WO2022078523A1 - 抗体药物偶联物及其应用 - Google Patents

抗体药物偶联物及其应用 Download PDF

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WO2022078523A1
WO2022078523A1 PCT/CN2021/124698 CN2021124698W WO2022078523A1 WO 2022078523 A1 WO2022078523 A1 WO 2022078523A1 CN 2021124698 W CN2021124698 W CN 2021124698W WO 2022078523 A1 WO2022078523 A1 WO 2022078523A1
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seq
chain variable
variable region
sequence
antibody
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PCT/CN2021/124698
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English (en)
French (fr)
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胡朝红
李虎
陈博
徐刚
王莹
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上海美雅珂生物技术有限责任公司
康诺亚生物医药科技（成都）有限公司
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Application filed by 上海美雅珂生物技术有限责任公司, 康诺亚生物医药科技（成都）有限公司 filed Critical 上海美雅珂生物技术有限责任公司
Priority to MX2023004236A priority Critical patent/MX2023004236A/es
Priority to CR20230207A priority patent/CR20230207A/es
Priority to AU2021359562A priority patent/AU2021359562A1/en
Priority to IL302122A priority patent/IL302122A/en
Priority to US18/031,731 priority patent/US20230381336A1/en
Priority to EP21879564.9A priority patent/EP4230225A1/en
Priority to CA3198667A priority patent/CA3198667A1/en
Publication of WO2022078523A1 publication Critical patent/WO2022078523A1/zh
Priority to CONC2023/0005979A priority patent/CO2023005979A2/es

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    • A—HUMAN NECESSITIES
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6835—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
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    • 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
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    • A61K47/6863—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from stomach or intestines cancer cell
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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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of medicinal chemistry, in particular, to antibody drug conjugates and applications thereof.
  • Gastric cancer is one of the most common cancers worldwide, with higher incidences in East Asia, Eastern Europe, and South America, and lower incidences in North America and Africa.
  • the standard initial treatment for advanced or recurrent gastric cancer is chemotherapy.
  • the prognosis of gastric cancer patients has improved significantly due to the development of surgical techniques and perioperative treatment, the 5-year overall survival rate is only 10-15%.
  • Targeted therapy has brought new hope for the treatment of recurrent/advanced gastric cancer.
  • Combining trastuzumab with chemotherapy can bring certain benefits to patients with HER2 positive, but only 15% of patients have HER2 positive expression, and the beneficiary population limited.
  • the inventor of the present application has prepared an anti-Claudin 18.2 antibody drug conjugate through a lot of experiments and creative work, and confirmed that it has good biological activity, thus completing the present invention.
  • the present invention provides an antibody drug conjugate, a pharmaceutically acceptable salt, solvate or a solvate of said salt thereof, said antibody drug conjugate having the formula The structure shown in I,
  • Ab is an anti-Claudin 18.2 antibody
  • the anti-Claudin 18.2 antibody comprises a heavy chain and a light chain
  • the variable region CDR1 of the heavy chain comprises a group selected from SEQ ID NOs: 2, 10, 18, 26, 34, 42, 68, 76, 84 , 92, 100, 108 or 116 or a mutant thereof
  • the heavy chain variable region CDR2 comprises a sequence selected from the group consisting of SEQ ID NO: 3, 11, 19, 27, 35, 43, 69, 77, 85, 93
  • the heavy chain variable region CDR3 comprises a sequence selected from SEQ ID NO: 4, 12, 20, 28, 36, 44, 70, 78, 86, 94, 102, 110
  • the light chain variable region CDR1 comprises a sequence selected from SEQ ID NO: 50, 58, 124 or 132 or a mutant thereof
  • the light chain variable region CDR2 comprises
  • D is a cytotoxic agent
  • L is a linker for connecting the anti-Claudin 18.2 antibody and the cytotoxic agent
  • p is 2.0-8.0 (eg 2.0-7.0, 2.0-6.0, 2.0-5.0, 2.0-4.0, 3.0-7.0, 3.0-6.0, 3.0-5.0 or 3.0-4.0, or eg 3.0, 4.0, 5.0, 6.0 or 7.0 ).
  • the antibody drug conjugate of the present invention has good tumor cell growth inhibition activity both in vivo and in vitro, and has a good application prospect.
  • the antibody-drug conjugate of the present invention is formed by connecting anti-Claudin 18.2 antibody and dolastatin derivative MMAE through MC-vc-PAB linker, and its anti-tumor action mechanism is as follows: the antibody-drug conjugate has the After Claudin 18.2 is bound, it enters tumor cells through endocytosis and transports to lysosomes, and then is degraded by protease in lysosomes to release MMAE. After entering the cytoplasm, MMAE binds to tubulin and inhibits its polymerization, thereby blocking tubulin. It participates in various physiological functions of cells including mitosis, thereby inhibiting tumor cell proliferation and leading to tumor cell death.
  • the "antibody drug conjugate” is a composition containing ADC molecules with the same or different DAR values.
  • the present invention provides compositions comprising a plurality of ADC molecules.
  • the multiple ADCs each comprise the same number of drug molecules in the composition.
  • the multiple ADCs each comprise a different number of drug molecules in the composition.
  • the above drug-to-antibody ratio refers to the number of drug molecules (eg, p in Formula I) conjugated to the antibody.
  • the number of drug molecules contained in the antibody-drug conjugate of the present invention (for example, p in formula I) is usually an integer, when the number of drug molecules contained in the antibody-drug conjugate of the present invention is (eg, p in Formula I) is a fraction, which refers to the average number of drug molecules conjugated per antibody in a composition comprising a plurality of ADC molecules.
  • the above drug-to-antibody ratios can be verified by conventional means, such as mass spectrometry, ELISA assays, HIC and HPLC.
  • the quantitative distribution of ADCs with respect to p can also be determined.
  • the separation, purification and validation of a homogeneous ADC for which p is a certain value from ADCs with other drug loads can be accomplished by means such as reverse phase HPLC or electrophoresis.
  • the heavy chain variable region CDR1 of the anti-Claudin 18.2 antibody comprises a sequence selected from the group consisting of SEQ ID NO: 2, 10, 18, 26, 34 or 42 or a mutant thereof
  • the heavy chain variable The region CDR2 comprises a sequence selected from SEQ ID NO: 3, 11, 19, 27, 35 or 43 or a mutant thereof
  • the heavy chain variable region CDR3 comprises a sequence selected from SEQ ID NO: 4, 12, 20, 28,
  • the light chain variable region CDR1 comprises a sequence selected from SEQ ID NO: 50 or 58 or a mutant thereof
  • the light chain variable region CDR2 comprises a sequence selected from SEQ ID NO:
  • the light chain variable region CDR3 comprises a sequence selected from the sequence shown in SEQ ID NO: 52 or 60 or a mutant thereof.
  • the heavy chain variable region CDR1 of the anti-Claudin 18.2 antibody comprises a sequence selected from the group consisting of SEQ ID NO: 68, 76, 84, 92, 100, 108 or 116 or a mutant thereof, heavy chain
  • the variable region CDR2 comprises a sequence selected from SEQ ID NO: 69, 77, 85, 93, 101, 109 or 117 or a mutant thereof
  • the heavy chain variable region CDR3 comprises a sequence selected from SEQ ID NO: 70, 78,
  • the light chain variable region CDR1 comprises a sequence selected from SEQ ID NO: 124 or 132 or a mutant thereof
  • the light chain variable region CDR2 comprises The light chain variable region CDR3 is selected from the sequence shown in SEQ ID NO: 125 or 133 or a mutant thereof
  • the light chain variable region CDR3 comprises a sequence selected from the sequence shown in SEQ ID NO: 126 or 134
  • the heavy chain variable region CDR1, CDR2, CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:
  • SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4,
  • the light chain variable region CDR1, CDR2 and CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:
  • SEQ ID NO: 50 SEQ ID NO: 51, SEQ ID NO: 52,
  • the heavy chain variable region CDR1, CDR2, CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:
  • SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4,
  • the light chain variable region CDR1, CDR2 and CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:
  • SEQ ID NO: 50 SEQ ID NO: 51, SEQ ID NO: 52,
  • the heavy chain variable region CDR1, CDR2, CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:
  • the light chain variable region CDR1, CDR2 and CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:
  • the heavy chain variable region FR1 of the anti-Claudin 18.2 antibody comprises a variable region FR1 selected from the group consisting of SEQ ID NO: 5, 13, 21, 29, 37, 45, 71, 79, 87, 95, 103, 111 or The sequence shown in 119 or a mutant thereof, the variable region FR2 of the heavy chain comprises a sequence selected from SEQ ID NO: 6, 14, 22, 30, 38, 46, 72, 80, 88, 96, 104, 112 or 120.
  • heavy chain variable region FR4 comprises a sequence selected from SEQ ID NO: 8, 16, 24, 32, 40, 48, 74, 82, 90, 98, 106, 114 or 122 or a mutant thereof
  • the light chain variable region FR1 comprises a sequence selected from SEQ ID NO: 53, 61, 127 or 135 or a mutant thereof
  • the light chain variable region FR2 comprises a sequence selected from SEQ ID NO: 54, 62, 128 or 136
  • the sequence shown or a mutant thereof, the light chain variable region FR3 comprises a sequence selected from SEQ ID NO: 55, 63, 129 or 137 or a mutant thereof
  • the light chain variable region FR4 comprises a sequence selected from SEQ ID NO: : the sequence shown in 56, 64, 130 or 138 or a mutant thereof.
  • the heavy chain variable region FR1 of the anti-Claudin 18.2 antibody comprises a sequence selected from the group consisting of SEQ ID NO: 5, 13, 21, 29, 37 or 45 or a mutant thereof
  • the heavy chain variable Region FR2 comprises a sequence selected from SEQ ID NO: 6, 14, 22, 30, 38 or 46 or a mutant thereof
  • the heavy chain variable region FR3 comprises a sequence selected from SEQ ID NO: 7, 15, 23, 31, The sequence shown in 39 or 47 or a mutant thereof
  • the heavy chain variable region FR4 comprises a sequence selected from SEQ ID NO: 8, 16, 24, 32, 40 or 48 or a mutant thereof
  • the light chain variable region FR1 comprises a sequence selected from the group consisting of SEQ ID NO: 53 or 61, or a mutant thereof
  • the light chain variable region FR2 comprises a sequence selected from the group consisting of SEQ ID NO: 54 or 62, or a mutant thereof
  • the light chain variable region The region FR3 comprises a sequence selected from the sequence shown in SEQ ID NO: 55 or 63 or
  • the heavy chain variable region FR1 of the anti-Claudin 18.2 antibody comprises a sequence selected from the group consisting of SEQ ID NO: 71, 79, 87, 95, 103, 111 or 119 or a mutant thereof
  • heavy chain The variable region FR2 comprises a sequence selected from the group consisting of SEQ ID NO: 72, 80, 88, 96, 104, 112 or 120 or a mutant thereof
  • the heavy chain variable region FR3 comprises a sequence selected from the group consisting of SEQ ID NO: 73, 81, The sequence shown in 89, 97, 105, 113 or 121 or a mutant thereof
  • the heavy chain variable region FR4 comprises a sequence selected from SEQ ID NO: 74, 82, 90, 98, 106, 114 or 122 or the sequence shown in 122 Mutants
  • the light chain variable region FR1 comprises a sequence selected from SEQ ID NO: 127 or 135 or a mutant thereof
  • the light chain variable region FR2 comprises
  • the heavy chain variable regions FR1, FR2, FR3, FR4 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:
  • the light chain variable regions FR1, FR2, FR3 and FR4 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:
  • SEQ ID NO: 53 SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,
  • the heavy chain variable regions FR1, FR2, FR3, FR4 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:
  • the light chain variable regions FR1, FR2, FR3 and FR4 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:
  • SEQ ID NO: 53 SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,
  • the heavy chain variable regions FR1, FR2, FR3, FR4 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:
  • the light chain variable regions FR1, FR2, FR3 and FR4 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:
  • SEQ ID NO: 127 SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130,
  • the heavy chain variable region of the anti-Claudin 18.2 antibody is selected from the group consisting of SEQ ID NO: 1, 9, 17, 25, 33, 41, 67, 75, 83, 91, 99, 107, or 115 the sequence shown,
  • the light chain variable region of the anti-Claudin 18.2 antibody is selected from the sequence shown in SEQ ID NO: 49, 57, 123 or 131.
  • the heavy chain variable region of the anti-Claudin 18.2 antibody is selected from the sequence set forth in SEQ ID NO: 1, 9, 17, 25, 33 or 41,
  • the light chain variable region of the anti-Claudin 18.2 antibody is selected from the sequence shown in SEQ ID NO: 49 or 57.
  • the heavy chain variable region of the anti-Claudin 18.2 antibody is selected from the sequence set forth in SEQ ID NO: 67, 75, 83, 91, 99, 107 or 115,
  • the light chain variable region of the anti-Claudin 18.2 antibody is selected from the sequence shown in SEQ ID NO: 123 or 131.
  • the heavy chain variable region and light chain variable region of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:
  • sequences of the heavy chain variable region and light chain variable region of the anti-Claudin 18.2 antibody are SEQ ID NO: 41 and SEQ ID NO: 49, respectively.
  • the heavy chain constant region of the anti-Claudin 18.2 antibody is selected from human IgG (eg, IgGl, IgG2, IgG3, or IgG4), IgM, IgA, IgD, IgA constant regions, or mutants of the above constant regions , preferably human IgG1;
  • the light chain constant region of the anti-Claudin 18.2 antibody is selected from human lambda constant region, kappa constant region or mutants of the above constant regions, preferably human kappa constant region.
  • the amino acid sequence of the heavy chain of the anti-Claudin 18.2 antibody comprises the sequence set forth in SEQ ID NO:65, or comprises greater than 70% identity to the sequence set forth in SEQ ID NO:65, such as greater than 75%, 80%, 85%, 90%, 95%, 99% sequences;
  • the amino acid sequence of the light chain of the anti-Claudin 18.2 antibody comprises the sequence shown in SEQ ID NO: 66, or the identity with the sequence shown in SEQ ID NO: 66 is greater than 70%, such as greater than 75%, 80%, 85%, 90%, 95%, 99% sequences.
  • p is 3.0-4.0.
  • p is 3.0-3.8.
  • p is 3.0, 3.4, 3.5, or 3.8.
  • p is 3.8.
  • the cytotoxic agent is selected from the group consisting of SN-38, Gemcitabine, Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), maytansinoids (eg, Maytansine DM1, Maytansine DM4), Toxins such as calicheamicin, MGBA (eg duocarmycin), doxorubicin, ricin, diphtheria toxin, I131, interleukins, tumor necrosis factor, chemokines and nanoparticles.
  • MMAE Monomethyl auristatin E
  • MMAF Monomethyl auristatin F
  • maytansinoids eg, Maytansine DM1, Maytansine DM4
  • Toxins such as calicheamicin, MGBA (eg duocarmycin), doxorubicin, ricin, diphtheria toxin, I131, interleukins, tumor necrosis factor, chemokines and nanoparticles
  • the cytotoxic agent is MMAE.
  • MMAE The structure of MMAE is:
  • the linker is selected from the group consisting of 6-maleimidohexanoyl (MC), maleimidopropionyl (MP), N-succinimidyl 4-(2-pyridylthio) ) valerate (SPP), 4-(N-maleimidomethyl)-cyclohexane-1-formyl (MCC), N-succinimidyl(4-iodo-acetyl)aminobenzene formate (SIAB), and 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).
  • MC 6-maleimidohexanoyl
  • MP maleimidopropionyl
  • SPP N-succinimidyl 4-(2-pyridylthio) valerate
  • MCC 4-(N-maleimidomethyl)-cyclohexane-1-formyl
  • MIB N-succinimid
  • the linker is 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).
  • L-D described in formula I is MC-vc-PAB-MMAE, and its structure is shown in the following formula:
  • Ab includes:
  • L is MC-vc-PAB
  • the present invention provides a composition comprising the aforementioned antibody drug conjugate, a pharmaceutically acceptable salt, solvate or solvate of said salt.
  • the composition further comprises known chemotherapeutic drugs for the treatment of tumors, such as doxorubicin (Adriamycin), cyclophosphamide, taxanes [such as paclitaxel (Taxol) ), docetaxel (Taxotere)], capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (Arimidex, Furlong, Arnold New), 5-FU plus leucovorin, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin ), doxorubicin, prednisone, etc., or a combination thereof.
  • doxorubicin Adriamycin
  • cyclophosphamide taxanes
  • the composition further comprises a known immunotherapy drug for treating tumors, such as a PD-1 monoclonal antibody (such as pembrolizumab, nivolumab, etc.) ), PD-L1 monoclonal antibody (such as Atezolizumab), TIGIT monoclonal antibody, 4-1BB monoclonal antibody, VEGFR2 monoclonal antibody (such as Ramucirumab, apatinib), HER2 monoclonal antibody (such as trastuzumab, Trastuzumab biosimilar, Trastuzumab -dkst), etc., or a combination thereof.
  • a known immunotherapy drug for treating tumors such as a PD-1 monoclonal antibody (such as pembrolizumab, nivolumab, etc.) ), PD-L1 monoclonal antibody (such as Atezolizumab), TIGIT monoclonal antibody, 4-1BB monoclonal antibody, VEGFR2 monoclo
  • the composition further comprises an immunosuppressant selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as Cyclosporine and taupomycin, etc.; (3) anti-metabolites, such as azathioprine and 6-mercaptopurine, etc.; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3, etc. ; (5) Alkylating agents, such as cyclophosphamide.
  • an immunosuppressant selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as Cyclosporine and taupomycin, etc.; (3) anti-metabolites, such as azathioprine and 6-mercaptopurine, etc.; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3, etc.
  • the immunosuppressive agents are, for example, methylprednisolone, prednisone, azathioprine, prolacofol, xenipra, sule, cyclosporine, tacrolimus, rapamycin Fingolimod, mycophenolate mofetil, mizoribine, cyclophosphamide, etc.
  • the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention provides the aforementioned antibody drug conjugate, its pharmaceutically acceptable salt, solvate or solvate of said salt or the use of the aforementioned composition in the preparation of medicine , said medicament for the prevention and/or treatment of diseases associated with Claudin 18.2.
  • the disease associated with Claudin 18.2 is gastric cancer, gastroesophageal junction adenocarcinoma, pancreatic cancer.
  • the disease associated with Claudin 18.2 is gastric cancer.
  • the present invention provides a method for preventing and/or treating a disease associated with Claudin 18.2, comprising: administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of the aforementioned antibody drug conjugate compound, a pharmaceutically acceptable salt, solvate or solvate of said salt, or a composition of the foregoing.
  • the disease associated with Claudin 18.2 is gastric cancer, gastroesophageal junction adenocarcinoma, pancreatic cancer.
  • the disease associated with Claudin 18.2 is gastric cancer.
  • the present invention provides the aforementioned antibody drug conjugate, its pharmaceutically acceptable salt, solvate or solvate of said salt, or the aforementioned composition, which is used for prophylaxis and/or treatment of disorders associated with Claudin 18.2.
  • the disease associated with Claudin 18.2 is gastric cancer, gastroesophageal junction adenocarcinoma, pancreatic cancer.
  • the disease associated with Claudin 18.2 is gastric cancer.
  • Fig. 1 is the RT-PCR of the embodiment of the present invention showing that HEK293 stably transfected cell line expresses Claudin 18.1 and Claudin 18.2 respectively; HEK293-claudin 18.2 stably transfected cell line and control KATO III cells can amplify tight junction protein 18.1 and claudin 18.2 respectively; Connexin 18.2 has a specific 780bp characteristic band, while HEK293-claudin 18.1 can only amplify a common fragment of 504bp;
  • Fig. 2 is the result of screening HEK293-claudin 18.2 high-expressing stably-transformed cell lines by FACS experiment in the embodiment of the present invention, wherein the black dots are negative controls, and the gray dots are HEK293-claudin 18.2 high-expressing stably transfected cell lines;
  • Fig. 3 is the result of screening the NIH3T3-claudin 18.2 high-expressing stably transfected cell line by FACS experiment in the embodiment of the present invention, wherein the black line is the negative control, and the gray shade is the 3T3-claudin 18.2 stably transfected cell line; NO .32-H is the 3T3-claudin 18.2 stable transfection cell line with high expression, NO.18-M is the 3T3-claudin 18.2 stable transfection cell line with medium expression, and NO.6-L is the 3T3-claudin 18.2 with low expression. Claudin 18.2 stably transfected cell line;
  • Fig. 4 is the anti-Claudin18.2 antibody ADCC effect result diagram of the embodiment of the present invention.
  • FIG. 5 is a graph showing the results of the CDC effect of Anti-Claudin18.2 antibody according to an embodiment of the present invention.
  • Fig. 6 is the hydrophobic interaction chromatogram (HIC) of the antibody-drug conjugate of the embodiment of the present invention.
  • FIG. 7 is a graph showing the results of the cell killing effect of different CM311 ADCs in the LT-M11 cell line according to the embodiment of the present invention.
  • Figure 8 is a graph showing the results of the inhibitory activity of CM311-ADC-1 and control CM311-ADC-2 on the tumor growth inhibition of human gastric cancer nude mouse PDX model STO#025 according to the embodiment of the present invention
  • Figure 9 is a graph showing the effect of CM311-ADC-1 and control CM311-ADC-2 on the body weight of animals in the PDX model of human gastric cancer nude mice STO#025 according to the embodiment of the present invention.
  • Figure 10 is a graph showing the results of the inhibitory activity of CM311-ADC-1 and control CM311-ADC-2 on the tumor growth inhibition of human gastric cancer nude mouse PDX model STO#523 according to the embodiment of the present invention
  • Figure 11 is a graph showing the effect of CM311-ADC-1 and control CM311-ADC-2 on the body weight of animals in the PDX model of human gastric cancer nude mice STO#523 according to the embodiment of the present invention
  • Figure 12 is a graph showing the results of comparing the in vitro cell activity of the antibody-drug conjugate of the embodiment of the present invention and the antibody (QC Log transformation independent fitting graph).
  • Claudin protein is a skeletal protein that constitutes a tight junction structure. It is located on the top side of adjacent intercellular spaces. Its distribution is tissue-organ-specific. Its functions are mainly intercellular adhesion, maintenance of cell polarity, regulation of paracellular permeability, and participation in cell proliferation. Differentiation regulation. In tumors, the tight junctions between cells are destroyed and Claudin cannot perform its normal function.
  • Claudin l8 is a member of the Claudin family with 2 different first exons, so two isoforms can be generated by alternative splicing: Claudin l8.1 and Claudin 18.2, these two isoforms are in different tissues
  • Claudin l8.1 was mainly expressed in lung tissue, while Claudin l8.2 was specifically expressed in gastric tissue.
  • Claudin 18.2 (GenBank accession number: NM_001002026.3) is not expressed in other normal tissues except gastric mucosa, but it is significantly up-regulated in various tumors, including 80% of gastrointestinal adenomas, 60% of pancreas Tumors, and some tumors of the bile ducts, ovaries, and lungs.
  • Claudin 18.2-related disease refers to a disease in which the expression of Claudin 18.2 in tissue cells differs from (eg exceeds) normal levels. For example, the expression level of Claudin 18.2 in a certain tissue cell is higher than the expression level of Claudin 18.2 in the reference or control (ie, normal tissue cells), indicating that the tissue cell is derived from the object (especially the human) in the Claudin 18.2-related disease. exist.
  • any numerical range should be understood to include any value or any sub-range within the range.
  • the term “antibody” refers to an immunoglobulin molecule generally composed of two identical pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain.
  • the light chains of antibodies can be divided into two categories: kappa and lambda.
  • Heavy chains can be divided into five types: ⁇ , ⁇ , ⁇ , ⁇ or ⁇ , and antibodies can be divided into five types: IgM, IgD, IgG, IgA and IgE according to the difference of the heavy chain.
  • the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region ( VH ) and a heavy chain constant region ( CH ).
  • the heavy chain constant region consists of 3 domains ( CH1, CH2 and CH3 ) .
  • Each light chain consists of a light chain variable region ( VL ) and a light chain constant region ( CL ).
  • the light chain constant region consists of one domain, CL .
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and component C1q of the complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Definition by Chothia et al. (1989) Nature 342:878-883.
  • BLAST and BLAST 2.0 algorithms for determining sequence identity (homology) and percent sequence similarity are, for example, the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acid. Res. 25:3389- 3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410.
  • BLAST and BLAST 2.0 can be used to determine percent amino acid sequence identity of the present invention using, for example, those described in the literature or default parameters.
  • Software to perform 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 includes a polypeptide sequence that is substantially identical to the amino acid sequence, eg when using the methods described herein (eg, BLAST analysis using standard parameters) , contains 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 , 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.
  • the mutant of the amino acid sequence refers to the identity with the amino acid sequence of more than 70%, such as more than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of sequences, eg sequences with 3, 2 or 1 substitution, deletion or addition of amino acids.
  • amino acids eg sequences with 3, 2 or 1 substitution, deletion or addition of amino acids.
  • no more than 3 amino acids are substituted, added or deleted. More preferably, no more than 2 amino acids are substituted, added or deleted. Most preferably, no more than 1 amino acid is substituted, added or deleted.
  • substitutional variant is one in which at least one amino acid residue in the native sequence has been removed and a different amino acid inserted in its same position.
  • the substitutions can be single, wherein only one amino acid is substituted in the molecule, or multiple, wherein the same molecule has two or more amino acids substituted. Multiple substitutions can be made at consecutive sites.
  • one amino acid may be substituted by multiple residues, wherein such variants include both substitutions and insertions.
  • An “insertion” (or “additive") variant is one in which one or more amino acids are inserted into an amino acid immediately adjacent to a particular position in a native sequence. Immediately adjacent to an amino acid means attachment to the alpha-carboxyl or alpha-amino functional group of the amino acid.
  • a “deletion” variant is one in which one or more amino acids in the native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted in a specific region of their molecule.
  • less than the theoretical maximum of the drug moiety is conjugated to the antibody in the conjugation reaction.
  • antibodies do not contain many free and reactive cysteine thiol groups that can link drug moieties; in fact, most cysteine thiol groups in antibodies exist as disulfide bridges.
  • the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partially or fully reducing conditions to generate reactive cysteine thiol groups .
  • DTT dithiothreitol
  • TCEP tricarbonylethylphosphine
  • the pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt
  • the inorganic acid salt is hydrochloride, hydrobromide, hydroiodide, nitrate, bicarbonate Salts and carbonates, sulfates or phosphates
  • the organic acid salts being formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartaric acid salt, citrate, ascorbate, alpha-ketoglutarate, alpha-glycerophosphate, alkylsulfonate or arylsulfonate
  • the alkylsulfonate is methanesulfonic acid salt or ethyl sulfonate
  • the aryl sulfonate is benzene sulfonate or p-toluene sulfonate.
  • compositions can be obtained using standard procedures well known in the art, eg, by reacting a sufficient amount of a basic compound with a suitable acid to provide a pharmaceutically acceptable anion.
  • prodrug refers to a derivative that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide a compound of the present invention. Prodrugs only undergo this reaction under biological conditions to become the active compound, or they are active in their unreacted form. Prodrugs can generally be prepared using well-known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Edited by Manfred E. Wolff, 5th ed.).
  • solvates refer to these forms of the antibody-drug conjugates of the present invention: the complexes of the antibody-drug conjugates in solid or liquid form formed by coordinating with solvent molecules. Hydrates are a specific form of solvates, which have coordinated water molecules. In the present invention, hydrates are the preferred solvates.
  • compositions containing amounts of active ingredients are known, or will be apparent to those skilled in the art in light of the present disclosure.
  • methods of preparing such pharmaceutical compositions include incorporating suitable pharmaceutical excipients, carriers, diluents, etc., which It is not toxic to cells or mammals to which it is exposed at the doses and concentrations employed.
  • the pharmaceutical composition of the present invention may comprise a pH buffered aqueous solution.
  • 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 immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, sucrose, Trehalose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN TM , polyethylene glycol (PEG ) and PLURONICS TM .
  • the pharmaceutical formulations of the present invention are manufactured by known methods, including conventional mixing, dissolving or lyophilization methods.
  • the compounds of the present invention can be formulated into pharmaceutical compositions and administered to patients by various routes suitable for the chosen mode of administration, for example, orally or parenterally (by intravenous, intramuscular, topical or subcutaneous routes).
  • the compounds of the present invention can be administered systemically, eg, orally, in combination with a pharmaceutically acceptable carrier such as an inert diluent or an assimilable edible carrier. They can be enclosed in hard or soft shell gelatin capsules and can be compressed into tablets.
  • a pharmaceutically acceptable carrier such as an inert diluent or an assimilable edible carrier.
  • the active compound may be incorporated with one or more excipients and presented in the form of swallowable tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and the like use.
  • Such compositions and preparations should contain at least 0.1% active compound.
  • the proportions of such compositions and formulations may, of course, vary and may range from about 1% to about 99% by weight of a given unit dosage form.
  • the active compound is in an amount such that an effective dosage level can be obtained.
  • Tablets, troches, pills, capsules, etc. may also contain: a binder, such as tragacanth, acacia, cornstarch, or gelatin; an excipient, such as dicalcium hydrogen phosphate; a disintegrant, such as cornstarch, Potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, fructose, lactose, or aspartame; or flavoring agents, such as peppermint, oil of wintergreen, or cherry flavor.
  • a binder such as tragacanth, acacia, cornstarch, or gelatin
  • an excipient such as dicalcium hydrogen phosphate
  • a disintegrant such as cornstarch, Potato starch, alginic acid, etc.
  • lubricants such as magnesium stearate
  • sweeteners such as sucrose, fructose, lactose, or aspartame
  • flavoring agents such as pepper
  • any materials may be present, as coatings, or otherwise alter the physical form of the solid unit dosage form.
  • tablets, pills or capsules may be coated with gelatin, wax, shellac or sugar and the like.
  • a syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl or propyl paraben as a preservative, a dye and a flavoring (such as cherry flavor or orange flavor).
  • any materials used in the preparation of any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts to be used.
  • the active compounds can be incorporated into sustained release formulations and sustained release devices.
  • the active compound may also be administered intravenously or intraperitoneally by infusion or injection.
  • Aqueous solutions of the active compounds or salts thereof can be prepared, optionally mixed with nontoxic surfactants.
  • Dispersions in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils can also be prepared. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injection or infusion can include sterile aqueous solutions or dispersions of the active ingredient (optionally encapsulated in liposomes) containing the active ingredient suitable for extemporaneous preparation in sterile injectable or infusible solutions or dispersions. or sterile powder.
  • the liquid carrier can be a solvent or liquid dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, nontoxic glycerides, and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the desired particle size in the case of dispersions, or by the use of surfactants.
  • Prevention of microorganisms can be brought about by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases it is preferred to include isotonic agents such as sugars, buffers or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use of agents which delay absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compound in the required amount in an appropriate solvent with various of the other ingredients enumerated above as required, followed by filtered sterilization.
  • the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional required ingredients previously present in sterile-filtered solutions .
  • Useful solid carriers include pulverized solids (eg, talc, clays, microcrystalline cellulose, silica, alumina, and the like).
  • Useful liquid carriers include water, ethanol or ethylene glycol, or water-ethanol/ethylene glycol mixtures, in which the compounds of the present invention may be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants eg, fragrances
  • additional antimicrobial agents can be added to optimize properties for a given use.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified inorganic materials can also be used with liquid carriers to form coatable pastes, gels, ointments , soap, etc., directly on the user's skin.
  • unit dosage form which are physically discrete units containing unitary dosages, suitable for administration to the human and other mammalian bodies.
  • the unit dosage form can be a capsule or tablet, or a number of capsules or tablets.
  • the amount of active ingredient in a unit dose may vary or be adjusted from about 0.1 to about 1000 mg or more.
  • milk liposomes such as milk liposomes, microspheres and nanospheres
  • microparticle dispersion systems including polymeric micelles, nanoemulsion, submicroemuls
  • Pharmaceutical preparations such as microcapsules, microspheres, liposomes and niosomes (also known as nonionic surfactant vesicles).
  • treating generally refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms; and/or therapeutic in terms of partial or complete stabilization or cure of the disease and/or side effects due to the disease.
  • Treatment encompasses any treatment of a disease in a patient, including: (a) prevention of disease or symptoms in a patient susceptible to a disease or condition but not yet diagnosed; (b) suppression of symptoms of disease, That is, preventing its development; or (c) alleviating the symptoms of the disease, ie, causing the disease or symptoms to regress.
  • 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.
  • the mammal refers to a human.
  • an “effective amount” refers to an amount effective to achieve the desired therapeutic or prophylactic effect at the dose and time necessary.
  • 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 in which any toxic or detrimental consequences of the substance/molecule are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective at the dose and time necessary to achieve the desired prophylactic effect.
  • a prophylactically effective amount will be less than a therapeutically effective amount because the prophylactic dose is administered to the subject prior to the onset of the disease or at an early stage of the disease.
  • a therapeutically effective amount of the drug reduces the number of cancer cells; shrinks the tumor size; inhibits (ie, slows to some extent, preferably stops) infiltration of cancer cells into surrounding organs; inhibits (ie, slows to some extent, preferably stops) ) tumor metastasis; some degree of inhibition of tumor growth; and/or some degree of alleviation of one or more symptoms associated with cancer.
  • chimeric antibody refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody of antibodies.
  • Humanized antibodies refer to non-human (eg, mouse) forms of antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins.
  • the humanized antibody is a human immunoglobulin (recipient antibody) in which the complementarity determining region (CDR) residues of the recipient antibody are derived from a non-human species with the desired specificity, affinity and capacity ( donor antibody) such as mouse, rat or rabbit CDR residue substitutions.
  • CDR complementarity determining region
  • telomeres can be mutated amino acid residues within the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (eg, affinity) of the antibody .
  • PCR-mediated mutagenesis can be performed to introduce mutations whose effect on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions.
  • the plasmid gene claudin 18.1-puc57-Amp (Suzhou Hongxun Biology, SynbioTech) containing the full-length sequence of human claudin 18.1 (UniProtKB-P56856) was synthesized.
  • the upstream primer 5'-ttggcaaagaattgctagatgtccaccaccacatgcc-3' (SEQ ID NO: 171)
  • the downstream primer 5'-tgttcgggccctcctcgattacacatagtcgtgcttgg-3' (SEQ ID NO: 172)
  • the full length of human tight junction protein 18.1 was amplified by PCR Fragment (Met1-Val261).
  • the amplified product was enzymatically linked by NEBuilder HiFi DNA Assembly Master Mix (NEB, Cat: M0530L), and then cloned into the eukaryotic expression plasmid system. Similar to this, a plasmid gene Claudin 18.2-puc57-Amp (Suzhou Hongxun Biology, SynbioTech) containing the full-length sequence of human Claudin 18.2 (UniProtKB-P56856-2) was synthesized.
  • the upstream primer 5'-ttggcaaagaattgctagatggccgtgactgcctgtc-3' (SEQ ID NO: 173)
  • the downstream primer 5'-tgttcgggccctcctcgattacacatagtcgtgcttgg-3' (SEQ ID NO: 174)
  • the full length of human tight junction protein 18.2 was amplified by PCR Fragment (Met1-Val261).
  • the amplified product was enzymatically linked by NEBuilder HiFi DNA Assembly Master Mix (NEB, Cat: M0530L), and then cloned into the eukaryotic expression plasmid system.
  • NIH3T3 and HEK293 cells were electroporated with this plasmid, and 1-10 ⁇ g/mL Puromycin (Gibco, Cat: A1113803) was used for stepwise pressurization to screen for stable expression cell lines.
  • HEK293-Claudin 18.1 and HEK293-Claudin 18.2 stably transfected cell lines were first verified by RT-PCR method.
  • Total RNA was extracted from positive cloned cells with Trizol RNA extraction kit, reverse transcription kit (SuperScriptTM First-Strand Synthesis System, Cat: 18080051) was used, and cDNA library was obtained by reverse transcription with Oligo (dT) primer. Since the two splice fragments of Claudin 18.1 and 18.2 differ from the nitrogen terminus to the first extracellular region (Loop1), the primer KNB14 (5'-tgtgcgccaccatggccgtg-3' (SEQ ID NO: 175) was designed.
  • KNB15 (5'-tggaaggataagattgtacc-3' (SEQ ID NO: 176)
  • KNB16 5'-tgggtgccattggcctcctg-3' (SEQ ID NO: 177)
  • the full-length fragment (780 bp) between them was used as a positive control in KATO III cells (ATCC HTB-103) expressing Claudin 18.2.
  • RT-PCR showed that the HEK293 stably transfected cell line expressed Claudin 18.1 and Claudin 18.2, respectively.
  • Both HEK293-Claudin 18.2 stably transfected cell line and control KATO III cells can amplify a characteristic band of 780 bp specific for Claudin 18.2, while HEK293-Claudin 18.1 can only amplify a common fragment of 504 bp.
  • the constructed stable transfected cell lines HEK293-Claudin 18.2 cells and NIH3T3-Claudin 18.2 cells were digested and collected, washed twice with PBS, and 100 ⁇ L of 1:200 diluted primary antibody rabbit anti-Claudin 18.2 (Abcam, EPR19202, Cat. No.: ab222512), incubate at 4°C for 60 minutes, wash off excess primary antibody solution with 0.5% BSA/PBS, and add 50 ⁇ L of secondary antibody goat anti-rabbit IgGFc-AF647 (Jackson ImmunoResearch, Cat.
  • Figure 2 shows the results of detecting HEK293-Claudin 18.2 stably transfected cell lines transfected with the full-length Claudin 18.2 gene by FACS experiment.
  • the black dots are untransfected HEK293 cells
  • the gray dots are HEK293-Claudin 18.2 stable transfected cell lines
  • HEK293 cells do not express Claudin 18.2
  • HEK293-Claudin 18.2 stable transfected strains highly express tight junctions on the cell membrane surface Protein 18.2.
  • Figure 3 shows the results of detecting the high expression of Claudin 18.2 in NIH3T3-Claudin 18.2 stably transfected cell lines by FACS experiment.
  • the black line is the negative control, and the gray shading is the 3T3-claudin 18.2 stably transfected cell line.
  • NO.32-H is the 3T3-Claudin 18.2 stably transfected cell line with high expression of Claudin 18.
  • NO.18-M is the 3T3-Claudin 18.2 stably transfected cell line expressed in Claudin 18.2
  • NO. 6-L is a 3T3-Claudin 18.2 stably transfected cell line with low expression of Claudin 18.2.
  • the positive stably transfected cell lines that have grown and grown were collected and frozen, and the NIH3T3-claudin 18.2 stably transfected cell line was used to immunize animals.
  • 1 ⁇ 10 6 3T3-Claudin 18.2 stably transduced cells were taken each time and mixed with Freund's adjuvant or non-Freund's adjuvant, then injected into the thigh root and footpad, and again at different sites two weeks later. immunity.
  • mice Three days later, the mice were sacrificed, and the popliteal, inguinal, and iliac lymph nodes were collected and ground in DMEM to obtain a B cell-rich suspension; the mouse spleen was removed, ground in DMEM, and centrifuged to obtain a spleen cell suspension. An appropriate amount of lymph node and spleen cell suspension was mixed with SP2/0, and the cells were fused using an electrofusion apparatus.
  • the antibody fragments in the light chain library were double digested with BspQI and SfiI, and then ligated into the heavy chain library to form a mouse chimeric Fab phage display library based on filamentous phage M13, with a library capacity of 1.2 ⁇ 10 10 .
  • HEK293-Claudin 18.1, HEK293-Claudin 18.2, and HEK293 were pre-stained with 5 ⁇ M, 0.5 ⁇ M, and 0 ⁇ M Cell Tracker Green CMFDA Dye (Thermo, Cat. No. C2925), respectively, according to the instructions for live cell prestaining.
  • press 1 :1:1 mix After washing off the dye, press 1 :1:1 mix, add to 96-well plate (2x10 5 cells/well), and combine with hybridoma supernatant or bacterial induction supernatant, after ice bath incubation for 1 hour, add AlexaFluro647-labeled secondary antibody anti-mouse IgG Fc or Anti-human IgG F(ab)'2 (Jackson ImmunoResearch), incubated on ice for 45 minutes.
  • the screened antibody binds to HEK293-Claudin 18.2 stably transfected cells with high affinity and specificity, and does not bind to HEK293-Claudin 18.1 stably transfected cells and HEK293 cells.
  • the positive clones were screened from the phage library, the plasmids were extracted for sequencing, and the variable region sequences were cloned into the heavy chain and light chain constant region vectors respectively for full-length IgG expression.
  • the positive cells obtained from the hybridoma were lysed by adding 1 mL of TRNzol, and total RNA was extracted by the guanidine isothioate method. Using this as a template, after synthesizing the first-strand cDNA, the first-strand cDNA is used as a subsequent template to amplify the DNA sequence of the variable region corresponding to the hybridoma cells. After sequencing the amplified products, the variable region sequences of the heavy and light chains of the candidate hybridomas were obtained, as shown below.
  • underlined parts are respectively CDR1 (SEQ ID NO: 140), CDR2 (SEQ ID NO: 141), CDR3 (SEQ ID NO: 142) from left to right;
  • FR1 SEQ ID NO: 143
  • FR2 SEQ ID NO: 144
  • FR3 SEQ ID NO: 145
  • FR4 SEQ ID NO: 1466 from left to right.
  • underlined parts are respectively CDR1 (SEQ ID NO: 148), CDR2 (SEQ ID NO: 149), CDR3 (SEQ ID NO: 150) from left to right;
  • the underlined parts are FR1 (SEQ ID NO: 151), FR2 (SEQ ID NO: 152), FR3 (SEQ ID NO: 153), FR4 (SEQ ID NO: 154) from left to right.
  • underlined parts are respectively CDR1 (SEQ ID NO: 156), CDR2 (SEQ ID NO: 157), CDR3 (SEQ ID NO: 158) from left to right;
  • FR1 SEQ ID NO: 159
  • FR2 SEQ ID NO: 160
  • FR3 SEQ ID NO: 161
  • FR4 SEQ ID NO: 162
  • underlined parts are respectively CDR1 (SEQ ID NO: 164), CDR2 (SEQ ID NO: 165), CDR3 (SEQ ID NO: 166) from left to right;
  • FR1 SEQ ID NO: 167
  • FR2 SEQ ID NO: 168
  • FR3 SEQ ID NO: 169
  • FR4 SEQ ID NO: 170
  • the above heavy and light chain variable region sequence fragments were PCR amplified, and the heavy chain variable region was cloned into a vector containing the human heavy chain constant region to express the intact IgGl heavy chain in mammalian cells. Similarly, the light chain variable region was cloned into a vector containing the human light chain constant region to express the complete kappa light chain in mammalian cells. After correct sequencing, it was transfected into HEK293-6E mammalian cells, IgG1 was expressed and secreted into the medium, and the supernatant was collected and filtered for purification.
  • the IgG was purified by Protein A chromatography, and the culture supernatant was loaded onto an appropriate size Protein A column, washed with 50 mM Tris-HCl pH 8.0, 250 mM NaCl, and the bound IgG was washed with 0.1 M Glycine-HCl, pH 3.0 wash off.
  • the protein was concentrated by ultrafiltration using a concentration tube (Millipore), OD280 was detected, and the concentration of IgG was determined by spectrophotometry. The purity of IgG was analyzed by SDS-PAGE.
  • HEK293-Claudin 18.1 cells, HEK293-Claudin 18.2 cells and HEK293 cells in logarithmic growth phase add 5 ⁇ 10 4 cells/100 ⁇ L to a U-shaped 96-well plate after digestion, centrifuge at 1100 rpm for 3 minutes, and discard the supernatant , lightly tap to disperse cells, add 50 ⁇ L of serially diluted antibody to each well (antibody concentration starts from 100nM, 5-fold dilution for 8 gradients), and incubate at 4°C for 1 hour.
  • Different heavy chain derivatives and light chain derivatives were obtained by design. After the full sequence synthesis of the light and heavy chain derivatives, they were cloned into a vector containing the constant region Ckappa of the antibody kappa chain or the constant region CH1-CH3 of human IgG1. The same After the parent-derived light and heavy chain derivative plasmids were combined and paired, HEK293.6E cells were transfected, expressed for 5-6 days, and the supernatant was collected and purified on a Protein A column.
  • the sequence of the humanized antibody is as follows:
  • CDR1 SEQ ID NO: 2
  • CDR2 SEQ ID NO: 3
  • CDR3 SEQ ID NO: 4
  • the underlined parts are FR1 (SEQ ID NO: 5), FR2 (SEQ ID NO: 6), FR3 (SEQ ID NO: 7), FR4 (SEQ ID NO: 8) from left to right.
  • underlined parts are CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11), CDR3 (SEQ ID NO: 12) from left to right;
  • the underlined parts are FR1 (SEQ ID NO: 13), FR2 (SEQ ID NO: 14), FR3 (SEQ ID NO: 15), FR4 (SEQ ID NO: 16) from left to right.
  • underlined parts are CDR1 (SEQ ID NO: 18), CDR2 (SEQ ID NO: 19), CDR3 (SEQ ID NO: 20) respectively from left to right;
  • the underlined parts are FR1 (SEQ ID NO: 21), FR2 (SEQ ID NO: 22), FR3 (SEQ ID NO: 23), FR4 (SEQ ID NO: 24) from left to right.
  • underlined parts are CDR1 (SEQ ID NO: 26), CDR2 (SEQ ID NO: 27), CDR3 (SEQ ID NO: 28) respectively from left to right;
  • the underlined parts are FR1 (SEQ ID NO: 29), FR2 (SEQ ID NO: 30), FR3 (SEQ ID NO: 31), FR4 (SEQ ID NO: 32) from left to right.
  • the underlined parts are CDR1 (SEQ ID NO: 34), CDR2 (SEQ ID NO: 35), CDR3 (SEQ ID NO: 36) respectively from left to right;
  • the underlined parts are FR1 (SEQ ID NO: 37), FR2 (SEQ ID NO: 38), FR3 (SEQ ID NO: 39), FR4 (SEQ ID NO: 40) from left to right.
  • underlined parts are CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO: 43), CDR3 (SEQ ID NO: 44) respectively from left to right;
  • the underlined parts are FR1 (SEQ ID NO: 45), FR2 (SEQ ID NO: 46), FR3 (SEQ ID NO: 47), FR4 (SEQ ID NO: 48) respectively from left to right.
  • underlined parts are CDR1 (SEQ ID NO: 50), CDR2 (SEQ ID NO: 51), CDR3 (SEQ ID NO: 52) respectively from left to right;
  • the underlined parts are FR1 (SEQ ID NO: 53), FR2 (SEQ ID NO: 54), FR3 (SEQ ID NO: 55), FR4 (SEQ ID NO: 56) from left to right.
  • underlined parts are CDR1 (SEQ ID NO: 58), CDR2 (SEQ ID NO: 59), CDR3 (SEQ ID NO: 60) respectively from left to right;
  • the underlined parts are FR1 (SEQ ID NO: 61), FR2 (SEQ ID NO: 62), FR3 (SEQ ID NO: 63), FR4 (SEQ ID NO: 64) from left to right.
  • sequences of preferred humanized antibodies are as follows:
  • underlined parts are CDR1 (SEQ ID NO: 68), CDR2 (SEQ ID NO: 69), CDR3 (SEQ ID NO: 70) from left to right;
  • FR1 SEQ ID NO: 71
  • FR2 SEQ ID NO: 72
  • FR3 SEQ ID NO: 73
  • FR4 SEQ ID NO: 74
  • underlined parts are CDR1 (SEQ ID NO: 76), CDR2 (SEQ ID NO: 77), CDR3 (SEQ ID NO: 78) respectively from left to right;
  • the underlined parts are FR1 (SEQ ID NO: 79), FR2 (SEQ ID NO: 80), FR3 (SEQ ID NO: 81), FR4 (SEQ ID NO: 82) from left to right.
  • the underlined parts are CDR1 (SEQ ID NO: 84), CDR2 (SEQ ID NO: 85), CDR3 (SEQ ID NO: 86) respectively from left to right;
  • FR1 SEQ ID NO: 87
  • FR2 SEQ ID NO: 88
  • FR3 SEQ ID NO: 89
  • FR4 SEQ ID NO: 90
  • the underlined parts are CDR1 (SEQ ID NO: 92), CDR2 (SEQ ID NO: 93), CDR3 (SEQ ID NO: 94) respectively from left to right;
  • FR1 SEQ ID NO: 95
  • FR2 SEQ ID NO: 96
  • FR3 SEQ ID NO: 97
  • FR4 SEQ ID NO: 98
  • the underlined parts are respectively CDR1 (SEQ ID NO: 100), CDR2 (SEQ ID NO: 101), CDR3 (SEQ ID NO: 102) from left to right;
  • FR1 SEQ ID NO: 103
  • FR2 SEQ ID NO: 104
  • FR3 SEQ ID NO: 105
  • FR4 SEQ ID NO: 106
  • the underlined parts are respectively CDR1 (SEQ ID NO: 108), CDR2 (SEQ ID NO: 109), CDR3 (SEQ ID NO: 110) from left to right;
  • the underlined parts are FR1 (SEQ ID NO: 111), FR2 (SEQ ID NO: 112), FR3 (SEQ ID NO: 113), FR4 (SEQ ID NO: 114) from left to right.
  • underlined parts are respectively CDR1 (SEQ ID NO: 116), CDR2 (SEQ ID NO: 117), CDR3 (SEQ ID NO: 118) from left to right;
  • FR1 SEQ ID NO: 119
  • FR2 SEQ ID NO: 120
  • FR3 SEQ ID NO: 121
  • FR4 SEQ ID NO: 122
  • underlined parts are respectively CDR1 (SEQ ID NO: 124), CDR2 (SEQ ID NO: 125), CDR3 (SEQ ID NO: 126) from left to right;
  • the underlined parts are FR1 (SEQ ID NO: 127), FR2 (SEQ ID NO: 128), FR3 (SEQ ID NO: 129), FR4 (SEQ ID NO: 130) from left to right.
  • underlined parts are respectively CDR1 (SEQ ID NO: 132), CDR2 (SEQ ID NO: 133), CDR3 (SEQ ID NO: 134) from left to right;
  • FR1 SEQ ID NO: 135)
  • FR2 SEQ ID NO: 136
  • FR3 SEQ ID NO: 137
  • FR4 SEQ ID NO: 138
  • 18D10 chimera, 18A9 chimera, anti-claudin 18.2 humanized antibodies 18D10, 18A9 (ie CM311, CM311 is the collective name of humanized antibodies 18D10 or 18A9) and the dilution of the control antibody anti-KLH (40, 20 , 10, 5, 2.5, 1.25 ⁇ g/mL), 25 ⁇ L/well. Incubate for 30 min at 37 °C, 5% CO 2 . Take out the well plate and add Kato III cells, 8 ⁇ 10 3 cells/well, 25 ⁇ L/well.
  • the Kato III cells in logarithmic growth phase were resuspended to 1 ⁇ 10 7 cells/mL.
  • CFSE (Sigma, 87444-5MG-F) was added to a final concentration of 1 ⁇ M. Incubate at room temperature for 10 minutes, and add 3 volumes of medium to stop the reaction. Centrifuge at 1000 rpm at 4°C for 5 minutes, discard the supernatant, resuspend the medium, and inoculate the cells into a 96-well plate, 1*10 5 cells/well, 50 ⁇ L/well.
  • CM311 antibody Take 10 mg of CM311 antibody and replace it with reducing buffer (25mM sodium borate, pH8.0, 25mM NaCl, 5mM EDTA) using a 15mL 30KD ultrafiltration device for a total of three replacements; the final volume is about 1mL, and transfer to a new Eppendorf centrifuge tube (weighing), and weighed; the protein concentration was detected, and the total protein was calculated.
  • Add 2.5 times moles of DTT to the antibody incubate at room temperature for 2 hours, and mix continuously; use 15mL of 30KD ultrafiltration device to replace it with coupling buffer (50mM Tris, pH7.2, 150mM NaCl, 5mM EDTA), a total of three times replacement.
  • Sampling was used to determine the protein concentration with A280, and weighed to calculate the total amount of protein; 10 ⁇ L of the sample was taken, and the number of free sulfhydryl groups was determined by Ellman's method;
  • vc-MMAE MC-vc-PAB-MMAE
  • DMSO DMSO
  • the drug loading (DAR) of the antibody drug conjugates was determined by hydrophobic interaction chromatography (HIC-HPLC).
  • the average drug loading DAR of the antibody drug conjugate CM311-18D10-VH3/VL2-ADC was 3.5.
  • the average drug loading DAR of the antibody drug conjugate CM311-18D10-VH6/VL2-ADC was 3.4.
  • the average drug loading DAR of the antibody drug conjugate CM311-18A9-VH7/VL2-ADC was 3.0.
  • the names of the above antibody drug conjugates are such as CM311-18D10-VH6/VL1-ADC, which means that the antibody used in the preparation of the antibody drug conjugate is CM311-18D10-VH6/VL1.
  • the antibody name CM311-18D10-VH6/VL1 indicates that the antibody used in the preparation of the aforementioned antibody-drug conjugates is 18D10 in this class of antibodies in which the variable region of the heavy chain is VHv6 and the variable region of the light chain is VLv1, corresponding to As can be seen from Table 2 above, the specific antibody used in the preparation of the aforementioned antibody-drug conjugate is h18D10.v16.
  • CM311-18D10-VH3/VL2-ADC The names of the remaining three antibody drug conjugates CM311-18D10-VH3/VL2-ADC, CM311-18D10-VH6/VL2-ADC and CM311-18A9-VH7/VL2-ADC can be understood by referring to the above.
  • the specific antibody used in the preparation of the antibody-drug conjugate CM311-18D10-VH3/VL2-ADC was h18D10.v23 in Table 2; the antibody used in the preparation of the antibody-drug conjugate CM311-18D10-VH6/VL2-ADC The specific antibody is h18D10.v26 in Table 2; the specific antibody used in preparing the antibody drug conjugate CM311-18A9-VH7/VL2-ADC is h18A9.v27 in Table 2.
  • CM311ADC conjugated by different CM311 monoclonal antibodies
  • Figure 7 is a representative graph of the killing of LT-M11 cells by different CM311 ADCs.
  • CM311 ADC The antitumor activity of CM311 ADC was tested in two gastric cancer PDX models, STO#025 and STO#523. Both gastric cancer models had higher Claudin 18 mRNA levels.
  • the process of establishing the PDX model of human gastric cancer in nude mice is as follows: tumor tissue with a volume of about 15-30 mm 3 is transplanted into the back of BALB/c nude mice subcutaneously. When the tumor volume reached 150-260 mm, the mice were randomly divided into groups, with 5 mice in each group, to ensure that the tumor volume between the groups was uniform and the body weight was taken into account. There were 4 groups in total, including the vehicle group, 1 mg/kg CM311-ADC- 1 administration group, 3 mg/kg CM311-ADC-1 administration group and 3 mg/kg CM311-ADC-2 (unconjugated control ADC) administration group.
  • CM311-ADC-1 refers to the antibody drug conjugate CM311-18D10-VH6/VL1-ADC; compared with CM311-ADC-1, the antibody of CM311-ADC-2 is a human IgG1 isotype control, which is closely related to the surface of tumor cells. target is not bound.
  • tumor volumes were measured twice a week.
  • Relative tumor proliferation rate T/C (%) (RTV in administration group/RTV in Vehicle group) ⁇ 100%.
  • Tumor growth inhibition rate TGI% (average tumor volume of vehicle group-average tumor volume of administration group)/average tumor volume of vehicle group ⁇ 100%. When T/C(%) ⁇ 40%, and P ⁇ 0.05, it is considered that the test article has a significant inhibitory effect on tumor growth.
  • CM311-ADC-1 3mg/kg
  • CM311-ADC-1 1mg/kg
  • CM311-ADC-2 3mg/kg
  • Significant antitumor activity Both CM311-ADC-1 and CM311-ADC-2 were well tolerated by tumor-bearing mice.
  • CM311-ADC-1 After administration of CM311-ADC-1 at 1 and 3 mg/kg, the relative tumor proliferation rate T/C (%) on Day 28 was 35.60% and 6.79%, respectively, and the tumor growth inhibition rate TGI% was 64.40% and 93.21%, respectively ; CM311-ADC-2 (3mg/kg) group T/C (%) was 114.81%, TGI% was -14.81%, the experimental results showed that CM311-ADC-1 (3mg/kg) and CM311-ADC-1 ( 1 mg/kg) had significant inhibitory activity against tumor growth, while CM311-ADC-2 (3 mg/kg) had no significant anti-tumor activity. Both CM311-ADC-1 and CM311-ADC-2 were well tolerated by tumor-bearing mice.
  • the KATO III cell line was plated at 5000 cells/well, and the samples to be tested were added after 24 hours.
  • the samples to be tested were diluted 9 times with a final concentration of 1000ng/mL 2.4 times for 96 hours, and the fluorescence value was read using a microplate reader after Presto-Blue color development for 60min.
  • the samples to be tested are as follows:
  • Sample2 Anti-Claudin-18.2-ADC, specifically CM311-18D10-VH6/VL1-ADC;
  • IgG-L1D1 is an ADC in which an IgG antibody is coupled with a L1D1 small molecule (ie vcMMAE, that is, MC-vc-PAB-MMAE) drug).
  • Anti-Claudin-18.2-ADC e.g. CM311-18D10-VH6/VL1-ADC
  • CM311 e.g. CM311- 18D10-VH6/VL1
  • IgG-L1D1 did not show obvious cell killing effect.

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Abstract

本发明涉及抗体药物偶联物及其应用，具体提供了抗体药物偶联物，其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，所述抗体药物偶联物具有式Ⅰ所示的结构，其中，Ab为抗Claudin 18.2抗体。本发明的抗体药物偶联物在体内和体外都具有良好的抑制肿瘤细胞生长活性，且细胞毒性低，具有良好的应用前景。 Ab-(L-D)p 式Ⅰ

Description

抗体药物偶联物及其应用 技术领域
本发明涉及药物化学领域，具体地，涉及抗体药物偶联物及其应用。
背景技术
胃癌是世界范围内最常见的癌症之一，其中东亚、东欧和南美洲胃癌发病率较高，北美和非洲发病率较低。晚期或复发胃癌标准初始治疗是化疗。尽管由于手术技术以及围手术治疗的发展，胃癌患者的预后已经显著改善，但是其5年总生存率仅为10-15％。靶向治疗为复发/晚期胃癌治疗带来了新的希望，将曲妥珠单抗联合化疗可为有HER2阳性的患者带来一定获益，但只有15％的患者有HER2阳性表达，受益人群有限。近年来，免疫治疗给复发/晚期胃癌治疗带来了新的希望；但是根据KEYNOTE-12研究的结果，适用该治疗的PD-L1阳性人群比率也仅占复发/晚期胃或胃食管结合部腺癌患者的40％左右。因此，开发新的胃癌治疗药物仍然迫在眉睫。
发明内容
本申请的发明人通过大量实验和创造性劳动，制备得到了抗Claudin 18.2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。
为此，在本发明的第一方面，本发明提供了抗体药物偶联物，其药学上可接受的盐、溶剂合物或所述盐的溶剂合物，所述抗体药物偶联物具有式Ⅰ所示的结构，
Ab-(L-D) p
式Ⅰ
其中：
Ab为抗Claudin 18.2抗体，所述抗Claudin 18.2抗体包含重链和轻链，重链可变区CDR1包含选自SEQ ID NO：2、10、18、26、34、42、68、76、84、92、100、108或116所示的序列或其突变体，重链可变区CDR2包含选自SEQ ID NO：3、11、19、27、35、43、69、77、85、93、101、109或117所示的序列或其突变体，重链可变区CDR3包含选自SEQ ID NO：4、12、20、28、36、44、70、78、86、94、102、110或118所示的序列或其突变体，轻链可变区CDR1包含选自SEQ ID NO：50、58、124或132所示的序列或其突变体，轻链可变区CDR2包含选自SEQ ID NO：51、59、125或133所示的序列或其突变体，轻链可变区CDR3包含选自SEQ ID NO：52、60、126或134所示的序列或其突变体；
D为细胞毒剂；
L为接头，用于连接所述抗Claudin 18.2抗体和所述细胞毒剂；
p为2.0-8.0(例如2.0-7.0、2.0-6.0、2.0-5.0、2.0-4.0、3.0-7.0、3.0-6.0、3.0-5.0或3.0-4.0，或者例如3.0、4.0、5.0、6.0或7.0)。
本发明的抗体药物偶联物在体内和体外都具有良好的抑制肿瘤细胞生长活性，具有良好的应用前景。本发明的抗体药物偶联物由抗Claudin 18.2抗体和海兔毒素的衍生物MMAE通过MC-vc-PAB链接体连接而成，其抗肿瘤作用机理为：抗体药物偶联物与肿瘤细胞表面的Claudin 18.2结合后，通过内吞进入肿瘤细胞内并运输到溶酶体，再经溶酶体内蛋白酶降解释放出MMAE，MMAE进入细胞质后与微管蛋白结合并抑制其聚合，从而阻断微管蛋白参与的包括有丝分裂在内的细胞各项生理功能，进而抑制肿瘤细胞增殖并导致肿瘤细胞死亡。
需要说明的是，所述“抗体药物偶联物”为含有相同或不同DAR值的ADC分子的组合物。具体地，本发明提供了包含多个ADC分子的组合物。在某些情况下，该组合物中，多个ADC分别包含相同数目的药物分子。在其他情况下，该组合物中，多个ADC分别包含不 同数目的药物分子。
上述药物抗体比(DAR)是指偶联到抗体的药物分子的个数(例如，式I中的p)。本发明所述的抗体药物偶联物中包含的药物分子的个数(例如，式I中的p)通常是整数，当本发明所述的抗体药物偶联物中包含的药物分子的个数(例如，式I中的p)是分数时，该分数指的是包含多个ADC分子的组合物中，每个抗体偶联的药物分子的平均数量。
上述药物抗体比(DAR)可以通过常规手段来验证，诸如质谱、ELISA测定法、HIC和HPLC。还可以测定ADC在p方面的定量分布。在有些情况中，将p为某数值的同质ADC从具有其它药物载荷的ADC中分离、纯化和验证可以通过诸如反相HPLC或电泳的手段来实现。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区CDR1包含选自SEQ ID NO：2、10、18、26、34或42所示的序列或其突变体，重链可变区CDR2包含选自SEQ ID NO：3、11、19、27、35或43所示的序列或其突变体，重链可变区CDR3包含选自SEQ ID NO：4、12、20、28、36或44所示的序列或其突变体，轻链可变区CDR1包含选自SEQ ID NO：50或58所示的序列或其突变体，轻链可变区CDR2包含选自SEQ ID NO：51或59所示的序列或其突变体，轻链可变区CDR3包含选自SEQ ID NO：52或60所示的序列或其突变体。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区CDR1包含选自SEQ ID NO：68、76、84、92、100、108或116所示的序列或其突变体，重链可变区CDR2包含选自SEQ ID NO：69、77、85、93、101、109或117所示的序列或其突变体，重链可变区CDR3包含选自SEQ ID NO：70、78、86、94、102、110或118所示的序列或其突变体，轻链可变区CDR1包含选自SEQ ID NO：124或132所示的序列或其突变体，轻链可变区CDR2包含选自SEQ ID NO：125或133所示的序列或其突变体，轻链可变区CDR3包含选自SEQ ID NO：126或134所示的序列或其突变体。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区CDR1、CDR2、CDR3选自如下序列组合：
(1)SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4，
(2)SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12，
(3)SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20，
(4)SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28，
(5)SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36，
(6)SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44，
(7)SEQ ID NO：68、SEQ ID NO：69、SEQ ID NO：70，
(8)SEQ ID NO：76、SEQ ID NO：77、SEQ ID NO：78，
(9)SEQ ID NO：84、SEQ ID NO：85、SEQ ID NO：86，
(10)SEQ ID NO：92、SEQ ID NO：93、SEQ ID NO：94，
(11)SEQ ID NO：100、SEQ ID NO：101、SEQ ID NO：102，
(12)SEQ ID NO：108、SEQ ID NO：109、SEQ ID NO：110，
(13)SEQ ID NO：116、SEQ ID NO：117、SEQ ID NO：118，
所述抗Claudin 18.2抗体的轻链可变区CDR1、CDR2、CDR3选自如下序列组合：
(1)SEQ ID NO：50、SEQ ID NO：51、SEQ ID NO：52，
(2)SEQ ID NO：58、SEQ ID NO：59、SEQ ID NO：60，
(3)SEQ ID NO：124、SEQ ID NO：125、SEQ ID NO：126，
(4)SEQ ID NO：132、SEQ ID NO：133、SEQ ID NO：134。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区CDR1、CDR2、CDR3选自如下序列组合：
(1)SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4，
(2)SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12，
(3)SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20，
(4)SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28，
(5)SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36，
(6)SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44，
所述抗Claudin 18.2抗体的轻链可变区CDR1、CDR2、CDR3选自如下序列组合：
(1)SEQ ID NO：50、SEQ ID NO：51、SEQ ID NO：52，
(2)SEQ ID NO：58、SEQ ID NO：59、SEQ ID NO：60。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区CDR1、CDR2、CDR3选自如下序列组合：
(1)SEQ ID NO：68、SEQ ID NO：69、SEQ ID NO：70，
(2)SEQ ID NO：76、SEQ ID NO：77、SEQ ID NO：78，
(9)SEQ ID NO：84、SEQ ID NO：85、SEQ ID NO：86，
(4)SEQ ID NO：92、SEQ ID NO：93、SEQ ID NO：94，
(5)SEQ ID NO：100、SEQ ID NO：101、SEQ ID NO：102，
(6)SEQ ID NO：108、SEQ ID NO：109、SEQ ID NO：110，
(7)SEQ ID NO：116、SEQ ID NO：117、SEQ ID NO：118，
所述抗Claudin 18.2抗体的轻链可变区CDR1、CDR2、CDR3选自如下序列组合：
(1)SEQ ID NO：124、SEQ ID NO：125、SEQ ID NO：126，
(2)SEQ ID NO：132、SEQ ID NO：133、SEQ ID NO：134。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区FR1包含选自SEQ ID NO：5、13、21、29、37、45、71、79、87、95、103、111或119所示的序列或其突变体，重链可变区FR2包含选自SEQ ID NO：6、14、22、30、38、46、72、80、88、96、104、112或120所示的序列或其突变体，重链可变区FR3包含选自SEQ ID NO：7、15、23、31、39、47、73、81、89、97、105、113或121所示的序列或其突变体，重链可变区FR4包含选自SEQ ID NO：8、16、24、32、40、48、74、82、90、98、106、114或122所示的序列或其突变体，轻链可变区FR1包含选自SEQ ID NO：53、61、127或135所示的序列或其突变体，轻链可变区FR2包含选自SEQ ID NO：54、62、128或136所示的序列或其突变体，轻链可变区FR3包含选自SEQ ID NO：55、63、129或137所示的序列或其突变体，轻链可变区FR4包含选自SEQ ID NO：56、64、130或138所示的序列或其突变体。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区FR1包含选自SEQ ID NO：5、13、21、29、37或45所示的序列或其突变体，重链可变区FR2包含选自SEQ ID NO：6、14、22、30、38或46所示的序列或其突变体，重链可变区FR3包含选自SEQ ID NO：7、15、23、31、39或47所示的序列或其突变体，重链可变区FR4包含选自SEQ ID NO：8、16、24、32、40或48所示的序列或其突变体，轻链可变区FR1包含选自SEQ ID NO：53或61、所示的序列或其突变体，轻链可变区FR2包含选自SEQ ID NO：54或62所示的序列或其突变体，轻链可变区FR3包含选自SEQ ID NO：55或63所示的序列或其突变体，轻链可变区FR4包含选自SEQ ID NO：56或64所示的序列或其突变体。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区FR1包含选自SEQ ID NO：71、79、87、95、103、111或119所示的序列或其突变体，重链可变区FR2包含选自SEQ ID NO：72、80、88、96、104、112或120所示的序列或其突变体，重链可变区FR3包含选自SEQ ID NO：73、81、89、97、105、113或121所示的序列或其突变体，重链可变区FR4包含选自SEQ ID NO：74、82、90、98、106、114或122所示的序列或其突变体，轻链可变区FR1包含选自SEQ ID NO：127或135所示的序列或其突变体，轻链可变区FR2包含选自SEQ ID NO：128或136所示的序列或其突变体，轻链可变区FR3包含选自SEQ ID NO：129或137所示的序列或其突变体，轻链可变区FR4包含选自SEQ ID NO：130或138所示的序列或其突变体。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区FR1、FR2、FR3、FR4选自如下序列组合：
(1)SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8，
(2)SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16，
(3)SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24，
(4)SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32，
(5)SEQ ID NO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40，
(6)SEQ ID NO：45、SEQ ID NO：46、SEQ ID NO：47、SEQ ID NO：48，
(7)SEQ ID NO：71、SEQ ID NO：72、SEQ ID NO：73、SEQ ID NO：74，
(8)SEQ ID NO：79、SEQ ID NO：80、SEQ ID NO：81、SEQ ID NO：82，
(9)SEQ ID NO：87、SEQ ID NO：88、SEQ ID NO：89、SEQ ID NO：90，
(10)SEQ ID NO：95、SEQ ID NO：96、SEQ ID NO：97、SEQ ID NO：98，
(11)SEQ ID NO：103、SEQ ID NO：104、SEQ ID NO：105、SEQ ID NO：106，
(12)SEQ ID NO：111、SEQ ID NO：112、SEQ ID NO：113、SEQ ID NO：114，
(13)SEQ ID NO：119、SEQ ID NO：120、SEQ ID NO：121、SEQ ID NO：122，
所述抗Claudin 18.2抗体的轻链可变区FR1、FR2、FR3、FR4选自如下序列组合：
(1)SEQ ID NO：53、SEQ ID NO：54、SEQ ID NO：55、SEQ ID NO：56，
(2)SEQ ID NO：61、SEQ ID NO：62、SEQ ID NO：63、SEQ ID NO：64，
(3)SEQ ID NO：127、SEQ ID NO：128、SEQ ID NO：129、SEQ ID NO：130，
(4)SEQ ID NO：135、SEQ ID NO：136、SEQ ID NO：137、SEQ ID NO：138。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区FR1、FR2、FR3、FR4选自如下序列组合：
(1)SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8，
(2)SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16，
(3)SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24，
(4)SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32，
(5)SEQ ID NO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40，
(6)SEQ ID NO：45、SEQ ID NO：46、SEQ ID NO：47、SEQ ID NO：48，
所述抗Claudin 18.2抗体的轻链可变区FR1、FR2、FR3、FR4选自如下序列组合：
(1)SEQ ID NO：53、SEQ ID NO：54、SEQ ID NO：55、SEQ ID NO：56，
(2)SEQ ID NO：61、SEQ ID NO：62、SEQ ID NO：63、SEQ ID NO：64。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区FR1、FR2、FR3、FR4选自如下序列组合：
(1)SEQ ID NO：71、SEQ ID NO：72、SEQ ID NO：73、SEQ ID NO：74，
(2)SEQ ID NO：79、SEQ ID NO：80、SEQ ID NO：81、SEQ ID NO：82，
(3)SEQ ID NO：87、SEQ ID NO：88、SEQ ID NO：89、SEQ ID NO：90，
(4)SEQ ID NO：95、SEQ ID NO：96、SEQ ID NO：97、SEQ ID NO：98，
(5)SEQ ID NO：103、SEQ ID NO：104、SEQ ID NO：105、SEQ ID NO：106，
(6)SEQ ID NO：111、SEQ ID NO：112、SEQ ID NO：113、SEQ ID NO：114，
(7)SEQ ID NO：119、SEQ ID NO：120、SEQ ID NO：121、SEQ ID NO：122，
所述抗Claudin 18.2抗体的轻链可变区FR1、FR2、FR3、FR4选自如下序列组合：
(1)SEQ ID NO：127、SEQ ID NO：128、SEQ ID NO：129、SEQ ID NO：130，
(2)SEQ ID NO：135、SEQ ID NO：136、SEQ ID NO：137、SEQ ID NO：138。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区选自SEQ ID NO：1、9、17、25、33、41、67、75、83、91、99、107或115所示的序列，
所述抗Claudin 18.2抗体的轻链可变区选自SEQ ID NO：49、57、123或131所示的 序列。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区选自SEQ ID NO：1、9、17、25、33或41所示的序列，
所述抗Claudin 18.2抗体的轻链可变区选自SEQ ID NO：49或57所示的序列。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区选自SEQ ID NO：67、75、83、91、99、107或115所示的序列，
所述抗Claudin 18.2抗体的轻链可变区选自SEQ ID NO：123或131所示的序列。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区、轻链可变区选自如下序列组合：
(1)SEQ ID NO：17和SEQ ID NO：57；
(2)SEQ ID NO：41和SEQ ID NO：49；
(3)SEQ ID NO：41和SEQ ID NO：57；
(4)SEQ ID NO：115和SEQ ID NO：131。
在一些实施方案中，所述抗Claudin 18.2抗体的重链可变区、轻链可变区的序列分别为SEQ ID NO：41和SEQ ID NO：49。
在一些实施方案中，所述抗Claudin 18.2抗体的重链恒定区选自人源性IgG(如IgG1、IgG2、IgG3或IgG4)、IgM、IgA、IgD、IgA恒定区或上述恒定区的突变体，优选为人源性IgG1；
所述抗Claudin 18.2抗体的轻链恒定区选自人源性lambda恒定区、kappa恒定区或上述恒定区的突变体，优选为人源性kappa恒定区。
在一些实施方案中，所述抗Claudin 18.2抗体的重链的氨基酸序列包含如SEQ ID NO：65所示的序列，或者包含与SEQ ID NO：65所示序列的同一性大于70％，例如大于75％、80％、85％、90％、95％、99％的序列；
所述抗Claudin 18.2抗体的轻链的氨基酸序列包含如SEQ ID NO：66所示的序列，或者包含与SEQ ID NO：66所示序列的同一性大于70％，例如大于75％、80％、85％、90％、95％、99％的序列。
在一些实施方案中，p为3.0-4.0。
在一些实施方案中，p为3.0-3.8。
在一些实施方案中，p为3.0、3.4、3.5或3.8。
在一些实施方案中，p为3.8。
在一些实施方案中，所述细胞毒剂选自SN-38、吉西他滨(Gemcitabine)、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)、MGBA(如duocarmycin)、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。
在一些实施方案中，所述细胞毒剂为MMAE。
MMAE的结构为：
Figure PCTCN2021124698-appb-000001
在一些实施方案中，所述接头选自6-马来酰亚氨基己酰基(MC)、马来酰亚氨基丙酰基(MP)、N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(SPP)、4-(N-马来酰亚氨基甲基)-环己烷-1-甲酰基(MCC)、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)。
在一些实施方案中，所述接头为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)。
在一些实施方案中，式I中所述的L-D为MC-vc-PAB-MMAE，其结构如下式所示：
Figure PCTCN2021124698-appb-000002
在一些实施方案中，
Ab包括：
(a)重链可变区CDR1、CDR2、CDR3和轻链可变区CDR1、CDR2、CDR3，其中，重链可变区CDR1的序列如SEQ ID NO：42所示，重链可变区CDR2的序列如SEQ ID NO：43所示，重链可变区CDR3的序列如SEQ ID NO：44所示，轻链可变区CDR1的序列如SEQ ID NO：50所示，轻链可变区CDR2的序列如SEQ ID NO：51所示，轻链可变区CDR3的序列如SEQ ID NO：52所示；
(b)重链可变区和轻链可变区，其中，重链可变区的序列如SEQ ID NO：41所示，轻链可变区的序列如SEQ ID NO：49所示；和/或
(c)重链和轻链，其中，重链的序列如SEQ ID NO：65所示，轻链的序列如SEQ ID NO：66所示；
L为MC-vc-PAB；和
D为MMAE。
在本发明的第二方面，本发明提供了组合物，其包含前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。
在一些实施方案中，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、紫杉烷类【如紫杉醇(Taxol)、多西他赛(Taxotere)】、卡培他滨(Xeloda)、吉西他滨(Gemzar)、长春瑞滨(Navelbine)、他莫昔芬、芳香酶抑制剂(瑞宁得、弗隆、阿诺新)、5-FU加亚叶酸、伊立替康(camptosar)、奥沙利铂、顺铂、卡铂、雌莫司汀、米托蒽醌(Novantrone)、泼尼松、长春新碱(Oncovin)、多柔比星、强的松等，或它们的组合。
在一些实施方案中，所述组合物中还包含已知的用于治疗肿瘤的免疫治疗药物，所述免疫治疗药物例如为PD-1单抗(例如帕博利珠单抗、纳武利尤单抗)、PD-L1单抗(例如Atezolizumab)、TIGIT单抗,4-1BB单抗，VEGFR2单抗(例如Ramucirumab、阿帕替尼)、HER2单抗(例如曲妥珠单抗、Trastuzumab biosimilar、Trastuzumab-dkst)等，或它们的组合。
在一些实施方案中，所述组合物中还包含免疫抑制剂，所述免疫抑制剂选自：(1)糖皮质激素类，如可的松和强的松；(2)微生物代谢产物，如环孢菌素和藤霉素等；(3)抗代谢物，如硫唑嘌呤和6-巯基嘌呤等；(4)多克隆和单克隆抗淋巴细胞抗体，如抗淋巴细胞球蛋白和OKT3等；(5)烷化剂类，如环磷酰胺。具体地，所述免疫抑制剂例如为甲基强的松龙，强的松，硫唑嘌呤，普乐可复，赛尼哌，舒莱，环孢菌素，他克莫司，雷帕霉素，霉酚酸酯，咪唑立宾，环磷酰胺，芬戈莫德等。
在一些实施方案中，所述组合物还包含药学上可接受的载体、稀释剂或赋形剂。
在本发明的第三方面，本发明提供了前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或者前述的组合物在制备药物中的用途，所述药物用于预防和/或治疗与Claudin 18.2相关的疾病。
在一些实施方案中，所述与Claudin 18.2相关的疾病为胃癌、胃食管交界腺癌、胰腺癌。
在一些实施方案中，所述与Claudin 18.2相关的疾病为胃癌。
在本发明的第四方面，本发明提供了预防和/或治疗与Claudin 18.2相关的疾病的方法，其包括：给予有需要的受试者预防和/或治疗有效量的前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，或者前述的组合物。
在一些实施方案中，所述与Claudin 18.2相关的疾病为胃癌、胃食管交界腺癌、胰腺癌。
在一些实施方案中，所述与Claudin 18.2相关的疾病为胃癌。
在本发明的第五方面，本发明提供了前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，或者前述的组合物，其用于预防和/或治疗与Claudin 18.2相关的疾病。
在一些实施方案中，所述与Claudin 18.2相关的疾病为胃癌、胃食管交界腺癌、胰腺癌。
在一些实施方案中，所述与Claudin 18.2相关的疾病为胃癌。
附图说明
图1是本发明实施例的RT-PCR显示HEK293稳转细胞株分别表达紧密连接蛋白18.1和紧密连接蛋白18.2；HEK293-紧密连接蛋白18.2稳转细胞株和对照KATO III细胞均可扩增出紧密连接蛋白18.2特异性的780bp特征条带，而HEK293-紧密连接蛋白18.1只能扩增出504bp的共同片段；
图2是本发明实施例的FACS实验筛选HEK293-紧密连接蛋白18.2高表达稳转细胞株的结果，其中黑色点为阴性对照，灰色点为HEK293-紧密连接蛋白18.2高表达稳转细胞株；
图3是本发明实施例的FACS实验筛选NIH3T3-紧密连接蛋白18.2高表达稳转细胞株的结果，其中黑色的线为阴性对照，灰色的阴影为3T3-紧密连接蛋白18.2稳转细胞株；NO.32-H为高表达的3T3-紧密连接蛋白18.2稳转细胞株，NO.18-M为中表达的3T3-紧密连接蛋白18.2稳转细胞株，NO.6-L为低表达的3T3-紧密连接蛋白18.2稳转细胞株；
图4是本发明实施例的Anti-Claudin18.2抗体ADCC效应结果图；
图5是本发明实施例的Anti-Claudin18.2抗体CDC效应结果图；
图6是本发明实施例的抗体药物偶联物疏水作用色谱图(HIC)；
图7是本发明实施例的不同CM311ADC在LT-M11细胞株中的细胞杀伤作用结果图；
图8是本发明实施例的CM311-ADC-1和对照CM311-ADC-2对人胃癌裸小鼠PDX模型STO#025肿瘤生长抑制活性结果图；
图9是本发明实施例的CM311-ADC-1和对照CM311-ADC-2对人胃癌裸小鼠PDX模型STO#025的动物体重的影响结果图；
图10是本发明实施例的CM311-ADC-1和对照CM311-ADC-2对人胃癌裸小鼠PDX模型STO#523肿瘤生长抑制活性结果图；
图11是本发明实施例的CM311-ADC-1和对照CM311-ADC-2对人胃癌裸小鼠PDX模型STO#523的动物体重的影响结果图；
图12是本发明实施例的抗体药物偶联物与抗体的体外细胞活性比较结果图(QC Log变换独立拟合图)。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员 所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
Claudin蛋白是构成紧密连接结构的骨架蛋白，位于相邻细胞间隙顶侧，其分布具有组织器官特异性，功能主要为细胞间黏附、维持细胞极性、调节细胞旁通透性及参与细胞增殖、分化的调节。在肿瘤中，细胞间的紧密连接遭到破坏，Claudin无法发挥其正常功能。
Claudin l8是Claudin家族成员，具有2种不同的第一个外显子，因此通过选择性剪切可以产生两种亚型：Claudin l8.1和Claudin 18.2，这两种亚型分别在不同的组织中进行转录扩增，其中，Claudin l8.1主要表达在肺组织，而Claudin l8.2则特异性表达于胃组织。Claudin 18.2(GenBank登录号：NM_001002026.3)在除了胃黏膜之外的其他正常组织都不表达，但其在多种肿瘤中有显著上调，包括80％的胃肠道腺瘤、60％的胰腺肿瘤，以及部分胆管、卵巢和肺部等的肿瘤中。
术语“Claudin 18.2相关的疾病”指的是组织细胞中Claudin 18.2的表达不同于(如超过)正常水平的疾病。例如，某组织细胞中Claudin 18.2的表达水平高于参照或对照(即正常组织细胞)中Claudin 18.2的表达水平，则指示所述组织细胞所来源的对象(特别是人)中Claudin 18.2相关疾病的存在。
在本发明中，除非另有说明，否则任何数值范围应理解为包括范围内的任何值或任何子范围。
在本发明中，术语“抗体”是指通常由两对相同的多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体的轻链可分为κ和λ两类。重链可分为μ、δ、γ、α或ε五种，依据重链的不同可将抗体分为IgM、IgD、IgG、IgA和IgE五类。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(V H)和重链恒定区(C H)组成。重链恒定区由3个结构域(C H1、C H2和C H3)组成。各轻链由轻链可变区(V L)和轻链恒定区(C L)组成。轻链恒定区由一个结构域C L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。V H和V L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V H和V L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(V H和V L)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。
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，所述氨基酸序列的突变体指的是与所述氨基酸序列的同一性大于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个取代、缺失或添加氨基酸的序列。优选的是，取代、添加或缺失的氨基酸不超过3个氨基酸。更优选的是，取代、添加或缺失的氨基酸不超过2个氨基酸。最优选的是，取代、添加或缺失的氨基酸不超过1个氨基酸。
“取代型”变体是天然序列中至少一个氨基酸残基被除去并被不同的氨基酸插入其相同位置的变体。所述取代可为单个的，其中该分子中仅有一个氨基酸被取代；或可为多个的，其中该相同分子有两个或更多的氨基酸被取代。多个取代可位于连续的位点。同样，一个氨基酸可被多个残基取代，其中这样的变体包括取代和插入二者。“插入型”(或“添加型”)变体是一个或多个氨基酸被插入到紧邻一段天然序列某个特定位置处的氨基酸的变体。紧邻氨基酸意指与该氨基酸的α-羧基或α-氨基官能团连接。“缺失型”变体是天然氨基酸序列中一个或多个氨基酸被除去的变体。通常情况下，缺失型变体在其分子的特定区域内有一个或两个氨基酸被缺失。
在某些实施方案中，在偶联反应中少于理论最大值的药物模块偶联至抗体。一般而言，抗体不包含许多游离的和反应性的半胱氨酸硫醇基，其可连接药物模块；事实上，抗体中的大多数半胱氨酸硫醇基以二硫桥形式存在。在某些实施方案中，可以在部分或完全还原性条件下用还原剂诸如二硫苏糖醇(DTT)或三羰基乙基膦(TCEP)还原抗体以产生反应性半胱氨酸硫醇基。
在一些实施方案中，所述药学上可接受的盐为无机酸盐或有机酸盐，其中，所述无机酸盐为盐酸盐、氢溴酸盐、氢碘酸盐、硝酸盐、碳酸氢盐和碳酸盐、硫酸盐或磷酸盐，所述有机酸盐为甲酸盐、乙酸盐、丙酸盐、苯甲酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、柠檬酸盐、抗坏血酸盐、α-酮戊二酸盐、α-甘油磷酸盐、烷基磺酸盐或芳基磺酸盐；优选地，所述烷基磺酸盐为甲基磺酸盐或乙基磺酸盐；所述芳基磺酸盐为苯磺酸盐或对甲苯磺酸盐。
药学上可以接受的盐可使用本领域熟知的标准程序获得，例如，通过将足量的碱性化合物和提供药学上可以接受的阴离子的合适的酸反应。
如本文所使用，除非另外说明，术语“前药”是指可以在生物学条件(体外或体内)下水解、氧化或进行其他反应以提供本发明的化合物的衍生物。前药仅在生物学条件下经过该反应成为活性化合物，或者它们在它们不反应的形式中具有活性。通常可以使用公知的方法制备前药，例如Burger's Medicinal Chemistry and Drug Discovery(1995)172-178,949-982(Manfred E.Wolff编,第5版)中描述的那些方法。
在本发明中，溶剂合物表示这些形式的本发明的抗体药物偶联物：所述抗体药物偶联物通过与溶剂分子配位而形成的固态或液态形式的复合物。水合物是溶剂合物的一种具体形式，其具有配位的水分子。在本发明中，水合物是优选的溶剂合物。
制备各种含有一定量的活性成分的药物组合物的方法是已知的，或根据本发明的公开内容对于本领域技术人员是显而易见的。如REMINGTON’S PHARMACEUTICAL SCIENCES,Martin,E.W.,ed.,Mack Publishing Company,19th ed.(1995)所述，制备所述药物组合物的方法包括掺入适当的药学赋形剂、载体、稀释剂等，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。
本发明的药物组合物可以包含pH缓冲水溶液。或者包含，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TWEEN TM、聚乙二醇(PEG)和PLURONICS TM。
以已知的方法制造本发明的药物制剂，包括常规的混合、溶解或冻干方法。本发明的化合物可以制成药物组合物，并向患者以适于选定的施用方式的各种途径施用，例如，口 服或肠胃外(通过静脉内、肌内、局部或皮下途径)。
因此，本发明的化合物结合药学上可以接受的载体(如惰性稀释剂或可同化的可食用的载体)可以全身施用，例如，口服。它们可以封闭在硬或软壳的明胶胶囊中，可以压为片剂。对于口服治疗施用，活性化合物可以结合一种或多种赋形剂，并以可吞咽的片剂、颊含片剂、含片、胶囊剂、酏剂、悬浮剂、糖浆、圆片等的形式使用。这种组合物和制剂应该包含至少0.1％的活性化合物。这种组合物和制剂的比例当然可以变化，可以占给定的单位剂型重量的大约1％至大约99％。在这种治疗有用的组合物中，活性化合物的量使得能够获得有效剂量水平。
片剂、含片、丸剂、胶囊剂等也可以包含：粘合剂，如黄蓍胶、阿拉伯胶、玉米淀粉或明胶；赋形剂，如磷酸氢二钙；崩解剂，如玉米淀粉、马铃薯淀粉、藻酸等；润滑剂，如硬脂酸镁；和甜味剂，如蔗糖、果糖、乳糖或阿司帕坦；或调味剂，如薄荷、冬青油或樱桃香味。当单位剂型是胶囊时，除了上面类型的材料，它还可以包含液体载体，如植物油或聚乙二醇。各种其他材料可以存在，作为包衣，或以其他方式改变固体单位剂型的物理形式。例如，片剂、丸剂或胶囊剂可以用明胶、蜡、虫胶或糖等包衣。糖浆或酏剂可以包含活性化合物，蔗糖或果糖作为甜味剂，对羟苯甲酸甲酯或对羟苯甲酸丙酯作为防腐剂，染料和调味剂(如樱桃香料或桔子香料)。当然，用于制备任何单位剂型的任何材料应该是药学上可以接受的且以应用的量基本上无毒。此外，活性化合物可以掺入缓释制剂和缓释装置中。
活性化合物也可以通过输注或注射来静脉内或腹膜内施用。可以制备活性化合物或其盐的水溶液，任选地混和无毒的表面活性剂。也可以制备在甘油、液体聚乙二醇、甘油三乙酸酯及其混合物以及油中的分散剂。在普通的储存和使用条件下，这些制剂包含防腐剂以防止微生物生长。
适于注射或输注的药物剂型可以包括包含适于无菌的可注射或可输注的溶液或分散剂的即时制剂的活性成分(任选封装在脂质体中)的无菌水溶液或分散剂或无菌粉末。在所有情况下，最终的剂型在生产和储存条件下必须是无菌的、液体的和稳定的。液体载体可以是溶剂或液体分散介质，包括，例如水、乙醇、多元醇(例如，甘油、丙二醇、液体聚乙二醇等)、植物油、无毒的甘油酯及其合适的混合物。可以维持合适的流动性，例如，通过脂质体的形成，通过在分散剂的情况下维持所需的粒子大小，或通过表面活性剂的使用。可以通过各种抗细菌剂和抗真菌剂(如对羟苯甲酸酯、氯丁醇、苯酚、山梨酸、硫柳汞等)产生预防微生物的作用。在许多情况下，优选包括等渗剂，如糖、缓冲剂或氯化钠。通过使用延缓吸收剂的组合物(例如，单硬脂酸铝和明胶)可以产生可注射的组合物的延长吸收。
通过将合适的溶剂中的需要量的活性化合物与需要的上面列举的各种其他成分结合，然后进行过滤灭菌，制备无菌可注射溶液。在用于制备无菌注射溶液的无菌粉末的情况下，优选的制备方法是真空干燥和冷冻干燥技术，这会产生活性成分加上任何另外需要的以前无菌过滤溶液中存在的成分的粉末。
有用的固体载体包括粉碎的固体(如滑石、粘土、微晶纤维素、二氧化硅、氧化铝等)。有用的液体载体包括水、乙醇或乙二醇或水-乙醇/乙二醇混合物，本发明的化合物可以任选在无毒的表面活性剂的帮助下以有效含量溶解或分散在其中。可以加入佐剂(如香味)和另外的抗微生物剂来优化对于给定用途的性质。
增稠剂(如合成的聚合物、脂肪酸、脂肪酸盐和酯、脂肪醇、改性纤维素或改性无机材料)也可和液体载体用于形成可涂覆的糊剂、凝胶、软膏、肥皂等，直接用于使用者的皮肤上。
上述制剂可以以单位剂型存在，该单位剂型是含有单位剂量的物理分散单元，适于向人体和其它哺乳动物体给药。单位剂型可以是胶囊或片剂，或是很多胶囊或片剂。根据所涉及的具体治疗，活性成分的单位剂量的量可以在大约0.1到大约1000毫克或更多之间 进行变化或调整。
此外，还包括各种药物新剂型如乳脂质体、微球和纳米球的应用，如使用微粒分散体系包括聚合物胶束(polymeric micelles)、纳米乳(nanoemulsion)、亚微乳(submicroemuls微囊(microcapsule)、微球(microsphere)、脂质体(liposomes)和类脂囊泡(niosomes)(又称非离子表面活性剂囊泡)等制备的药剂。
本文使用的术语“治疗”一般是指获得需要的药理和/或生理效应。该效应根据完全或部分地预防疾病或其症状，可以是预防性的；和/或根据部分或完全稳定或治愈疾病和/或由于疾病产生的副作用，可以是治疗性的。本文使用的“治疗”涵盖了对患者疾病的任何治疗，包括：(a)预防易感染疾病或症状但还没诊断出患病的患者所发生的疾病或症状；(b)抑制疾病的症状，即阻止其发展；或(c)缓解疾病的症状，即，导致疾病或症状退化。
在本发明中，“受试者”指脊椎动物。在某些实施方案中，脊椎动物指哺乳动物。哺乳动物包括，但不限于，牲畜(诸如牛)、宠物(诸如猫、犬、和马)、灵长类动物、小鼠和大鼠。在某些实施方案中，哺乳动物指人。
在本发明中，“有效量”指在必需的剂量和时间上有效实现期望的治疗或预防效果的量。本发明的物质/分子的“治疗有效量”可根据诸如个体的疾病状态、年龄、性别和体重及该物质/分子在个体中引发期望应答的能力等因素而变化。治疗有效量还涵盖该物质/分子的治疗有益效果胜过任何有毒或有害后果的量。“预防有效量”指在必需的剂量和时间上有效实现期望的预防效果的量。通常而非必然，由于预防剂量是在疾病发作之前或在疾病的早期用于受试者的，因此预防有效量会低于治疗有效量。在癌症的情况中，药物的治疗有效量可减少癌细胞数；缩小肿瘤体积；抑制(即一定程度的减缓，优选停止)癌细胞浸润到周围器官中；抑制(即一定程度的减缓，优选停止)肿瘤转移；一定程度的抑制肿瘤生长；和/或一定程度的减轻与癌症有关的一种或多种症状。
在本发明中，20种常规氨基酸和其缩写遵从常规用法。参见Immunology-A Synthesis(第2版,E.S.Golub和D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991)),其通过引用合并入本文。
术语“嵌合抗体”是指这样的抗体，其中可变区序列源自一个物种，恒定区序列源自另一物种，如其中可变区序列源自小鼠抗体及恒定区序列源自人抗体的抗体。
“人源化”抗体是指非人(例如小鼠)抗体形式，其是嵌合的免疫球蛋白、免疫球蛋白链或者其片段(如Fv、Fab、Fab'、F(ab')2或者抗体的其它抗原结合亚序列)，含有源自非人免疫球蛋白的最小序列。优选地，人源化抗体是人免疫球蛋白(接受者抗体)，其中接受者抗体的互补决定区(CDR)的残基由来自具有希望的特异性、亲和性和能力的非人物种(供体抗体)如小鼠、大鼠或者兔的CDR残基置换。
此外，在人源化中，还可能对VH和/或VL的CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，由此改善抗体的一或多种结合特性(例如亲和性)。可进行例如PCR介导的突变引入突变，其对抗体结合或其它功能特性的影响可利用本文所述的体外或体内测试评估。通常，引入保守性突变。此类突变可为氨基酸取代、添加或缺失。另外，CDR内的突变通常不超过一个或两个。因此，本发明所述人源化抗体还涵盖CDR内包含1或2两个氨基酸突变的抗体。
下面结合具体实施例对本发明进行进一步的解释说明，但这些实施例并非限制本发明的范围。
实施例1 人源化抗体的制备
1、抗紧密连接蛋白18.2单克隆抗体的制备
多种策略同时应用于免疫Balb/c小鼠和筛选，以获得特异性只结合紧密连接蛋白18.2，不结合紧密连接蛋白18不同剪接体紧密连接蛋白18.1的单克隆抗体，具体如下：
1)构建紧密连接蛋白18.2稳转细胞株
合成含人紧密连接蛋白18.1(UniProtKB-P56856)全长序列的质粒基因紧密连接蛋白18.1-puc57-Amp(苏州泓迅生物，SynbioTech)。以该质粒为模板，上游引物5’-ttggcaaagaattgctagatgtccaccaccacatgcc-3’(SEQ ID NO：171)，下游引物5’-tgttcgggccctcctcgattacacatagtcgtgcttgg-3’(SEQ ID NO：172)，PCR扩增人紧密连接蛋白18.1全长片段(Met1-Val261)。扩增产物经NEBuilder HiFi DNA Assembly Master Mix(NEB，Cat：M0530L)酶连后，克隆到真核表达质粒系统。以此类似，合成含人紧密连接蛋白18.2(UniProtKB-P56856-2)全长序列的质粒基因紧密连接蛋白18.2-puc57-Amp(苏州泓迅生物，SynbioTech)。以该质粒为模板，上游引物5’-ttggcaaagaattgctagatggccgtgactgcctgtc-3’(SEQ ID NO：173)，下游引物5’-tgttcgggccctcctcgattacacatagtcgtgcttgg-3’(SEQ ID NO：174)，PCR扩增人紧密连接蛋白18.2全长片段(Met1-Val261)。扩增产物经NEBuilder HiFi DNA Assembly Master Mix(NEB，Cat：M0530L)酶连后，克隆到真核表达质粒系统。以此质粒分别电转NIH3T3和HEK293细胞，使用1-10μg/mL Puromycin(Gibco，Cat：A1113803)进行逐步加压筛选稳定表达细胞系。
得到的HEK293-紧密连接蛋白18.1和HEK293-紧密连接蛋白18.2稳转细胞系首先用RT-PCR方法进行验证。用Trizol RNA提取试剂盒从阳性克隆细胞中提取总RNA，用逆转录试剂盒(SuperScriptTM First-Strand Synthesis System，Cat：18080051)，利用Oligo(dT)引物逆转录得到cDNA文库。由于紧密连接蛋白的2个剪接片段紧密连接蛋白18.1和紧密连接蛋白18.2区别在于氮端到第一个胞外区(Loop1)，设计引物KNB14(5’-tgtgcgccaccatggccgtg-3’(SEQ ID NO：175))，KNB15(5’-tggaaggataagattgtacc-3’(SEQ ID NO：176))，可以扩增紧密连接蛋白18.1和紧密连接蛋白18.2的Loop1之后到碳端之间区域(504bp)；设计引物KNB16(5’-tgggtgccattggcctcctg-3’(SEQ ID NO：177))，特异性互补结合紧密连接蛋白18.2氮端，不结合紧密连接蛋白18.1氮端区域，只扩增紧密连接蛋白18.2的氮端到碳端之间全长片段(780bp)，以表达紧密连接蛋白18.2的KATO III细胞(ATCC HTB-103)作为阳性对照。结果见图1，RT-PCR显示HEK293稳转细胞株分别表达紧密连接蛋白18.1和紧密连接蛋白18.2。HEK293-紧密连接蛋白18.2稳转细胞株和对照KATO III细胞均可扩增出紧密连接蛋白18.2特异性的780bp特征条带，而HEK293-紧密连接蛋白18.1只能扩增出504bp的共同片段。
将构建的稳转细胞株HEK293-紧密连接蛋白18.2细胞和NIH3T3-紧密连接蛋白18.2细胞消化收集，PBS洗2次，每管加入100μL 1:200稀释的一抗兔抗紧密连接蛋白18.2(Abcam，EPR19202，货号：ab222512)，4℃孵育60分钟，用0.5％BSA/PBS洗去多余的一抗溶液，再加入50μL二抗山羊抗兔IgGFc-AF647(Jackson ImmunoResearch，货号：111-606-046)，4℃孵育45分钟，之后用0.5％BSA/PBS洗去多余的二抗，最后用100μL PBS溶液重悬细胞，立即上流式细胞仪器检测。结果见图2、图3。
图2显示了通过FACS实验，检测转染紧密连接蛋白18.2全长基因的HEK293-紧密连接蛋白18.2稳转细胞株的结果。其中黑色点为未转染的HEK293细胞，灰色点为HEK293-紧密连接蛋白18.2稳转细胞株，HEK293细胞不表达紧密连接蛋白18.2，HEK293-紧密连接蛋白18.2稳转株在细胞膜表面高表达紧密连接蛋白18.2。
图3显示了通过FACS实验，检测紧密连接蛋白18.2在NIH3T3-紧密连接蛋白18.2高表达稳转细胞株的结果。其中黑色的线为阴性对照，灰色的阴影为3T3-紧密连接蛋白18.2稳转细胞株。NO.32-H为紧密连接蛋白18.2高表达的3T3-紧密连接蛋白18.2稳转细胞株，NO.18-M为紧密连接蛋白18.2中表达的3T3-紧密连接蛋白18.2稳转细胞株，NO.6-L为紧密连接蛋白18.2低表达的3T3-紧密连接蛋白18.2稳转细胞株。
将扩增生长的阳性稳转细胞系收集冻存，其中NIH3T3-紧密连接蛋白18.2稳转细胞株用于免疫动物。
2)从杂交瘤制备抗紧密连接蛋白18.2单克隆抗体
取6～8周雌性Balb/c小鼠，以3T3-紧密连接蛋白18.2稳转细胞或编码紧密连接蛋白18.2的质粒分别进行免疫，或交替进行免疫。用细胞进行免疫时，每次取1×10 6 3T3-紧密连接蛋白18.2稳转细胞与弗氏佐剂或非弗氏佐剂混合后，注射大腿根部和足垫，两周后在不同部位再次免疫。采用DNA进行免疫时，取20μg质粒与1μg CpG混合后，用基因枪(Biorad)40psi直接打入小鼠腹部，每周免疫一次。准备融合前3天，采用HEK293-紧密连接蛋白18.2高表达的稳转细胞株1×10 6细胞/50μL/只，进行尾静脉注射冲击免疫。三天后处死小鼠，收集腘淋巴结、腹股沟淋巴结、髂淋巴结，在DMEM中研磨得到富含B细胞的悬浮液；取出小鼠脾脏，在DMEM中研磨后离心，得到脾细胞悬浮液。取适量的淋巴结和脾细胞的混悬液和SP2/0混合后，采用电融合仪进行细胞融合。
3)构建抗紧密连接蛋白18.2噬菌体抗体库
将收集的部分小鼠脾脏与外周淋巴结细胞悬液，用Trizol RNA提取试剂盒提取总RNA，用逆转录试剂盒(SuperScript First-Strand Synthesis System，货号18080051)，利用轻、重链特异性引物逆转录分别得到抗体轻、重链cDNA文库。以该cDNA为模板，利用轻、重链可变区引物PCR扩增抗体轻、重链可变区片段，酶切克隆到含有人抗体轻链恒定区Ckappa或人IgG1重链恒定区CH1的噬菌体质粒载体，分别形成嵌合体轻链库和重链库。将轻链库中的抗体片段以BspQI和SfiI双酶切后，连接进入重链库，形成基于丝状噬菌体M13的小鼠嵌合体Fab噬菌体展示文库，库容为1.2×10 10。取0.8mL噬菌体(滴度约1x10 13/mL)与200μL 5％BSA/PBS混合后，加入1x10 7HEK293-紧密连接蛋白18.1细胞，冰浴1小时；1000rpm离心10分钟，收集上清，将上清与1x10 6HEK293-紧密连接蛋白18.2细胞混合，冰浴1小时，1000rpm离心3分钟，弃上清；加入1mL 1％BSA/PBS，反复洗涤5-10次；加入1mL 100mM TEA(三乙胺)裂解细胞，室温10分钟，加入0.5mL 1M Tris-HCl,pH7.5中和后，感染10mL处于对数生长期的TG1E.coli.细胞，37℃静置30分钟。按照分子生物学常规流程，回收噬菌体，检测滴度并进行下一轮筛选。
2、抗紧密连接蛋白18.2特异性抗体的筛选和序列获取
将HEK293-紧密连接蛋白18.1、HEK293-紧密连接蛋白18.2和HEK293分别以5μM、0.5μM和0μM Cell Tracker Green CMFDA Dye(Thermo，货号C2925)按说明进行活细胞预染，洗去染料后，按1:1:1混合，加至96孔板(2x10 5细胞/孔)，并与杂交瘤上清或细菌诱导上清结合，冰浴孵育1小时后，加入AlexaFluro647标记的二抗抗mouse IgG Fc或抗人IgG F(ab)’2(Jackson ImmunoResearch)，冰上孵育45分钟。洗涤后，每孔加入100μL PBS重悬细胞，准备流式细胞仪(iQue Screener)，根据FL2通道荧光强度区别，分别圈出三种不同的细胞群后，检测FL4通道待测抗体与各细胞群的结合情况。筛选得到的抗体以高亲和力、特异性结合HEK293-紧密连接蛋白18.2稳转细胞，同时不结合HEK293-紧密连接蛋白18.1稳转细胞及HEK293细胞。从杂交瘤细胞中，通过FACS检测，共筛选出320个克隆可以结合紧密连接蛋白18.2，同时不结合紧密连接蛋白18.1；经过3轮panning，从噬菌体Fab库中，筛选得到62个高亲和力结合紧密连接蛋白18.2但不结合紧密连接蛋白18.1的克隆。
从噬菌体库筛选得到的阳性克隆，提取质粒进行测序，可变区序列分别克隆进入重链和轻链恒定区载体进行全长IgG表达。从杂交瘤得到的阳性细胞中，加入1mL TRNzol进行裂解，以异硫氢酸胍法提取总RNA。以此为模板，合成第一链cDNA后，以第一链cDNA为后续模板扩增杂交瘤细胞所对应的可变区DNA序列。将扩增产物测序后，得到候选杂交瘤重链和轻链可变区序列，如下所示。
克隆18D10：
重链
Figure PCTCN2021124698-appb-000003
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：140)、CDR2(SEQ ID NO：141)、CDR3(SEQ ID NO：142)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：143)、FR2(SEQ ID NO：144)、FR3(SEQ ID NO：145)、FR4(SEQ ID NO：146)。
核酸序列
Figure PCTCN2021124698-appb-000004
轻链
Figure PCTCN2021124698-appb-000005
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：148)、CDR2(SEQ ID NO：149)、CDR3(SEQ ID NO：150)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：151)、FR2(SEQ ID NO：152)、FR3(SEQ ID NO：153)、FR4(SEQ ID NO：154)。
核酸序列
Figure PCTCN2021124698-appb-000006
克隆18A9：
重链
Figure PCTCN2021124698-appb-000007
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：156)、CDR2(SEQ ID NO：157)、CDR3(SEQ ID NO：158)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：159)、FR2(SEQ ID NO：160)、FR3(SEQ ID NO：161)、FR4(SEQ ID NO：162)。
核酸序列
Figure PCTCN2021124698-appb-000008
轻链
Figure PCTCN2021124698-appb-000009
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：164)、CDR2(SEQ ID NO：165)、CDR3(SEQ ID NO：166)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：167)、FR2(SEQ ID NO：168)、FR3(SEQ ID NO：169)、FR4(SEQ ID NO：170)。
核酸序列
Figure PCTCN2021124698-appb-000010
Figure PCTCN2021124698-appb-000011
上述重链和轻链可变区序列片段PCR扩增，将重链可变区克隆入含有人重链恒定区的载体，以在哺乳动物细胞中表达完整的IgG1重链。类似地，将轻链可变区克隆入含有人轻链恒定区的载体，以在哺乳动物细胞中表达完整的kappa轻链。经测序正确后转染入HEK293-6E哺乳动物细胞中，IgG1经表达分泌入培养基中，合并收集上清，过滤后纯化。采用Protein A层析纯化IgG，将培养上清液加载于大小合适的Protein A柱子上，用50mM Tris-HCl pH8.0，250mM NaCl洗涤，用0.1M Glycine-HCl，pH3.0将结合的IgG洗脱下来。利用浓缩管(Millipore)将蛋白超滤浓缩，检测OD280，通过分光光度法测定IgG的浓度。采用SDS-PAGE分析IgG的纯度。
取对数生长期的HEK293-紧密连接蛋白18.1细胞、HEK293-紧密连接蛋白18.2细胞和HEK293细胞，消化后以5×10 4细胞/100μL加入U型96孔板，1100rpm离心3分钟，弃上清，轻轻拍散细胞，每孔加入50μL梯度稀释的抗体(抗体浓度从100nM起，5倍稀释8个梯度)，4℃孵育1小时。孵育结束后，每孔加入140μL 0.5％BSA洗3次，加30μL/孔二抗AlexaFluro647抗人IgG(Jackson ImmunoResearch，货号：109-606-170)，4℃孵育40分钟。孵育结束后，每孔加入140μL 0.5％BSA洗3次，最后每孔重悬于50μL PBS进行流式细胞仪(iQue Screener)检测。结果如表1所示，结果显示，得到的嵌合体紧密连接蛋白18.2IgG1抗体只识别转染了紧密连接蛋白18.2的HEK293-紧密连接蛋白18.2细胞，但与HEK293和HEK293-紧密连接蛋白18.1无结合。
表1：紧密连接蛋白18.2抗体与稳转细胞株的结合(N.B.表示未检测到结合)
Figure PCTCN2021124698-appb-000012
3、抗紧密连接蛋白18.2抗体的人源化
把选择的单克隆抗体可变区序列与人胚系抗体序列进行比对，找出同源性高的序列进行CDR移植；随后同时利用计算机进行同源建模，分析CDR区及其周边的框架氨基酸序列，考察其空间立体结合方式。通过计算静电力，范德华力，亲疏水性和熵值，分析各阳性单克隆抗体基因序列中可能与靶点相互作用以及维护空间构架的关键氨基酸个体，在此基础上设计回复突变位点。分析考察HLA-DR亲和性，选出免疫原性较低的人胚胎系框架序列。分析可能在发酵过程中发生修饰的氨基酸残基，并设计突变以降低发生修饰的可能性。
设计共得到不同的重链衍生物和轻链衍生物，将轻、重链衍生物分别进行全序列合成后，克隆到含有抗体kappa链恒定区Ckappa或人IgG1恒定区CH1-CH3的载体，同样母本来源的轻、重链衍生物质粒进行组合配对后，转染HEK293.6E细胞，表达5-6天，收取上清Protein A柱纯化。
人源化抗体的序列如下：
18D10：
重链可变区：
18D10VHv1：
Figure PCTCN2021124698-appb-000013
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：2)、CDR2(SEQ ID NO：3)、CDR3(SEQ ID NO：4)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：5)、FR2(SEQ ID NO：6)、FR3(SEQ ID NO：7)、FR4(SEQ ID NO：8)。
核酸序列
Figure PCTCN2021124698-appb-000014
18D10VHv2：
Figure PCTCN2021124698-appb-000015
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：10)、CDR2(SEQ ID NO：11)、CDR3(SEQ ID NO：12)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：13)、FR2(SEQ ID NO：14)、FR3(SEQ ID NO：15)、FR4(SEQ ID NO：16)。
核酸序列
Figure PCTCN2021124698-appb-000016
18D10VHv3：
Figure PCTCN2021124698-appb-000017
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：18)、CDR2(SEQ ID NO：19)、CDR3(SEQ ID NO：20)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：21)、FR2(SEQ ID NO：22)、FR3(SEQ ID NO：23)、FR4(SEQ ID NO：24)。
核酸序列
Figure PCTCN2021124698-appb-000018
18D10VHv4：
Figure PCTCN2021124698-appb-000019
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：26)、CDR2(SEQ ID NO：27)、CDR3(SEQ ID NO：28)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：29)、FR2(SEQ ID NO：30)、FR3(SEQ ID NO：31)、FR4(SEQ ID NO：32)。
核酸序列
Figure PCTCN2021124698-appb-000020
Figure PCTCN2021124698-appb-000021
18D10VHv5：
Figure PCTCN2021124698-appb-000022
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：34)、CDR2(SEQ ID NO：35)、CDR3(SEQ ID NO：36)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：37)、FR2(SEQ ID NO：38)、FR3(SEQ ID NO：39)、FR4(SEQ ID NO：40)。
核酸序列
Figure PCTCN2021124698-appb-000023
18D10VHv6：
Figure PCTCN2021124698-appb-000024
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：42)、CDR2(SEQ ID NO：43)、CDR3(SEQ ID NO：44)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：45)、FR2(SEQ ID NO：46)、FR3(SEQ ID NO：47)、FR4(SEQ ID NO：48)。
核酸序列
Figure PCTCN2021124698-appb-000025
轻链可变区：
18D10VLv1：
Figure PCTCN2021124698-appb-000026
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：50)、CDR2(SEQ ID NO：51)、CDR3(SEQ ID NO：52)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：53)、FR2(SEQ ID NO：54)、FR3(SEQ ID NO：55)、FR4(SEQ ID NO：56)。
核酸序列
Figure PCTCN2021124698-appb-000027
18D10VLv2：
Figure PCTCN2021124698-appb-000028
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：58)、CDR2(SEQ ID NO：59)、CDR3(SEQ ID NO：60)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：61)、FR2(SEQ ID NO：62)、FR3(SEQ ID NO：63)、FR4(SEQ ID NO：64)。
核酸序列
Figure PCTCN2021124698-appb-000029
优选的人源化抗体的序列如下：
重链氨基酸序列：
Figure PCTCN2021124698-appb-000030
重链核酸序列
Figure PCTCN2021124698-appb-000031
轻链氨基酸序列：
Figure PCTCN2021124698-appb-000032
轻链核酸序列
Figure PCTCN2021124698-appb-000033
Figure PCTCN2021124698-appb-000034
18A9：
重链可变区：
18A9VHv1：
Figure PCTCN2021124698-appb-000035
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：68)、CDR2(SEQ ID NO：69)、CDR3(SEQ ID NO：70)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：71)、FR2(SEQ ID NO：72)、FR3(SEQ ID NO：73)、FR4(SEQ ID NO：74)。
核酸序列
Figure PCTCN2021124698-appb-000036
18A9VHv2：
Figure PCTCN2021124698-appb-000037
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：76)、CDR2(SEQ ID NO：77)、CDR3(SEQ ID NO：78)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：79)、FR2(SEQ ID NO：80)、FR3(SEQ ID NO：81)、FR4(SEQ ID NO：82)。
核酸序列
Figure PCTCN2021124698-appb-000038
18A9VHv3：
Figure PCTCN2021124698-appb-000039
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：84)、CDR2(SEQ ID NO：85)、CDR3(SEQ ID NO：86)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：87)、FR2(SEQ ID NO：88)、FR3(SEQ ID NO：89)、FR4(SEQ ID NO：90)。
核酸序列
Figure PCTCN2021124698-appb-000040
18A9VHv4：
Figure PCTCN2021124698-appb-000041
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：92)、CDR2(SEQ ID NO：93)、CDR3(SEQ ID NO：94)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：95)、FR2(SEQ ID NO：96)、FR3(SEQ ID NO：97)、FR4(SEQ ID NO：98)。
核酸序列
Figure PCTCN2021124698-appb-000042
18A9VHv5：
Figure PCTCN2021124698-appb-000043
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：100)、CDR2(SEQ ID NO：101)、CDR3(SEQ ID NO：102)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：103)、FR2(SEQ ID NO：104)、FR3(SEQ ID NO：105)、FR4(SEQ ID NO：106)。
核酸序列
Figure PCTCN2021124698-appb-000044
18A9VHv6：
Figure PCTCN2021124698-appb-000045
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：108)、CDR2(SEQ ID NO：109)、CDR3(SEQ ID NO：110)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：111)、FR2(SEQ ID NO：112)、FR3(SEQ ID NO：113)、FR4(SEQ ID NO：114)。
核酸序列
Figure PCTCN2021124698-appb-000046
18A9VHv7：
Figure PCTCN2021124698-appb-000047
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：116)、CDR2(SEQ ID NO：117)、CDR3(SEQ ID NO：118)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：119)、FR2(SEQ ID NO：120)、FR3(SEQ ID NO：121)、FR4(SEQ ID NO：122)。
核酸序列
Figure PCTCN2021124698-appb-000048
轻链可变区：
18A9VLv1：
Figure PCTCN2021124698-appb-000049
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：124)、CDR2(SEQ ID NO：125)、CDR3(SEQ ID NO：126)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：127)、FR2(SEQ ID NO：128)、FR3(SEQ ID NO：129)、FR4(SEQ ID NO：130)。
核酸序列
Figure PCTCN2021124698-appb-000050
18A9VLv2：
Figure PCTCN2021124698-appb-000051
其中下划线部分从左向右依次分别为CDR1(SEQ ID NO：132)、CDR2(SEQ ID NO：133)、CDR3(SEQ ID NO：134)；
无下划线部分从左向右依次分别为FR1(SEQ ID NO：135)、FR2(SEQ ID NO：136)、FR3(SEQ ID NO：137)、FR4(SEQ ID NO：138)。
核酸序列
Figure PCTCN2021124698-appb-000052
实施例2 人源化抗体药理学研究
1、人源化抗体的亲和力测定(EC 50)
取对数生长期的细胞，3％BSA封闭30分钟以5×10 4细胞/100μL铺U型96孔板，1100rpm离心3分钟，弃上清，轻轻拍散细胞，每孔加入50μL梯度稀释的抗体(抗体浓度从100nM起，5倍稀释8个梯度)，4℃孵育1小时。孵育结束后，每孔加入140μL 0.5％BSA洗3次，加30μL/孔AF 647APC抗人二抗4℃孵育40分钟。孵育结束后，每孔加入140μL 0.5％BSA洗3次，最后每孔重悬于50μL PBS进行iQue(Intellicyt，USA)检测(见表2)。
表2：人源化抗体的亲和力测定结果
Figure PCTCN2021124698-appb-000053
Figure PCTCN2021124698-appb-000054
2、CM311抗体对肿瘤细胞的ADCC杀伤活性
取30mL鲜血至50mL离心管中，加入15mL 1×PBS，混和均匀，缓慢加入至已加入20mL Ficoll.Paque Plu的离心管中，使血液铺于Ficoll.Paque Plu表面。20℃，2000rpm离心30分钟，弃最上层血清，吸取白膜层(即PBMC)，分装至50mL离心管，每管10mL。每管加入不低于30mL的1×PBS，混合均匀。4℃，1300rpm离心10分钟，弃上清，加入10mL 1×PBS清洗并计数。
FBS/RPMI 1640培养基重悬细胞，37℃，5％CO 2培养2小时。1300rpm离心10分钟，弃上清，FBS/RPMI 1640培养基重悬，接种细胞于U型96孔板中，4*10 5个/孔，50μL/孔。加入前述18D10嵌合体，18A9嵌合体，抗紧密连接蛋白18.2人源化抗体18D10、18A9(即CM311，CM311为人源化抗体18D10或18A9的统称)以及对照抗体anti-KLH的稀释液(40、20、10、5、2.5、1.25μg/mL)，25μL/孔。37℃，5％CO 2孵育30分钟。取出孔板，加入Kato III细胞，8×10 3个/孔，25μL/孔。37℃5％CO 2孵育3.5小时，向靶细胞最大释放孔加入2μL 10*裂解液，37℃5％CO 2继续孵育30分钟。取出孔板，1000rpm离心3分钟，取上清至黑色酶标板，50μL/孔。加入LDH检测底物50μL/孔，室温孵育10分钟，加入终止液25μL/孔，终止反应。酶标仪(Biotek)检测。
结果计算如下：
Figure PCTCN2021124698-appb-000055
结果如图4所示，抗紧密连接蛋白18.2人源化抗体18D10、18A9具有较强的ADCC活性。
3、CM311抗体对肿瘤细胞的CDC细胞杀伤活性
取对数生长期的Kato III细胞重悬至1×10 7个/mL。加入CFSE(Sigma，87444-5MG-F)，终浓度为1μM。室温孵育10分钟，加入3倍体积培养基终止反应。4℃1000rpm离心5分钟，弃上清，培养基重悬，接种细胞至96孔板，1*10 5个/孔，50μL/孔。加入已稀释的前述18D10嵌合体，18A9嵌合体，抗紧密连接蛋白18.2人源化抗体18D10、18A9(CM311)以及对照抗体anti-KLH(稀释后浓度为30、10、3.33、1.11、0.37μg/mL)，50μL/孔。用培养基稀释补体至30％，加入孔板，50μL/孔。37℃，5％CO 2孵育2小时，1000rpm离心3分钟，弃上清。1：200稀释PI染液，与Sulfate latex(Invitrogen，S37227)混匀后加入96孔板中，100μL/孔。冰上孵育10分钟，FACS检测。结果如图5所示，抗紧密连接蛋白18.2人源化抗体18D10、18A9具有较强的CDC效应。
实施例3 抗体药物偶联物的制备和检测
1、抗体药物偶联物的制备
取10毫克的CM311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，共三次置换；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，共三次置换。取样以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 PCTCN2021124698-appb-000056
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即MC-vc-PAB-MMAE(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(即MC-vc-PAB-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组氨酸(Histidine),3％蔗糖(Sucrose),0.03％Tween-80,pH5.5)中，共三次置换，即得到抗体药物偶联物产物Anti-Claudin-18.2-ADC(人源化抗体18D10或18A9制备的抗体药物偶联物的统称)于4℃保存。
2、抗体药物偶联物的DAR测定
抗体药物偶联物的载药量(DAR)是用疏水作用色谱法(HIC-HPLC)检测而得的。
典型的抗体药物偶联物CM311-18D10-VH6/VL1-ADC的图谱见图6。根据该图谱峰面积计算得到平均载药数DAR为3.8。
类似地可得，抗体药物偶联物CM311-18D10-VH3/VL2-ADC的平均载药数DAR为3.5。
抗体药物偶联物CM311-18D10-VH6/VL2-ADC的平均载药数DAR为3.4。
抗体药物偶联物CM311-18A9-VH7/VL2-ADC的平均载药数DAR为3.0。
需要说明的是，以上抗体药物偶联物的名称如CM311-18D10-VH6/VL1-ADC，其表示制备该抗体药物偶联物时使用的抗体为CM311-18D10-VH6/VL1。进一步地，该抗体名称CM311-18D10-VH6/VL1表示制备前述抗体药物偶联物时使用的抗体为18D10这一类中重链可变区为VHv6且轻链可变区为VLv1的抗体，对应上表2可知，制备前述抗体药物偶联物时使用的具体抗体为h18D10.v16。其余三种抗体药物偶联物CM311-18D10-VH3/VL2-ADC、CM311-18D10-VH6/VL2-ADC、CM311-18A9-VH7/VL2-ADC的名称可参照前述进行理解。具体 地，制备抗体药物偶联物CM311-18D10-VH3/VL2-ADC时使用的具体抗体为表2中的h18D10.v23；制备抗体药物偶联物CM311-18D10-VH6/VL2-ADC时使用的具体抗体为表2中的h18D10.v26；制备抗体药物偶联物CM311-18A9-VH7/VL2-ADC时使用的具体抗体为表2中的h18A9.v27。
实施例4 抗体药物偶联物体外药效学研究
复苏后的细胞株经过1-2代细胞传代之后，先吸取上清液至15mL离心管内，离心机离心，弃上清；用5mL PBS润洗细胞培养瓶，再用2mL胰蛋白酶消化细胞，用培养基重悬至之前的15mL离心管内，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(LT-1C8细胞以5000或10000个细胞/孔,LT-M11细胞以5000个细胞/孔，BxPC-3以3000个细胞/孔)，培养24小时后，加入系列稀释浓度的抗体药物偶联物CM311ADC(由不同CM311单抗偶联而成)保温96小时，之后每孔加入CCK-8或者Presto-Blue显色试剂，用酶标仪检测并进行四参数拟合。
实验试剂及来源：
受试药物
Figure PCTCN2021124698-appb-000057
细胞株
Figure PCTCN2021124698-appb-000058
实验结果：
各CM311ADC的IC 50的平均值见表3。图7为不同CM311ADC对LT-M11细胞杀伤的代表性图。
表3：不同CM311ADC在所筛选的细胞株中的IC 50值
Figure PCTCN2021124698-appb-000059
Figure PCTCN2021124698-appb-000060
注：图7中：
Sample 1：CM311-18D10-VH3/VL2-ADC
Sample 2：CM311-18D10-VH6/VL1-ADC
Sample 3：CM311-18D10-VH6/VL2-ADC
Sample 4：CM311-18A9-VH7/VL2-ADC
从表3和图7的结果可以看出，不同CM311ADC在Claudin18.2中、高度表达的细胞株中均表现出显著的细胞杀伤活性，在Claudin18.2阴性的BxPC-3细胞株中无显著细胞杀伤活性。
实施例5 抗体药物偶联物体内药效学研究
CM311ADC的抗肿瘤活性在STO#025和STO#523两个胃癌PDX模型中进行了测试。这两个胃癌模型都有较高的Claudin 18 mRNA水平。
人胃癌裸小鼠PDX模型的建立过程为：将体积大小约为15-30mm 3的肿瘤组织移植于BALB/c裸小鼠背部皮下。当肿瘤体积达到150-260mm 3时，用随机区组法分组，每组5只小鼠，保证各组间肿瘤体积均一并兼顾体重，共4组，包括溶媒组、1mg/kg CM311-ADC-1给药组、3mg/kg CM311-ADC-1给药组和3mg/kg CM311-ADC-2(非结合对照ADC)给药组。其中，CM311-ADC-1指的是抗体药物偶联物CM311-18D10-VH6/VL1-ADC；CM311-ADC-2相比于CM311-ADC-1，抗体是人IgG1同型对照，与肿瘤细胞表面的靶点无结合。
数据分析：实验期间，每周测定两次肿瘤体积。肿瘤体积(Tumor Volume，TV)的计算公式为：TV＝l×w 2/2。其中l、w分别代表肿瘤测量长和宽。根据测量结果计算出相对肿瘤体积(relative tumor volume，RTV)，RTV＝V f/V0。其中V0为分组给药时(即Day0)测量所得肿瘤体积，V f为最后一天测量的肿瘤体积。相对肿瘤增殖率T/C(％)＝(给药组RTV/Vehicle组RTV)×100％。肿瘤生长抑制率TGI％＝(Vehicle组平均肿瘤体积-给药组平均肿瘤体积)/Vehicle组平均肿瘤体积×100％。当T/C(％)≤40％，且P<0.05时认为供试品对肿瘤生长有显著抑制作用。
实验结果：
1)CM311ADC在Claudin 18 mRNA高表达水平的人胃癌PDX模型STO#025中的疗效研究
实验结果如图8和图9所示。CM311-ADC-1按1和3mg/kg剂量给药后，Day 28的相对肿瘤增殖率T/C(％)分别为29.86％和0％，肿瘤生长抑制率TGI％分别为70.13％和100％，分别有0/5和5/5的肿瘤完全消退，以及2/5和0/5的肿瘤部分消退；CM311-ADC-2(3mg/kg)组T/C(％)为67.24％，TGI％为32.76％，实验结果表明，CM311-ADC-1(3mg/kg)和CM311-ADC-1(1mg/kg)均具有显著抑制肿瘤生长的活性，CM311-ADC-2(3mg/kg)无显著抗肿瘤活性。荷瘤小鼠对CM311-ADC-1和CM311-ADC-2均有很好的耐受性。
2)CM311ADC在Claudin 18 mRNA高表达水平的人胃癌PDX模型STO#523中的疗效研究
实验结果如图10和图11所示。CM311-ADC-1按1和3mg/kg剂量给药后，Day 28的相对肿瘤增殖率T/C(％)分别为35.60％和6.79％，肿瘤生长抑制率TGI％分别为64.40％ 和93.21％；CM311-ADC-2(3mg/kg)组T/C(％)为114.81％，TGI％为-14.81％，实验结果表明，CM311-ADC-1(3mg/kg)和CM311-ADC-1(1mg/kg)均具有显著抑制肿瘤生长的活性，CM311-ADC-2(3mg/kg)无显著抗肿瘤活性。荷瘤小鼠对CM311-ADC-1和CM311-ADC-2均有很好的耐受性。
实施例6 抗体与抗体药物偶联物的体外细胞活性比较
测试方法：
KATO III细胞株以5000cells/孔铺板，24小时后加入待测样品。待测样品均以终浓度1000ng/mL 2.4倍稀释9次，作用96小时，Presto-Blue显色60min后使用酶标仪读取荧光值。
待测样品如下：
Sample1：CM311，具体为CM311-18D10-VH6/VL1；
Sample2：Anti-Claudin-18.2-ADC，具体为CM311-18D10-VH6/VL1-ADC；
Sample3：非结合对照ADC，具体为IgG-L1D1(IgG-L1D1即IgG抗体偶联了L1D1小分子(即vcMMAE，也就是MC-vc-PAB-MMAE)药物的ADC)。
测试结果如图12和表4所示。从图12中可以看出，Anti-Claudin-18.2-ADC(例如CM311-18D10-VH6/VL1-ADC)显示很强的细胞杀伤作用，EC 50值为16.37ng/mL；而CM311(例如CM311-18D10-VH6/VL1)和IgG-L1D1均未显示明显的细胞杀伤作用。
表4：抗体与抗体药物偶联物的体外细胞活性比较结果(4参数拟合结果)
Curve Fit：4-Parameter
Figure PCTCN2021124698-appb-000061
Figure PCTCN2021124698-appb-000062

Claims (15)

  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，所述抗体药物偶联物具有式Ⅰ所示的结构，
    Ab-(L-D) p
    式Ⅰ
    其中：
    Ab为抗Claudin 18.2抗体，所述抗Claudin 18.2抗体包含重链和轻链，重链可变区CDR1包含选自SEQ ID NO：2、10、18、26、34、42、68、76、84、92、100、108或116所示的序列或其突变体，重链可变区CDR2包含选自SEQ ID NO：3、11、19、27、35、43、69、77、85、93、101、109或117所示的序列或其突变体，重链可变区CDR3包含选自SEQ ID NO：4、12、20、28、36、44、70、78、86、94、102、110或118所示的序列或其突变体，轻链可变区CDR1包含选自SEQ ID NO：50、58、124或132所示的序列或其突变体，轻链可变区CDR2包含选自SEQ ID NO：51、59、125或133所示的序列或其突变体，轻链可变区CDR3包含选自SEQ ID NO：52、60、126或134所示的序列或其突变体；
    D为细胞毒剂；
    L为接头，用于连接所述抗Claudin 18.2抗体和所述细胞毒剂；
    p为2.0-8.0。
  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：
    所述抗Claudin 18.2抗体的重链可变区CDR1包含选自SEQ ID NO：2、10、18、26、34或42所示的序列或其突变体，重链可变区CDR2包含选自SEQ ID NO：3、11、19、27、35或43所示的序列或其突变体，重链可变区CDR3包含选自SEQ ID NO：4、12、20、28、36或44所示的序列或其突变体，轻链可变区CDR1包含选自SEQ ID NO：50或58所示的序列或其突变体，轻链可变区CDR2包含选自SEQ ID NO：51或59所示的序列或其突变体，轻链可变区CDR3包含选自SEQ ID NO：52或60所示的序列或其突变体；
    或者，所述抗Claudin 18.2抗体的重链可变区CDR1包含选自SEQ ID NO：68、76、84、92、100、108或116所示的序列或其突变体，重链可变区CDR2包含选自SEQ ID NO：69、77、85、93、101、109或117所示的序列或其突变体，重链可变区CDR3包含选自SEQ ID NO：70、78、86、94、102、110或118所示的序列或其突变体，轻链可变区CDR1包含选自SEQ ID NO：124或132所示的序列或其突变体，轻链可变区CDR2包含选自SEQ ID NO：125或133所示的序列或其突变体，轻链可变区CDR3包含选自SEQ ID NO：126或134所示的序列或其突变体；
    或者，所述抗Claudin 18.2抗体的重链可变区CDR1、CDR2、CDR3选自如下序列组合：
    (1)SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4，
    (2)SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12，
    (3)SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20，
    (4)SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28，
    (5)SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36，
    (6)SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44，
    (7)SEQ ID NO：68、SEQ ID NO：69、SEQ ID NO：70，
    (8)SEQ ID NO：76、SEQ ID NO：77、SEQ ID NO：78，
    (9)SEQ ID NO：84、SEQ ID NO：85、SEQ ID NO：86，
    (10)SEQ ID NO：92、SEQ ID NO：93、SEQ ID NO：94，
    (11)SEQ ID NO：100、SEQ ID NO：101、SEQ ID NO：102，
    (12)SEQ ID NO：108、SEQ ID NO：109、SEQ ID NO：110，
    (13)SEQ ID NO：116、SEQ ID NO：117、SEQ ID NO：118，
    所述抗Claudin 18.2抗体的轻链可变区CDR1、CDR2、CDR3选自如下序列组合：
    (1)SEQ ID NO：50、SEQ ID NO：51、SEQ ID NO：52，
    (2)SEQ ID NO：58、SEQ ID NO：59、SEQ ID NO：60，
    (3)SEQ ID NO：124、SEQ ID NO：125、SEQ ID NO：126，
    (4)SEQ ID NO：132、SEQ ID NO：133、SEQ ID NO：134；
    优选地，所述抗Claudin 18.2抗体的重链可变区CDR1、CDR2、CDR3选自如下序列组合：
    (1)SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4，
    (2)SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12，
    (3)SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20，
    (4)SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28，
    (5)SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36，
    (6)SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44，
    所述抗Claudin 18.2抗体的轻链可变区CDR1、CDR2、CDR3选自如下序列组合：
    (1)SEQ ID NO：50、SEQ ID NO：51、SEQ ID NO：52，
    (2)SEQ ID NO：58、SEQ ID NO：59、SEQ ID NO：60；
    或者优选地，所述抗Claudin 18.2抗体的重链可变区CDR1、CDR2、CDR3选自如下序列组合：
    (1)SEQ ID NO：68、SEQ ID NO：69、SEQ ID NO：70，
    (2)SEQ ID NO：76、SEQ ID NO：77、SEQ ID NO：78，
    (9)SEQ ID NO：84、SEQ ID NO：85、SEQ ID NO：86，
    (4)SEQ ID NO：92、SEQ ID NO：93、SEQ ID NO：94，
    (5)SEQ ID NO：100、SEQ ID NO：101、SEQ ID NO：102，
    (6)SEQ ID NO：108、SEQ ID NO：109、SEQ ID NO：110，
    (7)SEQ ID NO：116、SEQ ID NO：117、SEQ ID NO：118，
    所述抗Claudin 18.2抗体的轻链可变区CDR1、CDR2、CDR3选自如下序列组合：
    (1)SEQ ID NO：124、SEQ ID NO：125、SEQ ID NO：126，
    (2)SEQ ID NO：132、SEQ ID NO：133、SEQ ID NO：134。
  3. 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：
    所述抗Claudin 18.2抗体的重链可变区FR1包含选自SEQ ID NO：5、13、21、29、37、45、71、79、87、95、103、111或119所示的序列或其突变体，重链可变区FR2包含选自SEQ ID NO：6、14、22、30、38、46、72、80、88、96、104、112或120所示的序列或其突变体，重链可变区FR3包含选自SEQ ID NO：7、15、23、31、39、47、73、81、89、97、105、113或121所示的序列或其突变体，重链可变区FR4包含选自SEQ ID NO：8、16、24、32、40、48、74、82、90、98、106、114或122所示的序列或其突变体，轻链可变区FR1包含选自SEQ ID NO：53、61、127或135所示的序列或其突变体，轻链可变区FR2包含选自SEQ ID NO：54、62、128或136所示的序列或其突变体，轻链可变区FR3包含选自SEQ ID NO：55、63、129或137所示的序列或其突变体，轻链可变区FR4包含选自SEQ ID NO：56、64、130或138所示的序列或其突变体；
    优选地，所述抗Claudin 18.2抗体的重链可变区FR1包含选自SEQ ID NO：5、13、21、29、37或45所示的序列或其突变体，重链可变区FR2包含选自SEQ ID NO：6、14、 22、30、38或46所示的序列或其突变体，重链可变区FR3包含选自SEQ ID NO：7、15、23、31、39或47所示的序列或其突变体，重链可变区FR4包含选自SEQ ID NO：8、16、24、32、40或48所示的序列或其突变体，轻链可变区FR1包含选自SEQ ID NO：53或61、所示的序列或其突变体，轻链可变区FR2包含选自SEQ ID NO：54或62所示的序列或其突变体，轻链可变区FR3包含选自SEQ ID NO：55或63所示的序列或其突变体，轻链可变区FR4包含选自SEQ ID NO：56或64所示的序列或其突变体；
    或者优选地，所述抗Claudin 18.2抗体的重链可变区FR1包含选自SEQ ID NO：71、79、87、95、103、111或119所示的序列或其突变体，重链可变区FR2包含选自SEQ ID NO：72、80、88、96、104、112或120所示的序列或其突变体，重链可变区FR3包含选自SEQ ID NO：73、81、89、97、105、113或121所示的序列或其突变体，重链可变区FR4包含选自SEQ ID NO：74、82、90、98、106、114或122所示的序列或其突变体，轻链可变区FR1包含选自SEQ ID NO：127或135所示的序列或其突变体，轻链可变区FR2包含选自SEQ ID NO：128或136所示的序列或其突变体，轻链可变区FR3包含选自SEQ ID NO：129或137所示的序列或其突变体，轻链可变区FR4包含选自SEQ ID NO：130或138所示的序列或其突变体；
    或者优选地，所述抗Claudin 18.2抗体的重链可变区FR1、FR2、FR3、FR4选自如下序列组合：
    (1)SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8，
    (2)SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16，
    (3)SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24，
    (4)SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32，
    (5)SEQ ID NO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40，
    (6)SEQ ID NO：45、SEQ ID NO：46、SEQ ID NO：47、SEQ ID NO：48，
    (7)SEQ ID NO：71、SEQ ID NO：72、SEQ ID NO：73、SEQ ID NO：74，
    (8)SEQ ID NO：79、SEQ ID NO：80、SEQ ID NO：81、SEQ ID NO：82，
    (9)SEQ ID NO：87、SEQ ID NO：88、SEQ ID NO：89、SEQ ID NO：90，
    (10)SEQ ID NO：95、SEQ ID NO：96、SEQ ID NO：97、SEQ ID NO：98，
    (11)SEQ ID NO：103、SEQ ID NO：104、SEQ ID NO：105、SEQ ID NO：106，
    (12)SEQ ID NO：111、SEQ ID NO：112、SEQ ID NO：113、SEQ ID NO：114，
    (13)SEQ ID NO：119、SEQ ID NO：120、SEQ ID NO：121、SEQ ID NO：122，
    所述抗Claudin 18.2抗体的轻链可变区FR1、FR2、FR3、FR4选自如下序列组合：
    (1)SEQ ID NO：53、SEQ ID NO：54、SEQ ID NO：55、SEQ ID NO：56，
    (2)SEQ ID NO：61、SEQ ID NO：62、SEQ ID NO：63、SEQ ID NO：64，
    (3)SEQ ID NO：127、SEQ ID NO：128、SEQ ID NO：129、SEQ ID NO：130，
    (4)SEQ ID NO：135、SEQ ID NO：136、SEQ ID NO：137、SEQ ID NO：138；
    更优选地，所述抗Claudin 18.2抗体的重链可变区FR1、FR2、FR3、FR4选自如下序列组合：
    (1)SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8，
    (2)SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16，
    (3)SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：23、SEQ ID NO：24，
    (4)SEQ ID NO：29、SEQ ID NO：30、SEQ ID NO：31、SEQ ID NO：32，
    (5)SEQ ID NO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40，
    (6)SEQ ID NO：45、SEQ ID NO：46、SEQ ID NO：47、SEQ ID NO：48，
    所述抗Claudin 18.2抗体的轻链可变区FR1、FR2、FR3、FR4选自如下序列组合：
    (1)SEQ ID NO：53、SEQ ID NO：54、SEQ ID NO：55、SEQ ID NO：56，
    (2)SEQ ID NO：61、SEQ ID NO：62、SEQ ID NO：63、SEQ ID NO：64；
    或者更优选地，所述抗Claudin 18.2抗体的重链可变区FR1、FR2、FR3、FR4选自如下序列组合：
    (1)SEQ ID NO：71、SEQ ID NO：72、SEQ ID NO：73、SEQ ID NO：74，
    (2)SEQ ID NO：79、SEQ ID NO：80、SEQ ID NO：81、SEQ ID NO：82，
    (3)SEQ ID NO：87、SEQ ID NO：88、SEQ ID NO：89、SEQ ID NO：90，
    (4)SEQ ID NO：95、SEQ ID NO：96、SEQ ID NO：97、SEQ ID NO：98，
    (5)SEQ ID NO：103、SEQ ID NO：104、SEQ ID NO：105、SEQ ID NO：106，
    (6)SEQ ID NO：111、SEQ ID NO：112、SEQ ID NO：113、SEQ ID NO：114，
    (7)SEQ ID NO：119、SEQ ID NO：120、SEQ ID NO：121、SEQ ID NO：122，
    所述抗Claudin 18.2抗体的轻链可变区FR1、FR2、FR3、FR4选自如下序列组合：
    (1)SEQ ID NO：127、SEQ ID NO：128、SEQ ID NO：129、SEQ ID NO：130，
    (2)SEQ ID NO：135、SEQ ID NO：136、SEQ ID NO：137、SEQ ID NO：138。
  4. 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：
    所述抗Claudin 18.2抗体的重链可变区选自SEQ ID NO：1、9、17、25、33、41、67、75、83、91、99、107或115所示的序列，
    所述抗Claudin 18.2抗体的轻链可变区选自SEQ ID NO：49、57、123或131所示的序列；
    优选地，所述抗Claudin 18.2抗体的重链可变区选自SEQ ID NO：1、9、17、25、33或41所示的序列，
    所述抗Claudin 18.2抗体的轻链可变区选自SEQ ID NO：49或57所示的序列；
    或者优选地，所述抗Claudin 18.2抗体的重链可变区选自SEQ ID NO：67、75、83、91、99、107或115所示的序列，
    所述抗Claudin 18.2抗体的轻链可变区选自SEQ ID NO：123或131所示的序列。
  5. 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：
    所述抗Claudin 18.2抗体的重链可变区、轻链可变区选自如下序列组合：
    (1)SEQ ID NO：17和SEQ ID NO：57；
    (2)SEQ ID NO：41和SEQ ID NO：49；
    (3)SEQ ID NO：41和SEQ ID NO：57；
    (4)SEQ ID NO：115和SEQ ID NO：131；
    优选地，所述抗Claudin 18.2抗体的重链可变区、轻链可变区的序列分别为SEQ ID NO：41和SEQ ID NO：49。
  6. 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：
    所述抗Claudin 18.2抗体的重链恒定区选自人源性IgG(如IgG1、IgG2、IgG3或IgG4)、IgM、IgA、IgD、IgA恒定区或上述恒定区的突变体，优选为人源性IgG1；
    所述抗Claudin 18.2抗体的轻链恒定区选自人源性lambda恒定区、kappa恒定区或上述恒定区的突变体，优选为人源性kappa恒定区。
  7. 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：
    所述抗Claudin 18.2抗体的重链的氨基酸序列包含如SEQ ID NO：65所示的序列， 或者包含与SEQ ID NO：65所示序列的同一性大于70％，例如大于75％、80％、85％、90％、95％、99％的序列；
    所述抗Claudin 18.2抗体的轻链的氨基酸序列包含如SEQ ID NO：66所示的序列，或者包含与SEQ ID NO：66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，p为2.0-7.0、2.0-6.0、2.0-5.0、2.0-4.0、3.0-7.0、3.0-6.0、3.0-5.0或3.0-4.0，优选地，p为3.0-4.0，例如p为3.0-3.8，优选为3.0、3.4、3.5或3.8，更优选为3.8。
  9. 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，所述细胞毒剂选自SN-38、吉西他滨(Gemcitabine)、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)、MGBA(如duocarmycin)、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；
    优选地，所述细胞毒剂为MMAE。
  10. 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)、N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(SPP)、4-(N-马来酰亚氨基甲基)-环己烷-1-甲酰基(MCC)、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)；
    优选地，所述接头为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)。
  11. 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：
    Ab包括：
    (a)重链可变区CDR1、CDR2、CDR3和轻链可变区CDR1、CDR2、CDR3，其中，重链可变区CDR1的序列如SEQ ID NO：42所示，重链可变区CDR2的序列如SEQ ID NO：43所示，重链可变区CDR3的序列如SEQ ID NO：44所示，轻链可变区CDR1的序列如SEQ ID NO：50所示，轻链可变区CDR2的序列如SEQ ID NO：51所示，轻链可变区CDR3的序列如SEQ ID NO：52所示；
    (b)重链可变区和轻链可变区，其中，重链可变区的序列如SEQ ID NO：41所示，轻链可变区的序列如SEQ ID NO：49所示；和/或
    (c)重链和轻链，其中，重链的序列如SEQ ID NO：65所示，轻链的序列如SEQ ID NO：66所示；
    L为MC-vc-PAB；和
    D为MMAE。
  12. 组合物，其包含权利要求1-11任一项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；任选地，还包含已知的用于治疗肿瘤的化疗药物、免疫治疗药物和免疫抑制剂中的至少一种；或者任选地，还包含至少一种药学上可接受的载体、稀释剂或赋形剂。
  13. 权利要求1-11任一项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或者权利要求12所述的组合物在制备药物中的用途，所述药物用于预防和/或治疗与Claudin 18.2相关的疾病；
    优选地，所述与Claudin 18.2相关的疾病为胃癌、胃食管交界腺癌、胰腺癌；
    更优选地，所述与Claudin 18.2相关的疾病为胃癌。
  14. 预防和/或治疗与Claudin 18.2相关的疾病的方法，其包括：给予有需要的受试者预防和/或治疗有效量的权利要求1-11任一项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，或者权利要求12所述的组合物；
    优选地，所述与Claudin 18.2相关的疾病为胃癌、胃食管交界腺癌、胰腺癌；
    更优选地，所述与Claudin 18.2相关的疾病为胃癌。
  15. 权利要求1-11任一项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，或者权利要求12所述的组合物，其用于预防和/或治疗与Claudin 18.2相关的疾病；
    优选地，所述与Claudin 18.2相关的疾病为胃癌、胃食管交界腺癌、胰腺癌；
    更优选地，所述与Claudin 18.2相关的疾病为胃癌。
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