WO2016131409A1 - 抗体药物偶联物 - Google Patents

抗体药物偶联物 Download PDF

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WO2016131409A1
WO2016131409A1 PCT/CN2016/073844 CN2016073844W WO2016131409A1 WO 2016131409 A1 WO2016131409 A1 WO 2016131409A1 CN 2016073844 W CN2016073844 W CN 2016073844W WO 2016131409 A1 WO2016131409 A1 WO 2016131409A1
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cancer
solvate
tumor
antibody
pharmaceutically acceptable
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English (en)
French (fr)
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胡朝红
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上海美雅珂生物技术有限责任公司
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Priority to US15/550,995 priority Critical patent/US10792370B2/en
Priority to CN201680003611.9A priority patent/CN106999606B/zh
Publication of WO2016131409A1 publication Critical patent/WO2016131409A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001103—Receptors for growth factors
    • A61K39/001104—Epidermal growth factor receptors [EGFR]
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample

Definitions

  • the present invention relates to antibody drug conjugates, and in particular to anti-EGF receptor antibody drug conjugates.
  • the invention also relates to a composition comprising the antibody drug conjugate, and to the medical use of the antibody drug conjugate.
  • Epidermal Growth Factor Receptor (EGFR, also known as HER1, c-ErbB1) is a cell surface receptor of the epidermal growth factor family, which is composed of 1186 amino acid residues and has a molecular weight of 170 kD. Glycoprotein (Jorissen RN, Walker F, Pouliot N, et al. Epidermal growth factor receptor: mechanisms of activation and signaling. Exp Cell Res, 2003; 284: 31-53).
  • EGFR belongs to the type I tyrosine kinase receptor ErbB subfamily (ErbB 1-4) and has tyrosine kinase activity. EGFR is stably expressed in many epithelial tissues, including skin and hair follicles.
  • Abnormal expression of the epidermal growth factor receptor or activation by receptor mutations can lead to cancer.
  • Many solid tumors have been found to overexpress EGFR, such as colorectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, and esophageal cancer (Olayioye MA, Neve RM, Lane HA, et al.
  • the EerbB Signaling network receptor heterodimerzation in development and cancer.
  • Growth factors such as transforming growth factor alpha and epidermal growth factor are endogenous ligands for the epidermal growth factor receptor.
  • ligands bind to the epidermal growth factor receptor, activate the activity of the receptor intracellular tyrosine protein kinase, initiate multiple downstream signal transduction pathways, thereby regulating the growth and differentiation of normal cells, increasing the invasiveness and promotion of tumor cells.
  • Angiogenesis inhibition of tumor cell apoptosis (Ciardiello F, Tortola GA novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res, 2001; 7: 2958-2970).
  • the overexpression of epidermal growth factor receptor in tumors and its important role in tumor cell growth and differentiation make epidermal growth factor receptor a promising target for tumor therapy.
  • the effective rate (ORR) of treatment of colorectal cancer with Erbitux and irinotecan is 23%, and the effective rate of treatment of head and neck cancer with fluoropyrimidine and other chemotherapy drugs is 13%-30%. Due to the chimeric antibody of human and mouse, Erbitux produced antibody responses in 3.7% of patients in clinical trials.
  • panitumumab (Vectibix, panitumumab, Amgen), a fully humanized monoclonal antibody prepared using transgenic mouse technology, without a murine protein sequence.
  • the antibody targets the epidermal growth factor receptor (EGFR) and was approved by the FDA in September 2006. It is used in combination with fluoropyrimidine, oxaliplatin and irinotecan or after chemotherapy to treat EGFR-positive metastatic junctions. Rectal cancer. In 2006, the FDA approved its monotherapy for chemotherapy-resistant metastatic colorectal cancer (mCRC). However, panitumumab is an IgG2 subtype antibody.
  • antibody-based drugs having a higher biological activity of humanized anti-epidermal growth factor receptors, particularly antibody drugs that are effective against KRAS mutants, such as antibody drug conjugates, to further enhance Efficacy, reduce side effects.
  • ADCs antibody drug conjugates
  • Antibody drug conjugates generally consist of three parts:
  • linker of the small molecule drug and the monoclonal antibody, also called the linker.
  • Antibody drug conjugates use tumor target specificity and cytotoxicity of chemical drugs to kill tumor cells. Its mechanism of action is: (1) antibody drug conjugates specifically bind to target antigens on tumor cells using monoclonal antibodies; (2) complexes of antibody drug conjugates and target antigens are mediated through target antigens Endocytosis into cells; (3) antibody drug conjugates degrade in cells, releasing cytotoxic chemical drugs; (4) cytotoxic chemical drugs kill tumor cells.
  • antibody drug conjugates may seem simple, but whether an antibody drug conjugate can be a safe and effective drug is very complex and unpredictable, depending on many factors, such as:
  • the characteristics of the ADC whether the complex of the ADC and the target can be endocytosed, the stability of the ADC in the blood, what linker is used on the ADC and linking several chemicals, and the ADC kills the activity and biological toxicity of the cancer cells.
  • the inventors of the present invention prepared an anti-epidermal growth factor receptor antibody drug conjugate by a large amount of experiments and creative labor, and confirmed that it has a good biological activity, thereby completing the present invention.
  • a first aspect of the invention relates to an antibody drug conjugate, a pharmaceutically acceptable salt, solvate thereof or solvate thereof, comprising an anti-epidermal growth factor receptor antibody covalently linked to a cytotoxic agent.
  • the anti-EGF receptor antibody comprises a heavy chain and a light chain
  • the heavy chain variable region CDR1, CDR2, CDR3 comprises SEQ ID. NO: a sequence represented by 5 to 7 or a mutant thereof
  • the light chain variable region CDR1, CDR2, and CDR3 each include the sequence shown in SEQ ID NOS: 12 to 14, or a mutant thereof.
  • the anti-EGF receptor antibody light chain variable region CDR1, CDR2, CDR3 region comprises the sequences set forth in SEQ ID NOs: 12-14, respectively, or with SEQ ID NO
  • the sequence of 12 to 14 has an identity greater than 70%, such as greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the sequence, for example, a sequence having 3, 2 or 1 mutation, deletion or addition of an amino acid.
  • the anti-EGF receptor antibody heavy chain variable region CDR1, CDR2, CDR3 region comprises the sequence set forth in SEQ ID NOs: 5-7, respectively, or with SEQ ID NO
  • the sequence of 5 to 7 has an identity greater than 70%, such as greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the sequence, for example, a sequence having 3, 2 or 1 mutation, deletion or addition of an amino acid.
  • the anti-EGF receptor antibody heavy chain variable region FR1, FR2, FR3, FR4 region comprises the sequence set forth in SEQ ID NOs: 8-11, respectively, or a mutation thereof body.
  • the anti-EGF receptor antibody heavy chain variable region FR1, FR2, FR3, FR4 region comprises the sequences set forth in SEQ ID NOs: 8-11, respectively, or comprises The identity with the above sequence is greater than 70%, such as greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% the sequence of.
  • the anti-EGF receptor antibody light chain variable region FR1, FR2, FR3, FR4 region comprises the sequence set forth in SEQ ID NOs: 15-18, respectively, or a mutation thereof body.
  • the anti-EGF receptor antibody light chain variable region FR1, FR2, FR3, FR4 region comprises the sequences set forth in SEQ ID NOs: 15-18, respectively, or comprises The identity with the above sequence is greater than 70%, such as greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% the sequence of.
  • sequence of the heavy chain variable region of the anti-EGF receptor antibody is set forth in SEQ ID NO: 1.
  • the light chain of the anti-epidermal growth factor receptor antibody is The sequence of the variable region is shown in SEQ ID NO: 2.
  • the heavy chain constant region of the anti-epidermal growth factor receptor antibody is selected from the group consisting of a human IgG, IgM, IgA, IgD, IgA constant region or a mutant of the above constant region.
  • the IgG is selected from the group consisting of IgGl, IgG2, IgG3 and IgG4.
  • the amino acid sequence of the heavy chain constant region of the anti-EGF receptor antibody comprises the sequence set forth in SEQ ID NO: 3 or comprises the sequence set forth in SEQ ID NO: The identity is greater than 70%, such as sequences greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
  • the light chain constant region of the anti-EGF receptor antibody is a mutant of a human lambda constant region, a kappa constant region, or the above constant region.
  • the amino acid sequence of the light chain constant region of the anti-EGF receptor antibody comprises the sequence set forth in SEQ ID NO: 4 or comprises the sequence set forth in SEQ ID NO: The identity is greater than 70%, such as sequences greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
  • the antibody drug conjugate, a pharmaceutically acceptable salt, solvate thereof or solvate thereof has the structure of Formula I,
  • Ab represents an anti-epidermal growth factor receptor antibody
  • D represents a cytotoxic agent
  • p 1-8, such as 2-6, such as 3-5.
  • the cytotoxic agent is selected from the group consisting of a chemotherapeutic drug, a toxin (eg, a bacterial, fungal, plant, or animal-derived enzymatic toxin or fragment thereof), a radioisotope, a cytokine, an antibiotic, Enzymes, nanoparticles and bioactive peptides.
  • the cytotoxic agent is selected from the group consisting of Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), maytansinoid (eg, Maytansine DM1, Maytansine DM4), and calicheamicin. (calicheamicin), duocarmycin MGBA, doxorubicin, ricin, diphtheria toxin, etc. I131, interleukins, tumor necrosis factor, chemokines and nanoparticles.
  • MMAE Monomethyl auristatin E
  • MMAF Monomethyl auristatin F
  • maytansinoid eg, Maytansine DM1, Maytansine DM4
  • calicheamicin maytansinoid
  • duocarmycin MGBA doxorubicin
  • ricin ricin
  • diphtheria toxin etc. I131, interleukins, tumor necrosis factor, chemokines and nanoparticles.
  • the cytotoxic agent is MMAE.
  • the joint is cleavable or non-cleavable.
  • the linker is selected from the group consisting of 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (val- Cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) valerate (SPP), N -Succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl)aminobenzoic acid Ester (SIAB), and 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).
  • MC 6-maleimidocaproyl
  • MP maleimidopropionyl
  • val- Cit valine-citrulline
  • the linker is 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).
  • L-D as described in Formula I is vc-MMAE, the structure of which is as follows:
  • the antibody drug conjugate, pharmaceutically acceptable salt, solvate thereof or solvate of the salt is as follows:
  • Ab is an anti-epidermal growth receptor antibody and p is 1-8, such as 2-6, such as 3-5.
  • a second aspect of the invention relates to a composition (e.g., a pharmaceutical composition) comprising the antibody drug conjugate of any one of the first aspect of the invention, a pharmaceutically acceptable salt, solvate thereof or a solvate thereof
  • a pharmaceutically acceptable carrier, diluent or excipient is also included.
  • the composition further comprises a known method for treating a tumor Chemotherapeutic drugs such as Adriamycin, cyclophosphamide and taxanes [Taxol and Taxotere], capecitabine (Xeloda), gemcitabine ( Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (Rining, Furlong, Arnoldin), 5-FU plus folinic acid, irinotecan (camtosar), oxaliplatin , cisplatin, carboplatin, estramustine, novantrone, prednisone, vincristine (Oncovin), etc., or a combination thereof.
  • a tumor Chemotherapeutic drugs such as Adriamycin, cyclophosphamide and taxanes [Taxol and Taxotere], capecitabine (Xeloda), gemcitabine ( Gemzar), vinorelbine (Navelbine), tamoxifen
  • the present invention also relates to the antibody drug conjugate of any one of the first aspects of the present invention, a pharmaceutically acceptable salt, solvate thereof or solvate thereof, for the preparation of a prophylactic and/or therapeutic agent against surface growth factor Use in drugs for body (EGFR) related diseases.
  • EGFR drugs for body
  • the disease associated with surface growth factor receptor is a tumor associated with EGFR, such as a tumor associated with overexpression of EGFR, eg, selected from the group consisting of colon cancer, rectal cancer, Head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer and glioma.
  • a tumor associated with overexpression of EGFR eg, selected from the group consisting of colon cancer, rectal cancer, Head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer and glioma.
  • the tumor is a tumor of a KRAS gene mutation, such as colon cancer, rectal cancer, lung cancer or pancreatic cancer with a KRAS gene mutation.
  • a KRAS gene mutation such as colon cancer, rectal cancer, lung cancer or pancreatic cancer with a KRAS gene mutation.
  • the tumor is a tumor of a BRAF gene mutation, such as colon cancer, rectal cancer, and lung cancer selected from the group consisting of a BRAF gene mutation.
  • a BRAF gene mutation such as colon cancer, rectal cancer, and lung cancer selected from the group consisting of a BRAF gene mutation.
  • the present invention also relates to the antibody drug conjugate of any one of the first aspects of the present invention, a pharmaceutically acceptable salt thereof, a solvate thereof or a solvate of the salt, which inhibits tumor angiogenesis, delays tumor progression, and inhibits Use in tumor growth and drugs that inhibit tumor cell proliferation.
  • the tumor is selected from the group consisting of colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, Gastric cancer, pancreatic cancer and glioma.
  • the tumor is a tumor of a KRAS gene mutation, such as colon cancer, rectal cancer, lung cancer or pancreatic cancer with a KRAS gene mutation.
  • a KRAS gene mutation such as colon cancer, rectal cancer, lung cancer or pancreatic cancer with a KRAS gene mutation.
  • the tumor is a tumor of a BRAF gene mutation, such as colon cancer, rectal cancer, and lung cancer selected from the group consisting of a BRAF gene mutation.
  • a BRAF gene mutation such as colon cancer, rectal cancer, and lung cancer selected from the group consisting of a BRAF gene mutation.
  • the invention further relates to a method of preventing and/or treating a disease associated with epidermal growth factor receptor (EGFR), the method comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of any of the first aspects of the invention
  • Antibody drug conjugate, a pharmaceutically acceptable salt, solvate thereof or the like a solvate of a salt comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of any of the first aspects of the invention
  • Antibody drug conjugate, a pharmaceutically acceptable salt, solvate thereof or the like a solvate of a salt a pharmaceutically acceptable salt, solvate thereof or the like a solvate of a salt.
  • the disease associated with the epidermal growth factor receptor is a tumor associated with EGFR, such as a tumor associated with overexpression of EGFR, eg, selected from the group consisting of colon cancer, rectal cancer, Head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer and glioma.
  • a tumor associated with overexpression of EGFR eg, selected from the group consisting of colon cancer, rectal cancer, Head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer and glioma.
  • the tumor is a tumor of a KRAS gene mutation, such as colon cancer, rectal cancer, lung cancer or pancreatic cancer with a KRAS gene mutation.
  • a KRAS gene mutation such as colon cancer, rectal cancer, lung cancer or pancreatic cancer with a KRAS gene mutation.
  • the tumor is a tumor of a BRAF gene mutation, such as colon cancer, rectal cancer, and lung cancer selected from the group consisting of a BRAF gene mutation.
  • a BRAF gene mutation such as colon cancer, rectal cancer, and lung cancer selected from the group consisting of a BRAF gene mutation.
  • the invention further relates to a method of inhibiting tumor angiogenesis, delaying tumor progression, inhibiting tumor growth, inhibiting tumor cell proliferation, the method comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of any of the first aspects of the invention
  • An antibody drug conjugate of the invention, a pharmaceutically acceptable salt, solvate thereof or a solvate of the salt comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of any of the first aspects of the invention
  • the tumor is selected from the group consisting of colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, Gastric cancer, pancreatic cancer and glioma.
  • the tumor is a tumor of a KRAS gene mutation, such as colon cancer, rectal cancer, lung cancer or pancreatic cancer with a KRAS gene mutation.
  • a KRAS gene mutation such as colon cancer, rectal cancer, lung cancer or pancreatic cancer with a KRAS gene mutation.
  • the tumor is a tumor of a BRAF gene mutation, such as colon cancer, rectal cancer, and lung cancer selected from the group consisting of a BRAF gene mutation.
  • a BRAF gene mutation such as colon cancer, rectal cancer, and lung cancer selected from the group consisting of a BRAF gene mutation.
  • the invention further relates to an antibody drug conjugate according to any one of the first aspects of the invention, a pharmaceutically acceptable salt, solvate thereof or solvate thereof, for use in the prevention and/or treatment of epidermal growth Factor receptor (EGFR) related diseases.
  • EGFR epidermal growth Factor receptor
  • the disease associated with the epidermal growth factor receptor is a tumor associated with EGFR, such as a tumor associated with overexpression of EGFR, eg, selected from the group consisting of colon cancer, rectal cancer, Head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer and glioma.
  • a tumor associated with overexpression of EGFR eg, selected from the group consisting of colon cancer, rectal cancer, Head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer and glioma.
  • the tumor is a tumor of a KRAS gene mutation, such as colon cancer, rectal cancer, lung cancer or pancreatic cancer with a KRAS gene mutation.
  • a KRAS gene mutation such as colon cancer, rectal cancer, lung cancer or pancreatic cancer with a KRAS gene mutation.
  • the tumor is a tumor of a BRAF gene mutation, such as colon cancer, rectal cancer, and lung cancer selected from the group consisting of a BRAF gene mutation.
  • a BRAF gene mutation such as colon cancer, rectal cancer, and lung cancer selected from the group consisting of a BRAF gene mutation.
  • the invention further relates to an antibody drug conjugate according to any one of the first aspects of the invention, a pharmaceutically acceptable salt, solvate thereof or solvate thereof, for inhibiting tumor angiogenesis and delaying tumor progression Inhibition of tumor growth and inhibition of tumor cell proliferation.
  • the tumor is selected from the group consisting of colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, Gastric cancer, pancreatic cancer and glioma.
  • the tumor is a tumor of a KRAS gene mutation, such as colon cancer, rectal cancer, lung cancer or pancreatic cancer with a KRAS gene mutation.
  • a KRAS gene mutation such as colon cancer, rectal cancer, lung cancer or pancreatic cancer with a KRAS gene mutation.
  • the tumor is a tumor of a BRAF gene mutation, such as colon cancer, rectal cancer, and lung cancer selected from the group consisting of a BRAF gene mutation.
  • a BRAF gene mutation such as colon cancer, rectal cancer, and lung cancer selected from the group consisting of a BRAF gene mutation.
  • the anti-epidermal growth factor receptor antibody drug conjugate of the present invention a pharmaceutically acceptable salt thereof, a solvate thereof or a solvate of the salt has good tumor cell growth inhibiting activity in vivo and in vitro, in particular Tumor cells that are moderately and lowly expressed by EGFR also exhibit significant cytostatic activity and low cytotoxicity; more particularly, the anti-epidermal growth factor receptor antibody drug conjugate of the present invention is pharmaceutically acceptable
  • the salt, the solvate or the solvate of the salt also has a good curative effect on the KRAS gene mutation or the BRAF gene-mutated tumor, and thus has a good application prospect.
  • the term "antibody” refers to an immunoglobulin molecule usually composed of two identical pairs of polypeptide chains each having one "light” (L) chain and one "heavy” (H) chain.
  • the light chain of an antibody can be classified into two types, ⁇ and ⁇ .
  • the heavy chain can be divided into five types: ⁇ , ⁇ , ⁇ , ⁇ or ⁇ .
  • the antibody can be divided into five categories: IgM, IgD, IgG, IgA and IgE.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids.
  • Each heavy chain is comprised of a heavy chain variable region (V H) and a heavy chain constant region (C H) composition.
  • the heavy chain constant region is comprised of three domains (C H 1, C H 2 and C H 3) components.
  • Each light chain is comprised of a light chain variable region (V L) and a light chain constant region (C L) components.
  • the light chain constant region is comprised of one domain, C L composition.
  • the constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and component Clq of the complement system.
  • V H regions may be subdivided into hypervariability regions (termed complementarity determining regions (CDR)), interspersed with the more conserved regions referred to as framework regions (FR) of.
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.
  • antibody is not limited by any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibody may be a different type of antibody, for example, an IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.
  • the IgG heavy chain constant region comprises IgG1, IgG2, IgG3 or IgG4. In an embodiment of the invention, the IgG heavy chain constant region is of the IgGl type.
  • the kappa light chain constant region includes various isoforms such as Km1, Km1, 2 or Km3.
  • the heavy chain variable region amino acid sequence of the anti-EGF receptor antibody is SEQ ID NO: 1.
  • the light chain variable region amino acid sequence of the anti-EGF receptor antibody is SEQ ID NO:2.
  • amino acid sequence of the heavy chain variable region of the antibody of the invention has a sequence similarity to SEQ ID NO: 1 of at least 70%, preferably at least 75%, preferably at least 80%, preferably 85%. More preferably, it is at least 90%, and most preferably at least 95%.
  • amino acid sequence of the light chain variable region of the antibody of the invention has a sequence similarity to SEQ ID NO: 2 of at least 70%, preferably at least 75%, preferably at least 80%, preferably 85%. More preferably, it is at least 90%, and most preferably at least 95%.
  • amino acid sequences of the CDRs of the heavy and light chain variable regions of the anti-EGF receptor antibody are determined as follows:
  • amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain are SEQ ID NOS: 5-7, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are SEQ ID NOs: 12-14, respectively.
  • the CDRs of the heavy chain of the anti-EGF receptor antibody may comprise an amino acid sequence which is a mutation or addition or deletion of one or more amino acids at SEQ ID NOs: 5-7.
  • the amino acid that is mutated, added or deleted does not exceed 3 amino acids. More preferably, the amino acid that is mutated, added or deleted does not exceed 2 amino acids. Most preferably, the amino acid that is mutated, added or deleted does not exceed one amino acid.
  • the CDRs of the light chain of the anti-EGF receptor antibody may comprise an amino acid sequence that exhibits a mutation, addition or deletion of one or more amino acids at SEQ ID NOs: 12-14.
  • the amino acid that is mutated, added or deleted does not exceed 3 amino acids. More preferably, the amino acid that is mutated, added or deleted does not exceed 2 amino acids. Most preferably, the amino acid that is mutated, added or deleted does not exceed one amino acid.
  • amino acid sequences of the FRs of the heavy and light chain variable regions of the anti-EGF receptor antibody are determined as follows:
  • the sequences of the heavy chain variable regions FR1, FR2, FR3, FR4 are SEQ ID NOS: 8-11, respectively.
  • the sequences of the light chain variable regions FR1, FR2, FR3, FR4 are SEQ ID NOS: 15-18, respectively.
  • the amino acid sequence of the heavy or light chain variable region FR of an anti-epidermal growth factor receptor antibody may be one or more amino acids present on SEQ ID NOs: 8-11, SEQ ID NOs: 15-18 Mutation, addition or deletion.
  • the amino acid that is mutated, added or deleted does not exceed 3 amino acids. More preferably, the amino acid that is mutated, added or deleted does not exceed 2 amino acids. Most preferably, the amino acid that is mutated, added or deleted does not exceed one amino acid.
  • Variants after mutation, addition or deletion of an amino acid of the above antibody or CDR region or framework region still retain the ability to specifically bind to EGFR.
  • amino acid sequence of the heavy chain constant region of the anti-EGF receptor antibody is SEQ ID NO:3.
  • the light chain constant region amino acid sequence of the anti-EGF receptor antibody is SEQ ID NO:4.
  • amino acid sequence of the heavy chain constant region of the antibody of the invention has a sequence similarity to SEQ ID NO: 3 of at least 70%, preferably at least 75%, preferably at least 80%, preferably 85%, Still more preferably at least 90%, most preferably at least 95%, such as 96%, 97%, 98%, 99%.
  • amino acid sequence of the light chain constant region of the antibody of the invention has a sequence similarity to SEQ ID NO: 4 of at least 70%, preferably at least 75%, preferably at least 80%, preferably 85%, Still more preferably at least 90%, most preferably at least 95%, such as 96%, 97%, 98%, 99%.
  • the monoclonal antibody variants of the invention can be obtained by conventional genetic engineering methods. Those skilled in the art are fully aware of methods for engineering DNA molecules using nucleic acid mutations. In addition, nucleic acid molecules encoding heavy and light chain variants can also be obtained by chemical synthesis.
  • BLAST and BLAST 2.0 algorithms for determining sequence identity (homology) and percent sequence similarity are, for example, BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acid. Res. 25:3389, respectively. 3402 and Altschul et al. (1990) J. Mol. Biol. 215: 403-410.
  • BLAST and BLAST 2.0 can be used to determine the percent amino acid sequence identity of the invention, for example, as described in the literature or by default parameters.
  • Software for performing BLAST analyses is available to the public through the National Center for Biotechnology Information.
  • the amino acid sequence having at least 70% sequence identity to the amino acid sequence comprises a polypeptide sequence substantially identical to the amino acid sequence, for example when using the methods described herein (eg, BLAST analysis using standard parameters) Containing at least 70% sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% compared to the polypeptide sequence of the invention Those sequences of 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.
  • the toxin used for the antibody drug conjugate includes a diphtheria toxin A chain, a non-binding active fragment of diphtheria toxin, an exotoxin A chain (from Pseudomonas aeruginosa), and ricin ( Ricin) A chain, abrin A chain, modeccin A chain, ⁇ - quercin, aranthus (Aleutites fordii) toxic protein, dianthin Toxic protein, Phytolacaamericana toxic protein (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis Inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and cephalosporin ( Trichothecenes). See, for example, WO 93/21232, published October 28, 1993.
  • radionuclides can be used to generate antibody drug conjugates. Examples include 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
  • Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), iminosulfane (IT), imidate (such as dimethyl adipyl HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazide Compounds such as bis(p-azidobenzoyl)hexanediamine), double nitrogen derivatives (such as bis(p-diazobenzoyl)-ethylenediamine), diisothiocyanates (such as toluene 2) , a bifunctional derivative of a 6-diisocyanate), and a double active fluorine compound such as 1,5-difluoro-2,4-dinitrobenzene.
  • SPDP N-succinimidyl 3-(2-pyridyldithi
  • a ricin-containing immunotoxin prepared as described by Vitetta et al. (1987) Science, 238: 1098.
  • Carbon-14-labeled 1-isothiocyanate benzyl-3-methyldiethylenetriamine Acid is an exemplary chelating agent for coupling radionucleotides to antibodies (WO 94/11026).
  • the invention also encompasses antibodies and one or more small molecule toxins (such as calicheamicin, maytansinoid, dolastatin, auristatin, trichothecene) Trichothecene), and CC 1065 and conjugates of toxin-active derivatives of these toxins.
  • the antibody drug conjugate comprises an anti-epidermal growth factor receptor conjugated to dolastatin or dolastatin peptide analog and derivative auristatin (U.S. Patent No. 5,635,483; 5,780,588) antibody.
  • Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division activities (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anticancer activity. (US 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965).
  • the dolastatin or auristatin drug moiety can be attached to the antibody via the N (amino) terminus or C (carboxyl) terminus of the peptide drug moiety (WO 02/088172).
  • the enzyme as a cytotoxic agent may be a compound having nucleic acid degrading activity (for example, ribonuclease or DNA endonuclease such as deoxyribonuclease; DNase).
  • nucleic acid degrading activity for example, ribonuclease or DNA endonuclease such as deoxyribonuclease; DNase.
  • the drug loading is represented by p, i.e., the average number of drug modules (i.e., cytotoxic agents) per antibody in the molecule of Formula I: Ab-(LD) p .
  • the drug load can range from 1-20 drug modules per antibody (D).
  • An ADC of Formula I includes a collection of antibodies conjugated to a range of (1-20) drug modules.
  • the average number of drug modules per antibody in the ADC preparation from the coupling reaction can be verified by conventional means such as mass spectrometry, ELISA assays, and HPLC. It is also possible to determine the quantitative distribution of the ADC in terms of p. In some cases, separation, purification, and verification of a homologous ADC having a p value from other ADCs can be accomplished by means such as reverse phase HPLC or electrophoresis.
  • p may be limited by the number of attachment sites on the antibody.
  • the attachment site is a cysteine thiol
  • the antibody may have only one or several cysteine thiol groups, or there may be only one or several thiol groups with sufficient reactivity to attach the linker.
  • a higher drug load such as p > 5, can cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates.
  • the ADC of the present invention has a drug loading ranging from 1 to about 8; from about 2 to about 6; from about 3 to about 5; from about 4 to about 5; from about 3.5 to 4.5;
  • the optimal ratio of drug modules for each antibody that has been shown for some ADCs can be less than 8, and can range from about 2 to about 5. See US2005-0238649A1 (completely incorporated herein by reference).
  • a drug moiety that is less than a theoretical maximum in a coupling reaction is coupled to the antibody.
  • the antibody may comprise, for example, a lysine residue that does not react with a drug-linker intermediate or a linker reagent.
  • antibodies do not contain many free and reactive cysteine thiol groups that can be attached to a drug moiety; in fact, most of the cysteine thiol groups in the antibody exist as disulfide bridges.
  • the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or complete reducing conditions to produce a reactive cysteine thiol group.
  • the antibody is placed under denaturing conditions to expose a reactive nucleophilic group, such as lysine or cysteine.
  • the loading of the ADC can be controlled in different ways, for example by (i) limiting the number of moles of drug-linker intermediate or linker reagent relative to the antibody, and (ii) limiting the time or temperature of the coupling reaction, (iii) a cysteine thiol modified moiety or a limiting reducing condition, (iv) engineering the amino acid sequence of the antibody by recombinant techniques such that the number and position of cysteine residues are used to control linker-drug attachment The number and/or location of the changes.
  • the resulting product is a mixture of ADC compounds having one or more drug modules attached to the antibody.
  • the average number of drugs per antibody can be calculated from the mixture by a double ELISA antibody assay specific for antibodies and specific for the drug.
  • the various ADC molecules in the mixture can be identified by mass spectrometry and separated by HPLC, such as hydrophobic interaction chromatography.
  • a homogenous ADC having a single loading value can be separated from the coupling mixture by electrophoresis or chromatography.
  • the pharmaceutically acceptable salts of the antibody drug conjugates include acid addition salts of inorganic acids, carboxylic acids and sulfonic acids, such as the following acid salts: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonate. Acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalene disulfonic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, Tartaric acid, malic acid citric acid, fumaric acid, maleic acid and benzoic acid.
  • inorganic acids such as the following acid salts: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonate. Acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalene disulfonic acid,
  • the pharmaceutically acceptable salts of the antibody drug conjugates of the present invention also include salts of conventional bases such as, by way of example only and preferred, alkali metal salts (e.g., sodium and potassium), alkaline earth metal salts (e.g., calcium salts).
  • alkali metal salts e.g., sodium and potassium
  • alkaline earth metal salts e.g., calcium salts.
  • a magnesium salt and an ammonium salt derived from ammonia or an organic amine having 1 to 16 carbon atoms such as (exemplary and preferred only) ethylamine, diethylamine, triethylamine, ethyldi Isopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzamide, N-methylpiperidine, N-methylmorpholine, arginine Lysine and 1,2-ethylenediamine.
  • a solvate means an antibody drug conjugate of the present invention in these forms: a complex of the antibody drug conjugate formed in a solid or liquid form formed by coordination with a solvent molecule. Hydrates are a specific form of solvates that have coordinated water molecules. In the present invention, hydrate is a preferred solvate.
  • EGFR-associated tumors that can be preferably treated using the antibody drug conjugates of the invention include tumors that overexpress EGFR, respiratory tumors (eg, small cell carcinoma and non-small cell carcinoma, bronchial carcinoma), with non-small cells being particularly preferred.
  • respiratory tumors eg, small cell carcinoma and non-small cell carcinoma, bronchial carcinoma
  • non-small cells being particularly preferred.
  • Lung cancer tumors of the digestive tract (for example, esophagus, stomach, gallbladder, small intestine, large intestine, rectum), among which intestinal tumors; tumors of endocrine glands and exocrine glands (such as thyroid and parathyroid gland, pancreas and salivary gland), among which tumors are particularly preferred, are particularly preferred.
  • Pancreatic neoplasms eg, larynx, hypopharynx, nasopharynx, oropharynx, lips, mouth, tongue, and esophagus; and/or gliomas.
  • EGFR overexpression refers to an increase in the expression level of EGFR compared to the level of EGFR expression on the surface of normal epithelial cells; specifically, it can be divided into high expression, moderate expression and low expression, for example, DiFi cells are high in EGFR.
  • the expression cell line, LoVo cells are moderately expressed cell lines of EGFR
  • HT-29 cells are EGFR low expression cell lines (Wild, R., et al., Mol. Cancer Rher 2006: 5(1), p104-113 Cetuximab preclinical antitumor activity (monotherapy and combination based) is not predicted by relative total or activated epidermal growth factor receptor tumor expression levels).
  • the antibody drug conjugate of the present invention can be used in combination with a known chemotherapeutic agent for treating a tumor, such as Adriamycin, cyclophosphamide, and a taxane [Taxol).
  • Taxotere Xeloda
  • Gemzar Navelbine
  • Tamoxifen Aromatase Inhibitors (Rining, Furlong, Arnold New) , 5-FU plus leucovorin, camptosar (ocamptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), etc. , or a combination of them.
  • treatment refers to a clinical intervention that attempts to alter the natural course of the individual or cell being treated, either for prevention or in the course of clinical pathology.
  • the desired effects of treatment include prevention of the onset or recurrence of the disease, alleviation of symptoms, and any direct or indirect pathological consequences of the disease, Prevent metastasis, slow the rate of disease progression, improve or reduce disease status, and eliminate or improve prognosis.
  • an antibody or antibody drug conjugate of the invention is used to delay the onset of a disease or condition, or to slow the progression of a disease or condition.
  • efficacy can be measured, for example, by assessing the time to disease progression (TTP) and/or determining the response rate (RR).
  • a vertebrate refers to a mammal.
  • Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats.
  • a mammal refers to a human.
  • an effective amount means an amount effective to achieve a desired therapeutic or prophylactic effect at a necessary dose and time.
  • a “therapeutically effective amount” of a substance/molecule of the invention may vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the substance/molecule to elicit a desired response in the individual.
  • a therapeutically effective amount also encompasses an amount of therapeutic benefit of the substance/molecule that outweighs any toxic or detrimental effects.
  • prophylactically effective amount is meant an amount effective to achieve the desired prophylactic effect at the necessary dosages and times.
  • a therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the volume of the tumor; inhibit (ie, slow down, preferably stop) the infiltration of the cancer cells into the surrounding organs; inhibition (ie, a certain degree of slowing, preferably stop) a tumor metastasis; a degree of inhibition of tumor growth; and/or a degree of alleviation of one or more symptoms associated with cancer.
  • a suitable dose of the antibody drug conjugate of the invention (when used alone or in combination with one or more other therapeutic agents such as chemotherapeutic agents) will depend on the type of disease being treated, The type of antibody drug conjugate, the severity and progression of the disease, the administration of the antibody drug conjugate for preventive or therapeutic purposes, prior therapies, the patient's clinical history and reactivity to antibody drug conjugates, and the attending physician Teacher's judgment.
  • the antibody drug conjugate is administered to the patient once or through a series of treatments.
  • the initial candidate dose administered to the patient can be from about 1 [mu]g/kg to 100 mg/kg (eg, 0.1 mg/kg to 20 mg/kg) of the antibody drug conjugate, for example, one or more times. Apply separately or through continuous infusion. Typical daily doses may range from about 1 [mu]g/kg to 100 mg/kg or more, depending on the factors described above. For repeated administrations that last for several days or longer, depending on the condition, treatment is usually continued until the desired inhibition of the disease symptoms occurs. Exemplary dosages of antibody drug conjugates can range from about 0.05 mg/kg to about 10 mg/kg.
  • one or more doses of antibody drug conjugates of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) can be administered to a patient.
  • Such doses may be administered intermittently, for example weekly or every three weeks (eg, such that the patient receives from about 2 to about 20 doses, or for example about 6 doses of the antibody drug) Conjugate).
  • a higher initial loading dose can be administered followed by one or more lower doses.
  • the course of this therapy is easily monitored by conventional techniques and assays.
  • “Long-term” administration refers to the administration of the agent in a continuous mode as opposed to the short-term mode, thereby maintaining the initial therapeutic effect (activity) for a longer period of time.
  • Intermittent refers to treatment that is not continuously performed without interruption, but is essentially periodic.
  • Administration of "in combination" with one or more other therapeutic agents includes simultaneous (common) administration and sequential administration in any order.
  • “Pharmaceutically acceptable carrier” when used in the present invention, includes pharmaceutically acceptable carriers, excipients or stabilizers which are non-toxic to the cells or mammals to which they are exposed, at the dosages and concentrations employed.
  • the physiologically acceptable carrier is a pH buffered aqueous solution.
  • physiologically acceptable carriers include buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or Immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, nectar sugar, sucrose, trehalose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter ion, such as sodium; and / or nonionic surfactants such as TWEEN TM, polyethylene glycol (PEG), and PLURONICS TM.
  • buffers such as phosphates, citrates and other organic acids
  • antioxidants including ascorbic acid
  • the KRAS gene has the same meaning as the K-RAS gene, which is a member of the RAS gene family and encodes a K-ras protein, which is involved in the formation, proliferation, migration, proliferation, and angiogenesis of various tumors.
  • the common mutation sites are codon No. 12 and codon No. 13 of exon 2 of K-RAS gene, codon No. 61 of exon 3, and seven mutation hotspots: G12C, G12R, G12S, G12V G12D, G12A, G13V/D. These 7 mutations accounted for more than 90%.
  • the tumor is a tumor of a KRAS gene mutation associated with overexpression of EGFR.
  • the BRAF (v-raf murine sarcoma viral oncogene homolog B1) gene is a protooncogene and is a member of the RAF family.
  • About 8% of human tumors have BRAF mutations, and most of the mutations in BRAF gene mutations are BRAFV600E mutations, which lead to the continuous activation of the downstream MEK/ERK signaling pathway, which is essential for tumor growth, invasion and metastasis.
  • the tumor is a tumor of a BRAF gene mutation associated with EGFR overexpression.
  • Figure 1 is a HIC-HPLC image of the drug/antibody ratio of an antibody drug conjugate.
  • Figure 2 In vitro cell viability assay of monoclonal antibody and antibody drug conjugates, wherein ⁇ represents BA03 monoclonal antibody and ⁇ represents MYK-3 antibody drug conjugate.
  • Figure 3 shows the growth inhibitory activity of MYK-3 against colon cancer cell line HT-29, wherein ⁇ represents BA03 monoclonal antibody and ⁇ represents MYK-3 antibody drug conjugate.
  • Figure 4 shows the growth inhibitory activity of MYK-3 on glioma cancer cell line U87-MG, wherein ⁇ represents BA03 monoclonal antibody and ⁇ represents MYK-3 antibody drug conjugate.
  • Figure 5 shows the growth inhibitory activity of MYK-3 on lung cancer cell A549, wherein ⁇ represents BA03 monoclonal antibody and ⁇ represents MYK-3 antibody drug conjugate.
  • Figure 6 is a growth inhibitory activity of MYK-3 against KRAS mutant colon cancer cell line LoVo, wherein ⁇ represents an Erbitux monoclonal antibody and ⁇ represents a MYK-3 antibody drug conjugate.
  • Figure 7 Effect of monoclonal antibody and antibody drug conjugate on the volume of mouse HT-29 colon cancer xenografts. Data are shown as mean ⁇ standard deviation; * indicates P ⁇ 0.05, ** indicates P ⁇ 0.01, and *** indicates P ⁇ 0.001 compared to the buffer control group.
  • Figure 8 Effect of monoclonal antibody and antibody drug conjugate on body weight of mouse HT-29 colon cancer xenograft model.
  • Figure 9 Growth inhibitory activity of MYMC-3, mAb BA03 and BA03+ equimolar vcMMAE on colorectal cancer cell DiFi, wherein ⁇ represents the monoclonal antibody component BA03 of MYK-3, and ⁇ represents the monoclonal antibody component BA03 plus MYMM-3 drug molar number of vcMMAE, ⁇ represents MYK-3.
  • Figure 10 is a growth inhibition activity of MYK-3 on KRAS mutant colon cancer cell line LoVo in nude mice.
  • FIG. 11 HIC-HPLC analysis profiles of BA03-MCC-MMAE, BA03-MC-MMAE and MYK-3.
  • Figure 12 Comparison of in vitro cell viability of MYK-3 with BA03-MC-MMAE, BA03-MCC-MMAE, wherein ⁇ represents BA03-MC-MMAE, ⁇ represents BA03-MCC-MMAE, and ⁇ represents MYK-3.
  • the antibody BA03 in the present invention is BA03 in Chinese Patent Application No. CN 103772504A, and the preparation method thereof is referred to Example 3 in the patent application, and the sequence of each part of the antibody is as follows:
  • the heavy chain variable region sequence is:
  • underlined portions are CDR1 (SEQ ID NO: 5), CDR2 (SEQ ID NO: 6), and CDR3 (SEQ ID NO: 7), respectively;
  • the ununderlined portions are FR1 (SEQ ID NO: 8), FR2 (SEQ ID NO: 9), FR3 (SEQ ID NO: 10), and FR4 (SEQ ID NO: 11), respectively.
  • the light chain variable region sequence is:
  • EIVLTQSPDFQSVTPKEKVTITC RASQSIGTNIH WYQQKPDQSPKLLIK YASESIS GIPSRFSGSGSGTDFTLTINSLEAEDAATYYC QQNNEWPT SF GQGTKLEIK (SEQ ID NO: 2).
  • underlined portions are CDR1 (SEQ ID NO: 12), CDR2 (SEQ ID NO: 13), and CDR3 (SEQ ID NO: 14), respectively;
  • the ununderlined portions are FR1 (SEQ ID NO: 15), FR2 (SEQ ID NO: 16), FR3 (SEQ ID NO: 17), and FR4 (SEQ ID NO: 18), respectively. .
  • the heavy chain constant region sequence is:
  • the light chain constant region sequence is:
  • b cuvette optical path length (usually 1 cm).
  • the free mole number of moles is calculated based on the molar concentration of the free thiol group and the total protein solution volume.
  • vc-MMAE purchased by Shanghai Qianyuan Chemical Technology Co., Ltd., item number HY-15575
  • DMSO dimethyl methacrylate
  • N-acetylcysteine in an amount of 20 times the number of moles of vc-MMAE charged was added to the reaction mixture, and the mixture was allowed to stand and allowed to stand for 5 minutes.
  • the prepared antibody drug conjugate MYK-3 was subjected to HIC-HPLC analysis (Jun Ouyang, Drug-To-Antibody (DAR) Ratio and Drug Distribution by Hydrophobic Interaction Chromatography and Reverse Phase High Performance Chromatography, Laurent Ducry (ed.), Antibody Drug Conjugates, Chapter 17, Methods in Molecular Biology, Vol 1045, p275-283) to determine the drug/antibody ratio (DAR), see Figure 1, the average drug loading DAR is 4.1 based on the peak area of the map. .
  • the medium was decanted, rinsed with 5 mL of DPBS, and then digested with 3 mL of trypsin, resuspended in medium, centrifuged, and discarded. Then, the medium was resuspended again, and 0.5 mL was taken out and counted by a cell counter. Plates were plated on 96-well cell culture plates (10000 cells/well for DiFi cells, 5000 cells/well for HT-29 cells, 2000 cells/well for A549 cells, and 3000 cells/well for U87-MG cells, LoVo cells were cultured for 24 hours at 4000 cells/well.
  • MYK-3 has significantly increased cell growth inhibitory activity compared to monoclonal antibody BA03, and EC50 is reduced by about 10-fold (the EC50 of BA03 is 51.9 ng/ml, The EC50 of MYK-3 is 5.1 ng/ml), as shown in Figure 2.
  • KRAS mutant colon cancer cells LoVo moderately expressed in EGFR (Dunn EF, Ilda M, Myers RA, Hintz KA, Campbell DA, Armstrong EA, Li C and Wheeler DL. Dasatinib sensitizes KRAS mutant colorectal tumors To cetruximab. Oncogene 2011; 30: 561-574), it was found that MYK-3 showed significant tumor growth inhibitory activity against KRAS mutant colon cancer cell LoVo (as shown in Fig. 6, EC50 was 3.2 ⁇ g/ml). When BA03 was used alone, it had almost no inhibitory activity on the cell line.
  • HT-29 colon cancer cells are cell lines with low expression of EGFR and BRAF mutations.
  • the EGFR-targeted monoclonal antibody Erbitux which is currently marketed for the treatment of colorectal cancer, does not grow on HT-29 cell lines. Inhibition activity.
  • HT-29 cell transplantation model Tumor cells in logarithmic growth phase were collected, counted, resuspended in 1 ⁇ PBS, and adjusted to a cell suspension concentration of 3 ⁇ 10 7 /ml. Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 ml syringe (4 gauge needle), 3 ⁇ 10 6 /0.1 ml/mouse. When the tumor volume reached 150-200 mm 3 , they were grouped by random block method, 8 small in each group. The mice were guaranteed to have a uniform tumor volume and weight in the mice. The mean value of tumor volume of each group differed from the mean of tumor volume of all experimental animals by no more than ⁇ 10%. The tail vein was administered once every four days (days 1, 5, 9, and 13), and a total of four doses were administered, and the tumor volume and mouse body weight were measured periodically. There were 8 mice in each administration group.
  • Mouse HT-29 colon cancer xenograft experiment The experiment was divided into five groups, including buffer group (20 mM sodium citrate, 0.3% sodium chloride, 5% trehalose, 0.05% Tween 80, pH 6), BA03 mAb Group (5 mg/kg), MYK-3 group (1 mg/kg), MYK-3 group (5 mg/kg) and non-binding ADC group (5 mg/kg) (human IgG-vcMMAE conjugate, in which IgG is from Healthy human serum purified IgG prepared in the same manner as MYK-3).
  • the transplanted tumor volume of mice administered with MYK-3 was significantly lower than that of the control group, showing a significant anti-tumor growth effect (Fig. 7).
  • the tumor growth inhibition rate of MYK-3 in the 5 mg/Kg dose group was 54% compared with the buffer group, and the tumor growth inhibition rate was 46% compared with the same dose group of monoclonal antibody BA03. Compared with non-binding ADC, the tumor growth inhibition rate reached 42%.
  • Mouse body weight The body weight of mice administered with MYK-3 did not change from the control group (see Figure 8), indicating that MYK-3 did not have a toxic effect on reducing body weight in mice.
  • the growth inhibitory activity of MYK-3, monoclonal antibody BA03 and BA03+ equimolar vcMMAE on colorectal cancer cell DiFi was detected according to the method of Example 2.
  • the experimental results are shown in Fig. 9, wherein the EC50 values were 8.4 ng/mL and 65.8 ng, respectively. /mL and 68.2 ng/mL.
  • the monoclonal antibody BA03 showed a certain growth inhibitory activity in the colorectal cancer cell DiFi with high expression of EGFR; and BA03 plus free vcMMAE equivalent to the MYK-3 drug molar number and BA03 activity alone There was no significant difference; however, the growth inhibitory activity of MYK-3 formed by coupling BA03 with vcMMAE as an ADC molecule against DiFi colorectal cancer cells was much higher than that of monoclonal antibody BA03 itself and BA03 plus the equivalent of MYK-3 drug carrier. The activity of free vcMMAE increased the EC50 by about 8 times.
  • MYK-3 showed complete inhibition of LoVo cell tumor growth at a dose of 3 mg/Kg, and MYK-3 at 1 mg/Kg has been shown to be more than the marketed drug Erbitux at a dose of 3 mg/Kg. Strong activity.
  • the drug/antibody ratio was determined using HIC-HPLC analysis method. For details, see Example 1, MYK-3 (ie, BA03-vcMMAE), BA03-MC-MMAE. The drug/antibody ratio to BA03-MCC-MMAE was 3.9, see Figure 11.
  • Example 2 For the in vitro cell viability assay method, see Example 2; in short, a certain number of DiFi cell lines were inoculated into 96-well plates, and after 24 hours of culture, BA03-MC-MMAE, BA03-MCC- Three samples of MMAE, MYK-3. After further incubation for 96 hours, the CCK-8 detection reagent was added for color development, and the OD value of each well of the 96-well plate was read on the microplate reader to detect the number of viable cells of different concentrations of the sample, and the proliferation inhibition effect of the sample on the DiFi cell line was judged. .
  • the cell proliferation inhibitory activity of MYK-3 was significantly higher than that of BA03-MC-MMAE and BA03-MCC-MMAE; in fact, the cell proliferation inhibitory activities of BA03-MC-MMAE and BA03-MCC-MMAE were
  • the activity of the monoclonal antibody BA03 itself is similar. This suggests the use of the same monoclonal antibodies and cytotoxic small molecules, but the use of different linkers can cause significant differences in ADC activity.

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Abstract

提供了一种抗表皮生长因子受体抗体药物偶联物，还公开了包含该抗体药物偶联物的组合物和该抗体药物偶联物在制备预防和/或治疗与表皮生长因子受体有关的疾病的药物中的应用。

Description

抗体药物偶联物 技术领域
本发明涉及抗体药物偶联物，具体涉及抗表皮生长因子受体抗体药物偶联物。本发明还涉及包含该抗体药物偶联物的组合物，以及该抗体药物偶联物的医药用途。
背景技术
表皮生长因子受体(Epidermal Growth Factor Receptor，EGFR，也称为HER1，c-ErbB1)是表皮生长因子家族的细胞表面受体，是由1186个氨基酸残基构成，分子量为170kD的一种跨膜糖蛋白(Jorissen RN，Walker F，Pouliot N，et al.Epidermal growth factor receptor：mechanisms of activation and signaling.Exp Cell Res，2003；284：31-53)。EGFR属于I型酪氨酸激酶受体ErbB亚族(ErbB 1-4)，具有酪氨酸激酶的活性。EGFR稳定的表达于许多上皮组织，包括皮肤和毛囊。表皮生长因子受体异常表达或因受体突变而活化会导致癌变。有很多实体瘤发现过度表达表皮生长因子受体，如结直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌和食管癌等(Olayioye MA，Neve RM，Lane HA，et al.TheEerbB Signaling network：receptor heterodimerzation in development and cancer.The EMBO J，2000；19：3159-3167)。转化生长因子α和表皮生长因子等生长因子是表皮生长因子受体的内源性配体。这些配体与表皮生长因子受体结合，激活受体胞内酪氨酸蛋白激酶的活性，启动下游多条信号转导途径，从而调节正常细胞的生长和分化，增加肿瘤细胞的侵袭力、促进血管生成、抑制肿瘤细胞的凋亡(Ciardiello F，Tortora G.A novel approach in the treatment of cancer：targeting the epidermal growth factor receptor.Clin Cancer Res，2001；7：2958-2970)。表皮生长因子受体在肿瘤中的过表达及其在肿瘤细胞生长、分化中起重要作用的这些特点，使表皮生长因子受体成为具有良好前景的肿瘤治疗靶点。
目前市场上已有两个抗表皮生长因子受体的治疗性抗体，一个是人鼠嵌合抗体C225抗体(爱必妥，Erbitux或Cetuximab，ImClone(现在Eli Lilly)公司)，与表皮生长因子受体有特异亲和性，可阻断EGF和TGFα等配体与表皮生长因子受体的结合，抑制其磷酸化和下游信号传导，从而抑制肿瘤细胞的生长，诱导凋亡，减少基质金属蛋白酶和血管上皮生长因子的 产生。2004年美国FDA批准爱必妥治疗结直肠癌，2006年批准其治疗头颈部癌，目前有更多临床试验用于其它肿瘤适应症。在临床上，爱必妥和伊立替康合用治疗结直肠癌的有效率(ORR)为23％，与氟嘧啶等化疗药物合用治疗头颈癌的有效率为13％-30％。由于是人鼠嵌合抗体，爱必妥在临床试验中有3.7％的病人产生了抗体反应。
另一个抗表皮生长因子受体的治疗性抗体是帕尼单抗(Vectibix，panitumumab，Amgen公司)，是采用转基因小鼠技术制备的全人化单克隆抗体，无鼠源蛋白序列。该抗体靶向作用于表皮生长因子受体(EGFR)，2006年9月被FDA批准上市，与氟嘧啶、奥沙利铂和伊立替康合用或在化疗后用于治疗EGFR阳性的转移性结直肠癌。2006年FDA批准其单药治疗化疗耐受的转移性结直肠癌(mCRC)。然而帕尼单抗是IgG2亚型抗体，与IgG1相比，IgG2的CDC活性及ADCC等生物学活性明显减低；另外，IgG2亚型抗体稳定性较差，这可能是临床效果上全人抗体帕尼单抗与嵌合抗体爱必妥相比没有明显优势的主要原因。在临床上治疗结直肠癌的总体生存率(OR)才达到8％，非进展生存期只延长了3.6个月。
目前大量的临床数据显示爱必妥单抗和帕尼单抗只对EGFR表达的KRAS野生型(KRAS wild type)有疗效，而对KRAS突变体没有肿瘤生长抑制活性，因此，美国临床肿瘤协会发表的指导原则中明确指出抗EGFR单抗药物只适用于KRAS野生型的结肠癌病人(Allegra CJ，Jessup JM，Somerield MR，Hamilton SR，Hammond EH，Hayes DF，et al.American Soceitey of Clinical Oncology provisional clinical opinion：testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor monoclonal antibody therapy.J.Clin Oncol.2009；27：2091-2096；Bardelli A，Siena S.Molecular mechanisms of resistance to cetuximab and panitumumab in colorectal cancer.J Clin Oncol.2010；28：1254-1261)。
因此，本领域亟需具有更高生物学活性的人源化抗表皮生长因子受体的抗体类药物，尤其是对KRAS突变体有疗效的抗体类药物，例如抗体药物偶联物，以进一步提高疗效，降低副作用。
由于单抗癌症药物的引领，近年来在欧美快速发展的抗体药物偶联物(antibody drug conjugate，ADC)成为目前最前沿的医药技术之一。
抗体药物偶联物一般由三个部分组成：
1.专一性结合靶点的单抗；
2.具有细胞毒性的小分子化学药物；
3.链接小分子药物和单抗的链接体，也叫接头。
抗体药物偶联物利用单抗的靶点专一性和化学药物的细胞毒性来杀死肿瘤细胞。它的作用机制是：(1)抗体药物偶联物利用单抗特异地结合到肿瘤细胞上的靶点抗原；(2)抗体药物偶联物和靶点抗原的复合物通过靶点抗原介导内吞进入细胞；(3)抗体药物偶联物在细胞内降解，释放具有细胞毒的化学药物；(4)具有细胞毒的化学药物杀死肿瘤细胞。
抗体药物偶联物的作用机制看似简单，但是一个抗体药物偶联物是否能成为安全有效的药物是非常复杂和不可预测的，依赖多种因素，例如：
1)靶点的特性：靶点是否能内吞噬(internalize)，靶点的表达水平，靶点是否在癌细胞和正常细胞中有足够的表达水平差异，靶点是否会脱落细胞外部分(extracellular domain，ECD)到血液中；
2)单抗的特性：单抗对靶点的特异性是否足够好(与其它蛋白无交叉反应)，单抗的稳定性如何，单抗和靶点的复合物是否能内吞噬入细胞；
3)小分子化学药物的特性：小分子化学药物的细胞毒性是否足够强，其在血液里的稳定性，以及其成为ADC后在体内的代谢产物的毒性；
4)接头的特性：接头是可切割的(cleavable)还是不可切割的(non-cleavable)，接头在血液里的稳定性；
5)ADC的特性：ADC和靶点的复合物是否能内吞噬，ADC在血液里的稳定性，ADC上用什么链接体和链接几个化学药物，ADC杀死癌细胞的活性和生物毒性。
可以看出，ADC药物的研发需要大量的实验摸索和验证，其安全性和有效性是无法在实验前预测的。
发明内容
本发明的发明人通过大量实验和创造性劳动，制备得到了抗表皮生长因子受体抗体药物偶联物，并证实其具有良好的生物学活性，由此完成了本发明。
本发明第一方面涉及抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其包含与细胞毒剂共价连接的抗表皮生长因子受体抗体。
在本发明的一个实施方案中，其中所述的抗表皮生长因子受体抗体包括重链和轻链，其中重链可变区CDR1、CDR2、CDR3分别包含如SEQ ID  NO：5～7所示的序列或其突变体，轻链可变区CDR1、CDR2、CDR3分别包含如SEQ ID NO：12～14所示的序列或其突变体。
在本发明的一个实施方案中，其中所述的抗表皮生长因子受体抗体轻链可变区CDR1、CDR2、CDR3区分别包含如SEQ ID NO：12～14所示的序列或者与SEQ ID NO：12～14所示的序列的同一性大于70％，例如大于75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列，例如具有3个、2个或1个突变、缺失或添加氨基酸的序列。
在本发明的一个实施方案中，其中所述的抗表皮生长因子受体抗体重链可变区CDR1、CDR2、CDR3区分别包含如SEQ ID NO：5～7所示的序列或者与SEQ ID NO：5～7所示的序列的同一性大于70％，例如大于75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列，例如具有3个、2个或1个突变、缺失或添加氨基酸的序列。
在本发明的一个实施方案中，其中所述的抗表皮生长因子受体抗体重链可变区FR1、FR2、FR3、FR4区分别包含如SEQ ID NO：8～11所示的序列或其突变体。
在本发明的一个实施方案中，其中所述的抗表皮生长因子受体抗体重链可变区FR1、FR2、FR3、FR4区分别包含如SEQ ID NO：8～11所示的序列，或者包含与上述序列的同一性大于70％，例如大于75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列。
在本发明的一个实施方案中，其中所述的抗表皮生长因子受体抗体轻链可变区FR1、FR2、FR3、FR4区分别包含如SEQ ID NO：15～18所示的序列或其突变体。
在本发明的一个实施方案中，其中所述的抗表皮生长因子受体抗体轻链可变区FR1、FR2、FR3、FR4区分别包含如SEQ ID NO：15～18所示的序列，或者包含与上述序列的同一性大于70％，例如大于75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列。
在本发明的一个实施方案中，所述抗表皮生长因子受体抗体的重链可变区的序列如SEQ ID NO：1所示。
在本发明的一个实施方案中，所述抗表皮生长因子受体抗体的轻链可 变区的序列如SEQ ID NO：2所示。
在本发明的一个实施方案中，其中所述的抗表皮生长因子受体抗体的重链恒定区选自人源性IgG、IgM、IgA、IgD、IgA恒定区或上述恒定区的突变体。
在本发明的一个实施方案中，其中所述IgG选自IgG1、IgG2、IgG3和IgG4。
在本发明的一个实施方案中，其中所述的抗表皮生长因子受体抗体重链恒定区的氨基酸序列包含如SEQ ID NO：3所示的序列，或者包含与SEQ ID NO：3所示序列的同一性大于70％，例如大于75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列。
在本发明的一个实施方案中，其中所述的抗表皮生长因子受体抗体的轻链恒定区为人源性lambda恒定区、kappa恒定区、或上述恒定区的突变体。
在本发明的一个实施方案中，其中所述的抗表皮生长因子受体抗体轻链恒定区的氨基酸序列包含如SEQ ID NO：4所示的序列，或者包含与SEQ ID NO：4所示序列的同一性大于70％，例如大于75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列。
在本发明的一个实施方案中，其中所述的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物具有式I所示的结构，
Ab-(L-D)p
式I
其中：
Ab代表抗表皮生长因子受体抗体；
L代表接头；
D代表细胞毒剂；
p代表1-8，例如2-6，例如3-5。
在本发明的一个实施方案中，其中所述的细胞毒剂选自化疗药物、毒素(例如细菌、真菌、植物、或动物源性的酶活性毒素或其片段)、放射性同位素、细胞因子、抗生素、酶、纳米颗粒和生物活性肽。
在本发明的一个实施方案中，其中所述的细胞毒剂选自Monomethyl auristatin E(MMAE)、Monomethyl auristatin F(MMAF)、美登木素生物碱(例如Maytansine DM1、Maytansine DM4)、卡奇霉素(calicheamicin)、duocarmycin MGBA、阿霉素(doxorubicin)、蓖麻毒素、白喉毒素等毒素、 I131、白介素类、肿瘤坏死因子、趋化因子和纳米颗粒等。
在本发明的一个实施方案中，其中所述的细胞毒剂为MMAE。
在本发明的一个实施方案中，其中所述的接头为可切割的或不可切割的。
在本发明的一个实施方案中，其中所述的接头选自6-马来酰亚氨基己酰基(MC)、马来酰亚氨基丙酰基(MP)、缬氨酸-瓜氨酸(val-cit)、丙氨酸-苯丙氨酸(ala-phe)、对氨基苄氧羰基(PAB)、N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)-环己烷-1-羧酸酯(SMCC)、N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)、和6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-vc-PAB)。
在本发明的一个实施方案中，其中所述的接头为6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-vc-PAB)。
在本发明的一个实施方案中，其中式I中所述的L-D为vc-MMAE，其结构如下式所示：
Figure PCTCN2016073844-appb-000001
在本发明的一个具体实施方案中，其中所述的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物如下式所示：
Figure PCTCN2016073844-appb-000002
其中Ab为抗表皮生长因子受体抗体，p为1-8，例如2-6，例如3-5。
本发明第二方面涉及组合物(例如药物组合物)，其含有本发明第一方面任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，任选地，还含有至少一种药学上可接受的载体、稀释剂或赋形剂。
在本发明的一个实施方案中，所述组合物中还包含已知的用于治疗肿瘤 的化疗药物，所述化疗药物例如为阿霉素(Adriamycin)、环磷酰胺和紫杉烷类[紫杉醇(Taxol)和多西他赛(Taxotere)]、卡培他滨(Xeloda)、吉西他滨(Gemzar)、长春瑞滨(Navelbine)、他莫昔芬、芳香酶抑制剂(瑞宁得、弗隆、阿诺新)、5-FU加亚叶酸、伊立替康(camptosar)、奥沙利铂、顺铂、卡铂、雌莫司汀、米托蒽醌(Novantrone)、泼尼松、长春新碱(Oncovin)等，或它们的组合。
本发明还涉及本发明第一方面任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物在制备预防和/或治疗与表面生长因子受体(EGFR)相关的疾病的药物中的用途。
在本发明的一个实施方案中，其中所述的与表面生长因子受体(EGFR)相关的疾病为与EGFR相关的肿瘤，例如与EGFR过表达相关的肿瘤，例如选自结肠癌、直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌、食管癌、乳腺癌、肾癌、前列腺癌、胃癌、胰腺癌和脑胶质瘤。
在本发明一个实施方案中，其中所述肿瘤为KRAS基因突变的肿瘤，例如为KRAS基因突变的结肠癌、直肠癌、肺癌或胰腺癌。
在本发明一个实施方案中，其中所述的肿瘤为BRAF基因突变的肿瘤，例如选自BRAF基因突变的结肠癌、直肠癌和肺癌。
本发明还涉及本发明第一方面任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物在制备抑制肿瘤血管生成、延缓肿瘤进展、抑制肿瘤生长、抑制肿瘤细胞增殖的药物中的用途。
在本发明的一个实施方案中，其中所述的肿瘤选自结肠癌、直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌、食管癌、乳腺癌、肾癌、前列腺癌、胃癌、胰腺癌和脑胶质瘤。
在本发明一个实施方案中，其中所述肿瘤为KRAS基因突变的肿瘤，例如为KRAS基因突变的结肠癌、直肠癌、肺癌或胰腺癌。
在本发明一个实施方案中，其中所述的肿瘤为BRAF基因突变的肿瘤，例如选自BRAF基因突变的结肠癌、直肠癌和肺癌。
本发明还涉及预防和/或治疗与表皮生长因子受体(EGFR)相关的疾病的方法，所述方法包括给予有需要的受试者预防和/或治疗有效量的本发明第一方面任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述 盐的溶剂合物。
在本发明的一个实施方案中，其中所述的与表皮生长因子受体(EGFR)相关的疾病为与EGFR相关的肿瘤，例如与EGFR过表达相关的肿瘤，例如选自结肠癌、直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌、食管癌、乳腺癌、肾癌、前列腺癌、胃癌、胰腺癌和脑胶质瘤。
在本发明一个实施方案中，其中所述肿瘤为KRAS基因突变的肿瘤，例如为KRAS基因突变的结肠癌、直肠癌、肺癌或胰腺癌。
在本发明一个实施方案中，其中所述的肿瘤为BRAF基因突变的肿瘤，例如选自BRAF基因突变的结肠癌、直肠癌和肺癌。
本发明还涉及抑制肿瘤血管生成、延缓肿瘤进展、抑制肿瘤生长、抑制肿瘤细胞增殖的方法，所述方法包括给予有需要的受试者预防和/或治疗有效量的本发明第一方面任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物。
在本发明的一个实施方案中，其中所述的肿瘤选自结肠癌、直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌、食管癌、乳腺癌、肾癌、前列腺癌、胃癌、胰腺癌和脑胶质瘤。
在本发明一个实施方案中，其中所述肿瘤为KRAS基因突变的肿瘤，例如为KRAS基因突变的结肠癌、直肠癌、肺癌或胰腺癌。
在本发明一个实施方案中，其中所述的肿瘤为BRAF基因突变的肿瘤，例如选自BRAF基因突变的结肠癌、直肠癌和肺癌。
本发明还涉及本发明第一方面任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其用于预防和/或治疗与表皮生长因子受体(EGFR)相关的疾病。
在本发明的一个实施方案中，其中所述的与表皮生长因子受体(EGFR)相关的疾病为与EGFR相关的肿瘤，例如与EGFR过表达相关的肿瘤，例如选自结肠癌、直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌、食管癌、乳腺癌、肾癌、前列腺癌、胃癌、胰腺癌和脑胶质瘤。
在本发明一个实施方案中，其中所述肿瘤为KRAS基因突变的肿瘤，例如为KRAS基因突变的结肠癌、直肠癌、肺癌或胰腺癌。
在本发明一个实施方案中，其中所述的肿瘤为BRAF基因突变的肿瘤，例如选自BRAF基因突变的结肠癌、直肠癌和肺癌。
本发明还涉及本发明第一方面任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其用于抑制肿瘤血管生成、延缓肿瘤进展、抑制肿瘤生长、抑制肿瘤细胞增殖。
在本发明的一个实施方案中，其中所述的肿瘤选自结肠癌、直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌、食管癌、乳腺癌、肾癌、前列腺癌、胃癌、胰腺癌和脑胶质瘤。
在本发明一个实施方案中，其中所述肿瘤为KRAS基因突变的肿瘤，例如为KRAS基因突变的结肠癌、直肠癌、肺癌或胰腺癌。
在本发明一个实施方案中，其中所述的肿瘤为BRAF基因突变的肿瘤，例如选自BRAF基因突变的结肠癌、直肠癌和肺癌。
本发明的抗表皮生长因子受体抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物在体内和体外都具有良好的抑制肿瘤细胞生长活性，特别是对EGFR中度和低度表达的肿瘤细胞也显示出明显的细胞生长抑制活性，且细胞毒性低；更特别地，本发明的抗表皮生长因子受体抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物对KRAS基因突变或BRAF基因突变的肿瘤也具有很好的疗效，因而具有良好的应用前景。
以下对本发明做进一步描述：
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本发明中，术语“抗体”是指通常由两对相同的多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体的轻链可分为κ和λ两类。重链可分为μ、δ、γ、α或ε五种，依据重链的不同可将抗体分为IgM、IgD、IgG、IgA和IgE五类。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的 恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和补体系统的组分C1q的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为骨架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda，Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196：901-917；Chothia等人(1989)Nature 342：878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同类型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
在本发明中，所述IgG重链恒定区包括IgG1、IgG2、IgG3或IgG4。在本发明的实施方案中，所述IgG重链恒定区为IgG1型。
在本发明中，所述κ轻链恒定区包括各种同种异型，如Km1、Km1，2或Km3。
关于抗体的氨基酸序列
在本发明的实施方案中，抗表皮生长因子受体抗体的重链可变区氨基酸序列是SEQ ID NO：1。在本发明的实施方案中，抗表皮生长因子受体抗体的轻链可变区氨基酸序列是SEQ ID NO：2。
另一方面，本发明所述的抗体的重链可变区的氨基酸序列与SEQ ID NO：1的序列相似性至少达70％，优选是至少75％，优选是至少80％，优选是85％，再优选是至少90％，最好的是至少95％。
另一方面，本发明所述的抗体的轻链可变区的氨基酸序列与SEQ ID NO：2的序列相似性至少达70％，优选是至少75％，优选是至少80％，优选是85％，再优选是至少90％，最好的是至少95％。
在本发明的实施方案中，抗表皮生长因子受体抗体的重链和轻链可变区的CDR的氨基酸序列确定如下：
重链的CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO：5-7；轻链的CDR1、CDR2和CDR3的氨基酸序列分别SEQ ID NO：12-14。
另一方面，抗表皮生长因子受体抗体的重链的CDR含有的氨基酸序列可能是在SEQ ID NO：5-7上出现一个或多个氨基酸的突变或增添或缺失。 优选的是，突变、添加或缺失的氨基酸不超过3个氨基酸。更优选的是，突变、添加或缺失的氨基酸不超过2个氨基酸。最优选的是，突变、添加或缺失的氨基酸不超过1个氨基酸。
另一方面，抗表皮生长因子受体抗体的轻链的CDR含有的氨基酸序列可能是在SEQ ID NO：12-14上出现一个或多个氨基酸的突变、增添或缺失。优选的是，突变、添加或缺失的氨基酸不超过3个氨基酸。更优选的是，突变、添加或缺失的氨基酸不超过2个氨基酸。最优选的是，突变、添加或缺失的氨基酸不超过1个氨基酸。
在本发明的实施方案中，抗表皮生长因子受体抗体的重链和轻链可变区的FR的氨基酸序列确定如下：
重链可变区FR1、FR2、FR3、FR4的序列分别为SEQ ID NO：8-11。轻链可变区FR1、FR2、FR3、FR4的序列分别为SEQ ID NO：15-18。
另一方面，抗表皮生长因子受体抗体的重链或轻链可变区FR的氨基酸序列可能是在SEQ ID NO：8-11、SEQ ID NO：15-18上出现一个或多个氨基酸的突变、增添或缺失。优选的是，突变、添加或缺失的氨基酸不超过3个氨基酸。更优选的是，突变、添加或缺失的氨基酸不超过2个氨基酸。最优选的是，突变、添加或缺失的氨基酸不超过1个氨基酸。
上述抗体或CDR区或框架区的氨基酸发生突变、添加或缺失之后的变异体仍然保留特异性结合EGFR的能力。
在本发明的实施方案中，抗表皮生长因子受体抗体重链恒定区氨基酸序列是SEQ ID NO：3。在本发明的实施方案中，抗表皮生长因子受体抗体的轻链恒定区氨基酸序列是SEQ ID NO：4。
另一方面，本发明所述的抗体的重链恒定区的氨基酸序列与SEQ ID NO：3的序列相似性至少达70％，优选是至少75％，优选是至少80％，优选是85％，再优选是至少90％，最好的是至少95％，如96％、97％、98％、99％。
另一方面，本发明所述的抗体的轻链恒定区的氨基酸序列与SEQ ID NO：4的序列相似性至少达70％，优选是至少75％，优选是至少80％，优选是85％，再优选是至少90％，最好的是至少95％，如96％、97％、98％、99％。
本发明所述的单抗变异体可以通过传统的基因工程方法获得。本领域的技术人员完全知晓利用核酸突变改造DNA分子的方法。另外，编码重链和轻链变异体的核酸分子也可以通过化学合成获得。
在本发明中，用于确定序列同一性(同源性)和序列相似性百分数的算法是例如BLAST和BLAST 2.0算法，它们分别描述在Altschul等(1977)Nucl.Acid.Res.25：3389-3402和Altschul等(1990)J.Mol.Biol.215：403-410。采用例如文献中所述或者默认参数，BLAST和BLAST 2.0可以用于确定本发明的氨基酸序列同一性百分数。执行BLAST分析的软件可以通过国立生物技术信息中心为公众所获得。
在本发明中，所述与氨基酸序列具有至少70％的序列同一性的氨基酸序列包括与所述氨基酸序列基本同一的多肽序列，例如当采用本文所述方法(例如采用标准参数的BLAST分析)时，与本发明多肽序列相比含有至少70％序列同一性、优选至少75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性的那些序列。
在本发明中，用于抗体药物偶联物的毒素包括白喉毒素A链、白喉毒素的非结合活性片段、外毒素A链(来自铜绿假单胞菌(Pseudomonas aeruginosa))、蓖麻毒蛋白(ricin)A链、相思豆毒蛋白(abrin)A链、蒴莲根毒蛋白(modeccin)A链、α-帚曲霉素(sarcin)、油桐(Aleutites fordii)毒蛋白、香石竹(dianthin)毒蛋白、美洲商陆(Phytolacaamericana)毒蛋白(PAPI、PAPII和PAP-S)、苦瓜(Momordica charantia)抑制物、麻疯树毒蛋白(curcin)、巴豆毒蛋白(crotin)、肥皂草(sapaonaria officinalis)抑制物、白树毒蛋白(gelonin)、丝林霉素(mitogellin)、局限曲菌素(restrictocin)、酚霉素(phenomycin)、依诺霉素(enomycin)、和单端孢菌素(trichothecenes)。参见例如1993年10月28日公布的WO 93/21232。
多种放射性核素可用于生成抗体药物偶联物。例子包括212Bi、131I、131In、90Y、和186Re。
抗体和细胞毒剂的偶联物可使用多种双功能蛋白质偶联剂来制备，诸如N-琥珀酰亚氨基3-(2-吡啶基二硫代)丙酸酯(SPDP)，亚氨基硫烷(IT)，亚氨酸酯(诸如盐酸己二酰亚氨酸二甲酯)、活性酯类(诸如辛二酸二琥珀酰亚氨基酯)、醛类(诸如戊二醛)、双叠氮化合物(诸如双(对-叠氮苯甲酰基)己二胺)、双重氮衍生物(诸如双(对-重氮苯甲酰基)-乙二胺)、二异硫氰酸酯(诸如甲苯2，6-二异氰酸酯)、和双活性氟化合物(诸如1，5-二氟-2，4-二硝基苯)的双功能衍生物。例如，可如Vitetta等在(1987)Science，238：1098中所述制备的含蓖麻毒的免疫毒素。碳-14标记的1-异硫氰酸苄基-3-甲基二亚乙基三胺五乙 酸(MX-DTPA)是用于将放射性核苷酸与抗体偶联的例示性螯合剂(WO94/11026)。本发明还涵盖抗体和一种或多种小分子毒素(诸如加利车霉素(calicheamicin)、美登素生物碱(maytansinoid)、多拉司他汀(dolastatin)、auristatin、单端孢霉素(trichothecene)、和CC 1065及这些毒素的具有毒素活性的衍生物的偶联物。
在有些实施方案中，抗体药物偶联物包含与多拉司他汀(dolastatin)或多拉司他汀肽类似物和衍生物auristatin(美国专利No.5,635,483；5,780,588)偶联的抗表皮生长因子受体抗体。多拉司他汀和auristatin已经显示出干扰微管动力学、GTP水解、及核和细胞分裂等活性(Woyke等(2001)Antimicrob.Agents and Chemother.45(12)：3580-3584)且具有抗癌(US 5,663,149)和抗真菌活性(Pettit等(1998)Antimicrob.Agents Chemother.42：2961-2965)。多拉司他汀或auristatin药物模块可经由肽药物模块的N(氨基)末端或C(羧基)末端附着于抗体(WO 02/088172)。
在本发明中，其中MMAE的结构为：
Figure PCTCN2016073844-appb-000003
在本发明中，其中MMAF的结构为：
Figure PCTCN2016073844-appb-000004
在本发明中，作为细胞毒剂的酶可以为具有核酸降解活性的化合物(例如核糖核酸酶或DNA内切核酸酶，诸如脱氧核糖核酸酶；DNA酶)。
在本发明中，药物载荷(loading)由p表示，即式I：Ab-(L-D)p的分子中每个抗体的平均药物模块(即细胞毒剂)数。药物载荷的范围可以为每个抗体1-20个药物模块(D)。通式I的ADC包括偶联有一定范围(1-20个)药物模块的抗体的集合。来自偶联反应的ADC制备物中每个抗体的平均药物模块数可以通过常规手段来验证，诸如质谱、ELISA测定法、和HPLC。还可以测定ADC在p方面的定量分布。在有些情况中，将p为某数值的同质ADC 从具有其它药物载荷的ADC中分离、纯化和验证可以通过诸如反相HPLC或电泳的手段来实现。
对于有些抗体-药物偶联物，p可能受到抗体上附着位点数目的限制。例如，若附着位点是半胱氨酸硫醇，抗体可能只有一个或数个半胱氨酸硫醇基，或者可能只有一个或数个有足够反应性的硫醇基，可附着接头。在某些实施方案中，较高的药物载荷，例如p＞5，可引起某些抗体-药物偶联物的聚集、不溶性、毒性、或丧失细胞通透性。
在某些实施方案中，本发明ADC的药物载荷范围为1到约8；约2到约6；约3到约5；约4到约5；约3.5到4.5；约4。事实上，对于某些ADC已经显示了每个抗体的药物模块的最佳比率可以为小于8，可以为约2到约5。参见US2005-0238649A1(完整收入本文作为参考)。
在某些实施方案中，在偶联反应中少于理论最大值的药物模块偶联至抗体。抗体可包含例如赖氨酸残基，其不与药物-接头中间物或接头试剂起反应。一般而言，抗体不包含许多游离的和反应性的半胱氨酸硫醇基，其可连接药物模块；事实上，抗体中的大多数半胱氨酸硫醇基以二硫桥形式存在。在某些实施方案中，可以在部分或完全还原性条件下用还原剂诸如二硫苏糖醇(DTT)或三羰基乙基膦(TCEP)还原抗体以产生反应性半胱氨酸硫醇基。在某些实施方案中，将抗体置于变性条件以暴露反应性亲核基团，诸如赖氨酸或半胱氨酸。
ADC的载荷(药物/抗体比率)可以以不同方式来控制，例如通过：(i)限制药物-接头中间物或接头试剂相对于抗体的摩尔数，(ii)限制偶联反应的时间或温度，(iii)半胱氨酸硫醇修饰的部分或限制还原性条件，(iv)通过重组技术对抗体的氨基酸序列进行工程改造，使得半胱氨酸残基的数目和位置为了控制接头-药物附着的数目和/或位置而进行改变。应当理解，若超过一个亲核基团与药物-接头中间物或者与接头试剂和接下来的药物模块试剂起反应，则所得产物是具有一个或多个药物模块附着于抗体的ADC化合物混合物。可以通过对抗体特异性和对药物特异性的双重ELISA抗体测定法自混合物计算每个抗体的平均药物数。混合物中的各种ADC分子可以通过质谱来鉴定，并通过HPLC来分离，例如疏水相互作用层析。在某些实施方案中，可以通过电泳或层析从偶联混合物中分离具有单一载荷值的同质ADC。
在本发明中，抗体药物偶联物的药学上可接受的盐包括无机酸、羧酸和磺酸的酸加成盐，例如以下酸的盐：盐酸、氢溴酸、硫酸、磷酸、甲烷磺酸、乙烷磺酸、苯磺酸、甲苯磺酸、萘二磺酸、乙酸、三氟乙酸、丙酸、乳酸、 酒石酸、苹果酸柠檬酸、富马酸、马来酸和苯甲酸。
本发明的抗体药物偶联物的药学上可接受的盐还包括常规碱的盐，例如(仅为示例性并优选)碱金属盐(例如钠盐和钾盐)、碱土金属盐(例如钙盐和镁盐)和衍生自氨或含有1-16个碳原子的有机胺的铵盐，所述有机胺例如(仅为示例性并优选)乙胺、二乙胺、三乙胺、乙基二异丙胺、单乙醇胺、二乙醇胺、三乙醇胺、二环己胺、二甲基氨基乙醇、普鲁卡因、二苯甲酰胺、N-甲基哌啶、N-甲基吗啉、精氨酸、赖氨酸和1，2-乙二胺。
在本发明中，溶剂合物表示这些形式的本发明的抗体药物偶联物：所述抗体药物偶联物通过与溶剂分子配位而形成的固态或液态形式的复合物。水合物是溶剂合物的一种具体形式，其具有配位的水分子。在本发明中，水合物是优选的溶剂合物。
可以优选地使用本发明的抗体药物偶联物进行治疗的与EGFR相关的肿瘤包括过表达EGFR的肿瘤、呼吸道肿瘤(例如小细胞癌和非小细胞癌、支气管癌)，其中特别优选非小细胞肺癌；消化道(例如食道、胃、胆囊、小肠、大肠、直肠)的肿瘤，其中特别优选肠肿瘤；内分泌腺和外分泌腺(例如甲状腺和甲状旁腺、胰腺和唾液腺)的肿瘤，其中特别优选胰腺肿瘤；头颈区域(例如喉、下咽、鼻咽、口咽、唇、口腔、舌和食道)的肿瘤；和/或神经胶质瘤。
在本发明中，EGFR过表达是指与正常上皮细胞表面的EGFR表达水平相比，EGFR的表达水平提高；其具体又可以分为高表达、中度表达和低表达，例如DiFi细胞是EGFR高表达细胞株，LoVo细胞是EGFR中度表达的细胞株，而HT-29细胞是EGFR低表达细胞株(Wild，R.，et al.，Mol.Cancer Rher 2006：5(1)，p104-113，Cetuximab preclinical antitumor activity(monotherapy and combination based)is not predicted by relative total or activated epidermal growth factor receptor tumor expression levels)。
本发明的抗体药物偶联物可以与已知的用于治疗肿瘤的化疗药物联用，所述化疗药物例如为阿霉素(Adriamycin)、环磷酰胺和紫杉烷类[紫杉醇(Taxol)和多西他赛(Taxotere)]、卡培他滨(Xeloda)、吉西他滨(Gemzar)、长春瑞滨(Navelbine)、他莫昔芬、芳香酶抑制剂(瑞宁得、弗隆、阿诺新)、5-FU加亚叶酸、伊立替康(camptosar)、奥沙利铂、顺铂、卡铂、雌莫司汀、米托蒽醌(Novantrone)、泼尼松、长春新碱(Oncovin)等，或它们的组合。
在本发明中，“治疗”指试图改变所治疗个体或细胞的自然进程的临床干预，可以是为了预防或在临床病理学的进程中进行。治疗的期望效果包括预防疾病的发生或复发、缓解症状、削弱疾病的任何直接或间接病理学后果、 预防转移、减缓疾病进展的速率、改善或减轻疾病状态、及免除或改善预后。在有些实施方案中，本发明的抗体或抗体药物偶联物用于延迟疾病或病症的发生，或用于减缓疾病或病症的进展。用于评估疾病的成功治疗和改善的上述参数可以容易的通过内科医师所熟悉的常规规程来测量。对于癌症治疗，可通过例如评估疾病进展前时间(TTP)和/或测定反应率(RR)来测量功效。
在本发明中，“受试者”指脊椎动物。在某些实施方案中，脊椎动物指哺乳动物。哺乳动物包括，但不限于，牲畜(诸如牛)、宠物(诸如猫、犬、和马)、灵长类动物、小鼠和大鼠。在某些实施方案中，哺乳动物指人。
在本发明中，“有效量”指在必需的剂量和时间上有效实现期望的治疗或预防效果的量。本发明的物质/分子的“治疗有效量”可根据诸如个体的疾病状态、年龄、性别和体重及该物质/分子在个体中引发期望应答的能力等因素而变化。治疗有效量还涵盖该物质/分子的治疗有益效果胜过任何有毒或有害后果的量。“预防有效量”指在必需的剂量和时间上有效实现期望的预防效果的量。通常而非必然，由于预防剂量是在疾病发作之前或在疾病的早期用于受试者的，因此预防有效量会低于治疗有效量。在癌症的情况中，药物的治疗有效量可减少癌细胞数；缩小肿瘤体积；抑制(即一定程度的减缓，优选停止)癌细胞浸润到周围器官中；抑制(即一定程度的减缓，优选停止)肿瘤转移；一定程度的抑制肿瘤生长；和/或一定程度的减轻与癌症有关的一种或多种症状。
对于疾病的预防或治疗，本发明的抗体药物偶联物的适宜剂量(在单独地或联合一种或多种其它别的治疗剂诸如化疗剂地使用时)会取决于待治疗疾病的类型、抗体药物偶联物的类型、疾病的严重程度和进程、施用抗体药物偶联物是出于预防还是治疗目的、先前的疗法、患者的临床病史和对抗体药物偶联物的反应性、及主治医师的判断。合适的是，一次性或通过一系列治疗将抗体药物偶联物施用于患者。根据疾病的类型和严重程度，施用于患者的初始候选剂量可以是约1μg/kg至100mg/kg(例如0.1mg/kg-20mg/kg)抗体药物偶联物，例如或是通过一次或多次分开施药或是通过连续输注。根据上文所述因素，典型日剂量的范围可以是约1μg/kg至100mg/kg或更多。对于持续数天或更长时间的重复施药，根据状况，通常持续治疗直至疾病症状发生期望的抑制。抗体药物偶联物的例示剂量的范围可以是约0.05mg/kg至约10mg/kg。如此，可以对患者施用一剂或多剂约0.5mg/kg、2.0mg/kg、4.0mg/kg或10mg/kg(或其任意组合)的抗体药物偶联物。此类剂量可间歇施用，例如每周或每三周(例如使得患者接受约2剂至约20剂，或例如约6剂抗体药物 偶联物)。可施用一剂较高的初始加载剂量，后续一剂或多剂较低剂量。这种疗法的进程易于通过常规技术和测定法来监测。“长期”施用指与短期模式相反，以连续模式施用药剂，从而将初始治疗效果(活性)维持较长一段时间。“间歇”施用指不是无间断连续进行的治疗，而是本质上周期性的。“联合”一种或多种其它治疗剂的施用包括同时(共同)施用和任意次序的序贯施用。
“药学上可接受的载体”在用于本发明时包括药学可接受的载体、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。通常，生理学可接受的载体是pH缓冲水溶液。生理学可接受载体的例子包括缓冲剂，诸如磷酸盐、柠檬酸盐和其它有机酸；抗氧化剂，包括抗坏血酸；低分子量(少于约10个残基)多肽；蛋白质，诸如血清清蛋白、明胶或免疫球蛋白；亲水性聚合物，诸如聚乙烯吡咯烷酮；氨基酸，诸如甘氨酸、谷氨酰胺、天冬酰胺、精氨酸或赖氨酸；单糖、二糖和其它碳水化合物，包括葡萄糖、甘露糖，蔗糖，海藻糖或糊精；螯合剂，诸如EDTA；糖醇，诸如甘露醇或山梨醇；成盐反荷离子，诸如钠；和/或非离子表面活性剂，诸如TWEENTM、聚乙二醇(PEG)和PLURONICSTM。
在本发明中，KRAS基因与K-RAS基因具有相同的含义，其为RAS基因家族成员之一，编码K-ras蛋白，与多种肿瘤的生成、增殖、迁移、扩散以及血管生成有关。其常见的突变位点为K-RAS基因2号外显子的12号密码子和13号密码子，3号外显子的61号密码子，其中有7个突变热点：G12C、G12R、G12S、G12V、G12D、G12A、G13V/D.这7种突变占到了90％以上。在本发明的一个实施方案中，所述肿瘤为与EGFR过表达相关的KRAS基因突变的肿瘤。
在本发明中，BRAF(v-raf murine sarcoma viral oncogene homologB1)基因是一种原癌基因，是RAF家族成员之一。大约8％的人类肿瘤发生BRAF突变，BRAF基因突变的绝大部分突变形式为BRAFV600E突变，该突变导致下游MEK/ERK信号通路持续激活，对肿瘤的生长增殖和侵袭转移至关重要。在本发明的一个实施方案中，所述肿瘤为与EGFR过表达相关的BRAF基因突变的肿瘤。
在本发明中，20种常规氨基酸和其缩写遵从常规用法。参见Immunology-A Synthesis(第2版，E.S.Golub和D.R.Gren，Eds.，Sinauer Associates，Sunderland，Mass.(1991))，其通过引用合并入本文。
附图说明
图1测定抗体药物偶联物的药物/抗体比例的HIC-HPLC图。
图2单抗及抗体药物偶联物的体外细胞活性测定，其中○代表BA03单克隆抗体，▲代表MYK-3抗体药物偶联物。
图3MYK-3对结肠癌细胞HT-29的生长抑制活性，其中○代表BA03单克隆抗体，▲代表MYK-3抗体药物偶联物。
图4MYK-3对脑胶质瘤癌细胞U87-MG的生长抑制活性，其中○代表BA03单克隆抗体，▲代表MYK-3抗体药物偶联物。
图5MYK-3对肺癌细胞A549的生长抑制活性，其中○代表BA03单克隆抗体，▲代表MYK-3抗体药物偶联物。
图6MYK-3对KRAS突变体结肠癌细胞LoVo的生长抑制活性，其中◇代表爱必妥单克隆抗体，▲代表MYK-3抗体药物偶联物。
图7单抗及抗体药物偶联物对小鼠HT-29结肠癌移植瘤体积的影响。图中数据用平均值±标准差表示；与缓冲液对照组相比，＊表示P＜0.05，＊＊表示P＜0.01，＊＊＊表示P＜0.001。
图8单抗及抗体药物偶联物对小鼠HT-29结肠癌移植瘤模型的体重的影响。
图9MYK-3、单抗BA03和BA03+等摩尔数vcMMAE对结直肠癌细胞DiFi的生长抑制活性，其中，□代表MYK-3的单抗组分BA03，△代表单抗组分BA03加上同等于MYK-3载药摩尔数的vcMMAE，◆代表MYK-3。
图10MYK-3对裸鼠中KRAS突变体结肠癌细胞LoVo的移植瘤生长抑制活性。
图11BA03-MCC-MMAE、BA03-MC-MMAE和MYK-3的HIC-HPLC分析图谱。
图12MYK-3与BA03-MC-MMAE、BA03-MCC-MMAE的体外细胞活性比较，其中，□代表BA03-MC-MMAE，△代表BA03-MCC-MMAE，◆代表MYK-3。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规 产品。
本发明中的抗体BA03即中国发明专利申请CN 103772504A中的BA03，其制备方法参考该专利申请中的实施例3，该抗体各部分的序列如下：
重链可变区序列为：
QVQLQESGPGLVKPSETLSLTCTVSGFSLSNYDVHWVRQAPGKGLEWLGVIWSGGNTDYNTPFTSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARALDYYDYEFAYWGQGTLVTVSS(SEQ ID NO：1)。
其中下划线部分分别为CDR1(SEQ ID NO：5)、CDR2(SEQ ID NO：6)、CDR3(SEQIDNO：7)；
未下划线部分分别为FR1(SEQIDNO：8)、FR2(SEQIDNO：9)、FR3(SEQIDNO：10)、FR4(SEQIDNO：11)。
轻链可变区序列为：
EIVLTQSPDFQSVTPKEKVTITCRASQSIGTNIHWYQQKPDQSPKLLIKYASESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQNNEWPT SFGQGTKLEIK(SEQ ID NO：2)。
其中下划线部分分别为CDR1(SEQ 1D NO：12)、CDR2(SEQ ID NO：13)、CDR3(SEQIDNO：14)；
未下划线部分分别为FR1(SEQ ID NO：15)、FR2(SEQ ID NO：16)、FR3(SEQIDNO：17)、FR4(SEQIDNO：18)。。
重链恒定区序列为：
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：3)。
轻链恒定区序列为：
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：4)。
实施例1抗体药物偶联物的制备方法
取10毫克的BA03抗体，使用15mL的30KD超滤装置置换到还原缓冲液(25mM硼酸钠，pH8.0，25mM NaCl，5mM EDTA)中，共置换三次；终体积约为1mL，转移至新的Eppendorf离心管(称重)中，并称重；检测蛋白浓度，计算蛋白总量。向抗体中加入2.5倍摩尔数的DTT，室温保温2小时，连续混匀；使用15ml的30KD超滤装置置换到偶联缓冲液(50mM Tris，pH7.2，150mM NaCl，5mM EDTA)中，共置换三次。取浓缩液，以A280测定蛋白浓度，并称重，计算蛋白总量；取10μl样品，以Ellman’s方法测定自由巯基数；
并以下列公式计算其自由巯基的摩尔浓度：
Figure PCTCN2016073844-appb-000005
b：比色皿光路长度(通常1cm)。
根据自由巯基的摩尔浓度和总蛋白溶液体积计算自由巯基摩尔数。
向还原后的抗体中加入1.1倍于自由巯基摩尔数的vc-MMAE(购上海皓元化学科技有限公司，货号HY-15575)(溶于DMSO)，混匀后室温反应2小时，间断混匀。向反应体系中加入20倍于所投入之vc-MMAE摩尔数的N-乙酰半胱氨酸于反应液中，混匀，静置5分钟。使用15ml的30KD超滤装置置换到偶联物储存液(20mM柠檬酸钠(Na-citrate)，0.3％NaCl，5％海藻糖(Trehalose)，0.05％TWeen-80，pH6.0)中，共置换三次，即得到抗体药物偶联物MYK-3；样品于4℃保存。
药物/抗体比例的测定：
将制备好的抗体药物偶联物MYK-3经过HIC-HPLC分析(Jun Ouyang，Drug-To-Antibody(DAR)Ratio and Drug Distributionby Hydrophobic Interaction Chromatography and Reverse Phase High Performance Chromatography，Laurent Ducry(ed.)，Antibody Drug Conjugates，Chapter 17，Methods in Molecular Biology，Vol 1045，p275-283)以测定药物/抗体比例(drug antibody ratio，DAR)，见图1，根据图谱峰面积计算得到平均载药数DAR为4.1。
实施例2抗体药物偶联物MYK-3体外细胞活性的测定
细胞活性检测方法：
1.1复苏后的细胞株经过3-4代细胞传代之后，先倒去培养基，用5mL DPBS润洗一遍，再用3mL胰蛋白酶消化细胞，分别用培养基重悬，离心机离心，弃上清，再用培养基再次重悬，取出0.5mL用细胞计数仪计数。在96孔细胞培养板上进行铺板(DiFi细胞以10000个细胞/孔，HT-29细胞以5000个细胞/孔，A549细胞以2000个细胞/孔，U87-MG细胞以3000个细胞/孔，LoVo细胞以4000个细胞/孔)，培养24小时后，加入系列稀释浓度的单克隆抗体BA03以及抗体药物偶联物MYK-3，保温72小时，之后每孔加入20μl CCK8显色试剂，在波长450-650nm用酶标仪检测OD450-650，并进行四参数拟合。
体外细胞活性实验结果：
以下各细胞株购自中国科学院上海细胞生物学研究所。
在EGFR高表达的DiFi细胞(人结直肠癌细胞)中的活性：MYK-3比单抗BA03有显著增高的细胞生长抑制活性，EC50降低了大约10倍(BA03的EC50是51.9ng/ml，MYK-3的EC50是5.1ng/ml)，如图2所示。
在EGFR中度表达和低度表达的其它肿瘤细胞中的活性：MYK-3相对于单抗本身对EGFR中度表达和低度表达的癌细胞(人结肠癌细胞HT29、人肺癌细胞A549、人脑星形胶质母细胞瘤细胞U87-MG)也显示了明显的细胞生长抑制活性(如图3、图4、图5所示)，其中HT-29的EC50是611ng/ml，A549的EC50是28.3μg/ml，U87-MG的EC50是5.3μg/ml。
另外，我们还测试了在EGFR中度表达的KRAS突变体结肠癌细胞LoVo(Dunn EF，Ilda M，Myers RA，Hintz KA，Campbell DA，Armstrong EA，Li C and Wheeler DL.Dasatinib sensitizes KRAS mutant colorectal tumors to cetruximab.Oncogene 2011；30：561-574)中的活性，发现MYK-3对KRAS突变体结肠癌细胞LoVo显示了明显的肿瘤生长抑制活性(如图6所示，EC50是3.2μg/ml)，而BA03单独使用时对该细胞株几乎没有抑制活性。
实施例3小鼠体内移植瘤实验
小鼠体内移植瘤实验方法：
HT-29结肠癌细胞是EGFR低表达并带有BRAF突变的细胞株，目前已在市场上销售的治疗结直肠癌的EGFR靶向单抗爱必妥(Erbitux)对HT-29细胞株没有生长抑制活性。
HT-29细胞移植模型：收集对数生长期的肿瘤细胞，计数后重悬于1×PBS，调整细胞悬液浓度至3×107/ml。用1ml注射器(4号针头)在裸鼠右侧背部皮下接种肿瘤细胞，3×106/0.1ml/鼠，当肿瘤体积达到150-200mm3后用随机区组法分组，每组8只小鼠，保证各组间肿瘤体积和小鼠体重均一。各组肿瘤体积的均值与所有实验动物肿瘤体积的均值差异不超过±10％。尾静脉给药，每四天给一次药(第1、5、9、13天)，一共给药4次，定期测量肿瘤体积和小鼠体重。每一个给药组分别有8只小鼠。
小鼠移植瘤实验结果
小鼠HT-29结肠癌移植瘤实验：实验共分五组，包括缓冲液组(20mM柠檬酸钠，0.3％氯化钠，5％海藻糖，0.05％吐温80，pH6)，BA03单抗组(5mg/kg)、MYK-3组(1mg/kg)、MYK-3组(5mg/kg)和非结合ADC组(5mg/kg)(人源IgG-vcMMAE偶联物，其中IgG是从健康人血清纯化的IgG，该偶联物制备方法与MYK-3相同)。MYK-3给药的小鼠移植肿瘤体积与对照组比较显著降低，显示了明显的抗肿瘤生长效应(图7)。在第18天，MYK-3在5mg/Kg的给药剂量组与缓冲液组相比肿瘤生长抑制率达到54％，与单抗BA03的同等剂量组相比肿瘤生长抑制率达到46％，与非结合ADC相比肿瘤生长抑制率达到42％.
小鼠体重：MYK-3给药的小鼠体重与对照组比较没有变化(见图8)，说明MYK-3没有使小鼠体重降低的毒性作用。
实施例4MYK-3、单抗BA03和BA03+等摩尔数vcMMAE对结直肠癌细胞DiFi的生长抑制活性
按照实施例2方法检测了MYK-3、单抗BA03和BA03+等摩尔数vcMMAE对结直肠癌细胞DiFi的生长抑制活性，实验结果见图9，其中的EC50值分别为8.4ng/mL，65.8ng/mL和68.2ng/mL。
从图中可以看出，单抗BA03在EGFR高表达的结直肠癌细胞DiFi中显示了一定的生长抑制活性；并且BA03加上同等于MYK-3载药摩尔数的游离vcMMAE与单独的BA03活性比较没有明显区别；然而，BA03与vcMMAE偶联形成的MYK-3作为ADC分子对DiFi结直肠癌细胞的生长抑制活性大大高于单抗BA03本身以及BA03加上同等于MYK-3载药摩尔数的游离vcMMAE的活性，EC50提高了大约8倍。
实施例5MYK-3对裸鼠中KRAS突变体结肠癌细胞LoVo的移植瘤生 长抑制活性
按照实施例3的模型构建(以2×106/0.1ml/鼠接种肿瘤细胞)和给药方法检测MYK-3对裸鼠中KRAS突变体结肠癌细胞LoVo的移植瘤生长抑制活性，实验共分六组，包括稀释缓冲液组(20mM柠檬酸钠，0.3％氯化钠，5％海藻糖，0.05％吐温80，pH6)，爱必妥单抗组(3mg/kg)、MYK-3组(0.3mg/kg、1mg/kg、3mg/kg三个剂量)和非结合对照ADC组(3mg/kg)，其中非结合对照ADC代表非结合的同等载药量的ADC对照(non-binding ADC control，为anti-CD20mAb-vcMMAE)，该偶联物制备方法与MYK-3相同。实验结果见图10。
从图中可以看出，MYK-3在3mg/Kg剂量下表现出对LoVo细胞肿瘤生长的完全抑制，并且MYK-3在1mg/Kg已显示出比上市药品Erbitux在3mg/Kg剂量下还更强的活性。
实施例6MYK-3与BA03-MC-MMAE、BA03-MCC-MMAE的体外细胞活性比较
MC-MMAE和MCC-MMAE的结构如下所示：
MC-MMAE：
Figure PCTCN2016073844-appb-000006
MCC-MMAE：
Figure PCTCN2016073844-appb-000007
MC-MMAE和MCC-MMAE的制备方法参见实施例1。
药物/抗体比例的测定：使用HIC-HPLC分析方法测定药物/抗体比例，具体参见实施例1，测得的MYK-3(即为BA03-vcMMAE)，BA03-MC-MMAE 和BA03-MCC-MMAE的药物/抗体比例均为3.9，参见图11。
体外细胞活性实验方法参见实施例2；简而言之，取一定数量的DiFi细胞株，接种于96孔板中，培养24小时后加入不同浓度梯度稀释的BA03-MC-MMAE，BA03-MCC-MMAE，MYK-3三个样品。再培养96小时，加入CCK-8检测试剂显色，在酶标仪上读取96孔板每个孔的OD值，检测不同浓度样品的活细胞数量，判断样品对DiFi细胞株的增殖抑制效果。实验结果见图12，其中BA03-MC-MMAE、BA03-MCC-MMAE和MYK-3的EC50值分别为72.6ng/mL，71.6ng/mL和9.8ng/mL。
由图中可见，MYK-3的细胞增殖抑制活性明显高于BA03-MC-MMAE和BA03-MCC-MMAE的活性；事实上，BA03-MC-MMAE和BA03-MCC-MMAE的细胞增殖抑制活性与单抗BA03本身的活性相似。这说明使用同样的单抗和细胞毒小分子，但使用不同的链接体可以造成ADC的活性有显著差异。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (34)

  1. 抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其包含与细胞毒剂共价连接的抗表皮生长因子受体抗体。
  2. 权利要求1的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的抗表皮生长因子受体抗体包括重链和轻链，其中重链可变区CDR1、CDR2、CDR3分别包含如SEQ ID NO：5～7所示的序列或其突变体，轻链可变区CDR1、CDR2、CDR3分别包含如SEQ ID NO：12～14所示的序列或其突变体。
  3. 权利要求2的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的抗表皮生长因子受体抗体重链可变区FR1、FR2、FR3、FR4区分别包含如SEQ ID NO：8～11所示的序列或其突变体。
  4. 权利要求2的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的抗表皮生长因子受体抗体轻链可变区FR1、FR2、FR3、FR4区分别包含如SEQ ID NO：15～18所示的序列或其突变体。
  5. 权利要求1-4任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的抗表皮生长因子受体抗体的重链恒定区选自人源性IgG、IgM、IgA、IgD、IgA恒定区或上述恒定区的突变体。
  6. 权利要求5的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述IgG选自IgG1、IgG2、IgG3和IgG4。
  7. 权利要求5的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的抗表皮生长因子受体抗体重链恒定区的氨基酸序列包含如SEQ ID NO：3所示的序列，或者包含与SEQ ID NO：3所示序列的同一性大于70％，例如大于75％、80％、85％、90％、95％、 99％的序列。
  8. 权利要求1-7任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的抗表皮生长因子受体抗体的轻链恒定区为人源性lambda恒定区、kappa恒定区、或上述恒定区的突变体。
  9. 权利要求8的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的抗表皮生长因子受体抗体轻链恒定区的氨基酸序列包含如SEQ ID NO：4所示的序列，或者包含与SEQ ID NO：4所示序列的同一性大于70％，例如大于75％、80％、85％、90％、95％、99％的序列。
  10. 权利要求1-9任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其具有式I所示的结构，
    Ab-(L-D)p
    式I
    其中：
    Ab代表抗表皮生长因子受体抗体；
    L代表接头；
    D代表细胞毒剂；
    p代表1-9，例如2-6，例如3-5。
  11. 权利要求1-10任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的细胞毒剂选自化疗药物、毒素、放射性同位素、细胞因子、抗生素、酶、纳米颗粒和生物活性肽。
  12. 权利要求11的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的细胞毒剂选自Monomethyl auristatin E(MMAE)、Monomethyl auristatin F(MMAF)、美登木素生物碱(例如Maytansine DM1、Maytansine DM4)、卡奇霉素(calicheamicin)、duocarmycin MGBA、阿霉素(doxorubicin)、蓖麻毒素、白喉毒素等毒素、1131、白介素类、肿瘤坏死因子、趋化因子和纳米颗粒等，优选为MMAE。
  13. 权利要求10的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的接头为可切割的或不可切割的。
  14. 权利要求13的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的接头选自6-马来酰亚氨基己酰基(MC)、马来酰亚氨基丙酰基(MP)、缬氨酸-瓜氨酸(val-cit)、丙氨酸-苯丙氨酸(ala-phe)、对氨基苄氧羰基(PAB)、N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)-环己烷-1-羧酸酯(SMCC)、N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)、和6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-vc-PAB)，优选为6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-vc-PAB)。
  15. 权利要求1-14任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其为：
    Figure PCTCN2016073844-appb-100001
    其中Ab为抗表皮生长因子受体抗体，p为1-8，例如2-6，例如3-5。
  16. 组合物，其含有权利要求1-15任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，任选地，还含有至少一种药学上可接受的载体、稀释剂或赋形剂。
  17. 权利要求1-15任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物在制备预防和/或治疗与表皮生长因子受体(EGFR)相关的疾病的药物中的用途。
  18. 权利要求16的用途，其中所述的与表皮生长因子受体(EGFR)相关的疾病为与EGFR相关的肿瘤，例如与EGFR过表达相关的肿瘤，例如选自结肠癌、直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌、食管癌、乳腺癌、肾癌、前列腺癌、胃癌、胰腺癌和脑胶质瘤。
  19. 权利要求1-15任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物在制备抑制肿瘤血管生成、延缓肿瘤进展、抑制肿瘤生长、抑制肿瘤细胞增殖的药物中的用途。
  20. 权利要求19的用途，其中所述的肿瘤选自结肠癌、直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌、食管癌、乳腺癌、肾癌、前列腺癌、胃癌、胰腺癌和脑胶质瘤。
  21. 权利要求18或20的用途，其中所述的肿瘤为KRAS基因突变的肿瘤，例如为KRAS基因突变的结肠癌、直肠癌、肺癌或胰腺癌。
  22. 权利要求18或20的用途，其中所述的肿瘤为BRAF基因突变的肿瘤，例如选自BRAF基因突变的结肠癌、直肠癌和肺癌。
  23. 预防和/或治疗与表皮生长因子受体(EGFR)相关的疾病的方法，所述方法包括给予有需要的受试者预防和/或治疗有效量的权利要求1-15任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物。
  24. 权利要求23的方法，其中所述的与表皮生长因子受体(EGFR)相关的疾病为与EGFR相关的肿瘤，例如与EGFR过表达相关的肿瘤，例如选自结肠癌、直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌、食管癌、乳腺癌、肾癌、前列腺癌、胃癌、胰腺癌和脑胶质瘤。
  25. 抑制肿瘤血管生成、延缓肿瘤进展、抑制肿瘤生长、抑制肿瘤细胞增殖的方法，所述方法包括给予有需要的受试者预防和/或治疗有效量的权利要求1-15任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物。
  26. 权利要求25的方法，其中所述的肿瘤选自结肠癌、直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌、食管癌、乳腺癌、肾癌、前列腺癌、胃癌、胰腺癌和脑胶质瘤。
  27. 权利要求24或26的方法，其中所述的肿瘤为KRAS基因突变的肿瘤，例如为KRAS基因突变的结肠癌、直肠癌、肺癌或胰腺癌。
  28. 权利要求24或26的方法，其中所述的肿瘤为BRAF基因突变的肿瘤，例如选自BRAF基因突变的结肠癌、直肠癌和肺癌。
  29. 权利要求1-15任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其用于预防和/或治疗与表皮生长因子受体(EGFR)相关的疾病。
  30. 权利要求29的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的与表皮生长因子受体(EGFR)相关的疾病为与EGFR相关的肿瘤，例如与EGFR过表达相关的肿瘤，例如选自结肠癌、直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌、食管癌、乳腺癌、肾癌、前列腺癌、胃癌、胰腺癌和脑胶质瘤。
  31. 权利要求1-15任一项的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其用于抑制肿瘤血管生成、延缓肿瘤进展、抑制肿瘤生长、抑制肿瘤细胞增殖。
  32. 权利要求31的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的肿瘤选自结肠癌、直肠癌、头颈部癌、肺癌、卵巢癌、宫颈癌、膀胱癌、食管癌、乳腺癌、肾癌、前列腺癌、胃癌、胰腺癌和脑胶质瘤。
  33. 权利要求30或32的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的肿瘤为KRAS基因突变的肿瘤，例如为KRAS基因突变的结肠癌、直肠癌、肺癌或胰腺癌。
  34. 权利要求30或32的抗体药物偶联物、其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，其中所述的肿瘤为BRAF基因突变的肿瘤，例如选自BRAF基因突变的结肠癌、直肠癌和肺癌。
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