100017
    Figure PCTCN2017112958-appb-100018
    Figure PCTCN2017112958-appb-100019
    Figure PCTCN2017112958-appb-100020
    Figure PCTCN2017112958-appb-100021
    Figure PCTCN2017112958-appb-100022
  13. 一种抗体-药物偶联物，其特征在于，所述的偶联物是用如权利要求5至权利要求12任一项所述的取代马来酰亚胺类连接子-药物缀合物，及其药学上可接受的盐或溶剂物与抗体进行偶联形成的。
  14. 如权利要求13所述的抗体-药物偶联物，其特征在于，所述的偶联物具有通式Ic和/或Id的结构：
    Figure PCTCN2017112958-appb-100023
    Figure PCTCN2017112958-appb-100024
    其中，Ar’、L1、L2、CTD如权利要求5所定义；
    m＝1.0～5.0；
    Ab选自下组：蛋白质、酶、抗体、抗体片段、多肽。
  15. 如权利要求13所述的抗体-药物偶联物，其特征在于，所述的抗体选自下组：单克隆抗体、双特异性抗体、嵌合抗体、人源化抗体、抗体片段。
  16. 如权利要求13所述的抗体-药物偶联物，其特征在于，所述抗体是能够与选自下组的肿瘤相关抗原结合的抗体：HER2、HER3、CD19、CD20、CD22、CD30、CD33、CD37、CD45、CD56、CD66e、CD70、CD74、CD79b、CD138、CD147、CD223、EpCAM、Mucin 1、STEAP1、GPNMB、FGF2、FOLR1、EGFR、EGFRvIII、Tissue factor、c-MET、Nectin 4、AGS-16、Guanylyl cyclase C、Mesothelin、SLC44A4、PSMA、EphA2、AGS-5、GPC-3、c-KIT、RoR1、PD-L1、CD27L、5T4、Mucin 16、NaPi2b、STEAP、SLITRK6、ETBR、BCMA、Trop-2、CEACAM5、SC-16、SLC39A6、Delta-like protein3、Claudin 18.2。
  17. 如权利要求16所述的抗体-药物偶联物，其特征在于，所述的HER2抗体选自下组：曲妥珠单抗、帕妥珠单抗，或者所述EGFR抗体选自Erbitux或Vectibix。
  18. 一种药物组合物，其特征在于，包括：(a)如权利要求13-17中任一项所述的抗体-药物偶联物；和(b)药学上可接受的稀释剂，载剂或赋形剂。
  19. 如权利要求13-17中任一项所述的抗体-药物偶联物在用于制备治疗肿瘤的药物的用途。
  20. 如权利要求12-17中任一项所述的抗体-药物偶联物的制备方法，其特征在于，包括步骤：
    (1)用抗体与还原试剂在缓冲液中反应，得到经还原后的抗体；
    (2)用连接子-药物缀合物与步骤(1)得到的经还原后的抗体在缓冲液与有机溶剂混合液中进行交联，得到抗体-药物偶联物。
  21. 如权利要求20所述抗体-药物偶联物的制备方法，其特征在于，所述方法的反应路线包括：
    Figure PCTCN2017112958-appb-100025
    其中R、Ab、Ar’、L1、L2、CTD、m如权利要求14的定义。
  22. 如权利要求1-4中任一项所述的取代马来酰胺类连接子片段的制备方法，包括如下步骤：通过中间体C与二卤代马来酸酐环合反应得中间体D，再与芳基硫酚进行取代反应后获 得连接子片断分子E，反应式如下：
    Figure PCTCN2017112958-appb-100026
    其中R、n如权利要求1的定义，X代表卤素，优选Br、Cl；U、V各自独立代表N或C。
  23. 根据权利要求22所述取代马来酰胺类连接子片段的制备方法，其特征在于，所述C通过B还原而得，反应式如下：
    Figure PCTCN2017112958-appb-100027
    其中R、n、U、V如权利要求22的定义。
  24. 根据权利要求23所述的取代马来酰胺类连接子片段的制备方法，其特征在于，所述B通过A与氟代硝基苯取代反应而得，反应式如下：
    Figure PCTCN2017112958-appb-100028
    其中R、n、U、V如权利要求22的定义。
  25. 根据权利要求24所述的取代马来酰胺类连接子片段的制备方法，其特征在于，所述B通过下式制备：
    Figure PCTCN2017112958-appb-100029
    其中R、n、U、V如权利要求22的定义。
  26. 根据权利要求25所述的取代马来酰胺类连接子片段的制备方法，其特征在于，A由n甘醇与卤代乙酸叔丁酯反应而得，反应式如下：
    Figure PCTCN2017112958-appb-100030
    其中n、X如权利要求22的定义。
  27. 如权利要求5至12中任一项所述的取代马来酰胺类连接子-药物缀合物的制备方法，包括取代马来酰胺类连接子与带有二肽/三肽-PAB细胞毒药物CTD进行缩合，分别得到F1或F’1，所述反应路线如下：
    Figure PCTCN2017112958-appb-100031
    其中R如权利要求1的定义，Rx代表卤素、C1-C4烷基、C1-C4烷氧基、三氟甲基、腈基或酰胺基，Ry代表H或烷基。
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