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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Publication number
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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Prior art keywords
seq
chain variable
variable region
sequence
antibody
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Ceased
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PCT/CN2021/124698
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English (en)
French (fr)
Chinese (zh)
Inventor
胡朝红
李虎
陈博
徐刚
王莹
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Miracogen Inc
Keymed Biosciences Co Ltd
Original Assignee
Shanghai Miracogen Inc
Keymed Biosciences Co Ltd
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Priority to CA3198667A priority Critical patent/CA3198667A1/en
Priority to EP21879564.9A priority patent/EP4230225A4/en
Priority to CR20230207A priority patent/CR20230207A/es
Priority to AU2021359562A priority patent/AU2021359562A1/en
Priority to IL302122A priority patent/IL302122A/en
Priority to MX2023004236A priority patent/MX2023004236A/es
Priority to JP2023547736A priority patent/JP7724866B2/ja
Priority to KR1020237014724A priority patent/KR102921110B1/ko
Priority to US18/031,731 priority patent/US20230381336A1/en
Application filed by Shanghai Miracogen Inc, Keymed Biosciences Co Ltd filed Critical Shanghai Miracogen Inc
Publication of WO2022078523A1 publication Critical patent/WO2022078523A1/zh
Priority to ZA2023/04381A priority patent/ZA202304381B/en
Anticipated expiration legal-status Critical
Priority to CONC2023/0005979A priority patent/CO2023005979A2/es
Priority to US18/910,550 priority patent/US20250032633A1/en
Priority to JP2025130431A priority patent/JP2025183203A/ja
Ceased legal-status Critical Current

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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6863—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
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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    • C07K16/30—Immunoglobulins [IG], 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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    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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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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